Lung cancer remains the leading cause of death in the United States. Although the incidence of cigarette smoking is decreasing in the United States it appears to be increasing worldwide. The five-year survival rate has not improved in cases with advanced disease, but several articles have indicated that survival can be improved in cases diagnosed early by sputum cytology and chest x-ray. In cases diagnosed while the lesion is in the in-situ stage or measures less than 1 cm in diameter, surgical excision and/or radiation therapy improves survival; therefore, the early diagnosis of high-risk patients should be vigorously pursued. A recent study at a community hospital in Grand Junction, Colorado, presented 45 lung cancer cases diagnosed with positive sputum cytology and negative chest x-ray, and indicates that early diagnosis does improve survival. This study has been conducted during the past six years; 16 cases have survived three years and six cases show five-year survival.

Even though the intrinsic soft tissue contrast sensitivity of magnetic resonance imaging (MRI) affords excellent visualization of anatomic detail, certain pathologic processes may be diagnosed earlier with the administration of a contrast-enhancing agent. At present there is one agent, gadopentetate dimeglumine, GdDTPA, that has received FDA approval for use in the MR scanning of the brain and spine in human patients. This paramagnetic chelate distributes throughout the extracellular fluid space as dictated by capillary permeability so that abnormal vascularity and sites of blood-CNS barrier breakdown are highlighted. Primary neoplastic disease, metastases, meningeal extension, residual and recurrent tumor have been found to be better distinguished in MR images acquired after administration of GdDTPA. Routine administration of GdDTPA for cranial imaging has resulted in the discovery of otherwise occult lesions in approximately 3 of patients. Although the clinical utility and high therapeutic safety index of the first approved magnetic resonance contrast agent, GdDTPA, have been well established, other contrast agents, having different physical, chemical and biological properties, may offer improved sensitivity and bio-specificity. Agents currently being evaluated in vivo include: low osmolal paramagnetic chelates, superparamagnetic particles, metalloporphyrins, liposome encapsulated agents, perfluorocarbons, intravascular macromolecular chelate complexes and labeled monoclonal antibodies. Concurrent with advances in the development of new compounds, innovations in scanning hardware, pulse sequence design and image post-processing are helping to extend the efficacy of contrast media. Additional clinical experience will indicate which contrast agents and which MR techniques can best facilitate the early detection of specific neoplastic lesions.

Malignant and transforming cells can be identified by biochemical parameters which can be used to localize lesions in situ for laser surgery. These cells express unique proteins, proteins in unusual quantities, or other biochemical alterations which can be utilized to image lesions of such cells. Several methods have been identified, both in vitro and in vivo, to identify such lesions. Several antibodies were examined for their properties of tissue identification, including CEA, F36/22, and AE1/AE3. F36/22, an antibody developed by M. T. Chu against human breast cancer cells, associated with two lines of oral cancer (KB and HCPC), and against two naturally occurring human oral squamous cell cancers. CEA, an antibody developed against human colon cancer, also reacted against both cell lines and both pathological samples. AE1/AE3, developed against normal fibrous components, also reacted against the samples, but in a much less regular manner. F36/22 associated with the histologically identifiably most dedifferentiated cells at the leading edge of the invading cancer. CEA, on the other hand, associated with more quiescent, older, established cancer cells. This demonstrates that antibodies developed against cancers of different organs can be used to identify a wide variety of cancers, and may have prognostic value. F36/22 coupled to fluorescein was used to identify oral cancer cells. Other properties of cancers and developing cancers can also be exploited to identify cancers, including their over-expression of tyrosine kinase and tyrosine kinase stimulating hormones such as Epidermal Growth Factor (EGF). A model of premalignant lesion produced in the hamster buccal cheek pouch with 6 week application of DMBA over-expresses constitutive tyrosine kinase which can be demonstrated biochemically. This initiated lesion can be promoted to frank cancer by growth factors released in response to laser surgery. Preliminary results suggest that these lesions can be identified by Photofrin II uptake. This work suggests that biochemical properties of cancers can be used to identify premalignant cells.

Two series of investigations utilizing laser-induced fluorescence (LIF) in characterizing diseased tissue are presented. In one in vitro investigation the fluorescence from normal and atherosclerotically diseased arteries are studied. In another clinical study the fluorescence in vivo from superficial urinary bladder malignancies in patients who had received a low-dose injection of Hematoporphyrin Derivative (HpD) is investigated. Additionally, the fluorescence properties of L-tryptophan, collagen-I powder, elastin powder, nicotinamide adenine dinucleotide and (beta) -carothene were investigated and compared with the spectra from the tissue samples. A nitrogen laser (337 nm) alone or in connection with a dye laser (405 nm) was used together with an optical multichannel analyzer (OMA) to study the fluorescence spectra. The fluorescence decay characteristics of atherosclerotic plaque were examined utilizing a mode locked argon ion laser, synchronously pumping a picosecond dye laser. A fast detection system based on photon counting was employed. The fluorescence decay curves were evaluated on a PC computer allowing up to three lifetime components to be determined. A fluorescence peak at 390 nm in fibrotic plaque was identified as due to collagen fibers, while a fluorescence peak at 520 nm was connected to (beta) -carotene. The in vivo measurements of urinary bladder malignancies were performed with the optical fiber of the OMA system inserted through the biopsy channel of a cystoscope during the diagnostical procedure. The spectral recordings from urinary bladders, obtained at 337 nm and 405 nm excitation, revealed fluorescence features which can be used to demarcate tumor areas from normal mucosa. The fluorescence emission might also be useful to characterize different degrees of dysplasia.

Carcinoma in situ and early lung cancer have been detected by fluorescence imaging, a ratio fluorometer, and a realtime fluorescence spectrum analyzer. Positive results have been obtained with all three methods, but there is less experience with the spectrum analyzer. The latest development is to ratio red-to-green in a pixel-by-pixel fashion, combining the advantages of the imaging technique and the red-to-green ratio fluorometer.

Laser induced fluorescence has been explored as an early detection scheme for two clinically important examples of neoplasia: colorectal dysplasia and transitional cell carcinoma in the urinary bladder. In both, it is desirable to detect microscopic and biochemical changes of pre-cancer in order to identify patients at risk for developing invasive carcinoma. This paper will compare the fluorescence obtained from these two pre-cancerous conditions, and discuss the connection between the fluorescence and the morphological/molecular changes occurring in the tissue. The similarities and differences in the fluorescence will be compared to determine the general features of pre-cancerous changes that might be utilized for detection of the disease.

An in vitro model with blood circulating in a silicon tubing system and including an artificial arterial bed is an important tool for evaluation of the pulse oximetry technique. The oxygen saturation was measured on an artificial finger using a pulse oximeter (SpO2) and on blood samples using a hemoximeter (SaO2). Measurements were performed at different blood flows and at different blood hematocrits. An increase in steady as well as in pulsatile blood flow was followed by an increase in pulse oximeter readings and a better agreement between SpO2 and SaO2 readings. After diluting the blood with normal saline (decreased hematocrit) the agreement was further improved. These results indicate that the pulse oximeter signal is related to blood hematocrit and the velocity of blood. The flow-related dependance of SpO2 was also evaluated in a human model. These results provided evidence that the pulse oximeter signal is dependent on vascular changes.

The growth of microscopic tumor lesions at or beyond treatment field lesions poses major problems in the diagnosis and curative treatment of numerous cancers. Early detection techniques which clearly define the extent of condemned or field spread of disease may improve the primary treatment of the disease. In vivo fluorescence photometry is a non-imaging technique which digitally displays relative fluorescence values in volts. The sensitivity of the instrument has allowed the detection of micrometastases in both pre-clinical and clinical studies using drug doses that are 80-90 lower than those used therapeutically. This technique is now being applied in preliminary experiments to the hamster cheek pouch models to (1) discern varying grades of dysplasia; (2) levels of uptake of the drug in normal growing and quiescent tumors. Results will be shown in two models in which this technique has shown to be efficacious preclinically in the Pollard rat adenocarcinoma model in which micrometastases in the lymph node have been detected, and preliminary studies involving the hamster cheek pouch model in which the pouch is painted with 9, 10 dimethyl-1, 2-benzanthracene (DMBA) for initiation and promotion of tumors. Clinically results will be shown in which fluorescence detection, confirmed by biopsy and histopathological examination, was capable of detecting the existence of micrometastatic involvement of less than 100 cells.

The authors studied the ability of Laser Induced Fluorescence Spectroscopy (LIFS) for the intraoperative identification of metastases using a photosensitizing agent Photofrin IIr to enhance spectroscopic detection. A He-Cd laser source (442 nm) was used to produce low-power illumination of tissue via a hand-held 400 micrometers fiberoptic probe. Through the same fiber, reflected and emitted light was returned to an optical multi-channel analyzer (OMA III) for analysis. Spectroscopic signals were displayed on a screen for immediate examination. Lobund Wistar rats, inoculated with Pollard rat adenocarcinoma cells, were used as an animal model. Photofrin IIr was administered intraperitoneal 24 or 48 hours prior to surgical exploration in doses varying from 0.75-7.5 mg/kg. Metastases detection was performed during abdominal exploration directed to ipsilateral and contralateral inguinal, iliac, para-aortic and renal lymph nodes. Nineteen tissue samples, identified as abnormal by LIFS, were removed for histologic analysis; 11 of these samples were larger than 5mm and histologic examination revealed malignancy in all cases. While LIFS signals showed malignancy in 8 tissue samples with dimensions less than 5mm, histology confirmed this in only 3. However, serial histologic sections were not performed. From the initial results, it was concluded that LIFS detection of malignant tissue is feasible and enhanced by the addition of Photofrin IIr. LIFS may be a promising technique for the intraoperative detection of primary malignant and metastatic tissue.

Over the last decade considerable interest has developed in the use of exogenously administered chromophores in combination with visible light for the treatment of human tumors. Whether cells are killed directly, or indirectly as a result of disruption of the tissue, microvasculature is unknown. The authors have developed methods to assess in short term culture the effects of PDT on precision cut tissue slices. The use of these tissue slices provide an important link between in vivo studies and in vitro isolated cultured cells for the following reasons: 1) slices contain all of the normal cells in their proper in vivo architectural arrangement; 2) since slices can be obtained relatively easily and in a very short period of time (a few minutes), animals can be treated with compounds in vivo, the tissue can be removed, sliced and mechanistic studies performed in vitro (without the several hours delay required to produce cultured cells); 3) in vitro comparisons between species, including human, can be readily made; and 4) mechanisms of PDT-induced cell killing can be studied in the absence of a functioning vascular system. Using this in vivo drug/in vitro light system, the results presented will detail the findings using normal rat liver and a transplantable rat tumor model.

The validity and utility of the measurement of the photodynamic threshold dose as an in vivo tool for quantitatively assessing the effectiveness of performing photodynamic therapy (PDT) using differing photosensitizers, irradiation protocols and adjuvant treatments was examined. The depth of the boundary between necrotic tissue and normal tissue following surface irradiation of normal rat liver was used to calculate the photodynamic threshold, a quantity independent of photosensitizer concentration, light fluence and wavelength. PDT was performed using Photofrin II to investigate the changes in photodynamic threshold using four different treatment conditions. Using a range of values for photosensitizer concentration and total light fluence, the photodynamic threshold was found to be independent of these parameters and estimated as (3.4 +/- 0.3) X 1018 photons cm-3. Using periodic light irradiation schedules to determine if diffusion of 3O2 affects the threshold measurement, the photodynamic thresholds were found not to be significantly different from those using normal irradiation which suggests that this model is not sensitive to this parameter. PDT was performed in conjunction with hyperthermia and significant reductions in the photodynamic threshold were obtained if hyperthermia followed PDT but not if hyperthermia preceded PDT. Finally, the effects of the radio-protecting agents WR-77913 and WR-2721 were examined and significant increases in the photodynamic threshold were obtained for PDT performed following injection of sufficient quantity of either radio-protecting drug.

Investigation is made of the optical attenuation depth in Fisher rat brain at different wavelengths and during PDT treatment. The attenuation depth gradually increased from 0.6 to 1.7 mm when the irradiating light wavelength increased from 450 nm to 710 nm. During PDT treatment (Photofrin II 48 hours prior to 633 nm, 5 mm diameter surface irradiation of 100 J/cm2 and 100 mW/cm2), the attenuation depth was found relatively constant (variation

Porphyrin-like compounds such as tetra-(4 sulfonatophenyl) porphine (TSPP), chlorin-e6 derivative (CED) and tetrasulfophthalocyanin (TSPC) are photosensitizing agents that absorbed light at 630 nm, 657 nm and 625 nm, respectively, and bind preferentially to atherosclerotic plaque. Porphyrin-treated human cadaveric aorta was compared with untreated aorta using several techniques: the absorptive spectrophotometry which has demonstrated the distinct absorptive peaks at 630 nm, 657 nm, 625 nm in porphyrin-treated plaque which were absent in untreated normal aorta; the fluorescence microscopy, which has shown that the treated atheroma acquired the characteristic fluorescence of porphyrin under ultraviolet light. Because of the porphyrin''s unique property, porphyrin-treated and untreated aorta was exposed to red laser radiation at a wavelength of 632.8 nm. It was found that enhanced photoalteration of porphyrin-treated compared with untreated atheroma. Histologic analysis of the depth of tissue penetration of equal amounts of laser radiation has demonstrated more pronounced photosensitizing effect in TSPC-treated plaque compared with CED-, TSPP-, and untreated plaques. This study demonstrates a potential of tetrasulfophthalocyanin for selective alteration of atheroma by He-Ne laser radiation.

Three tumor models were used to study the microlocalization of two photosensitizers, Photofrin and TPPS4 (mesotetra[p-sulphophenyl]porphyne). The three tumor types employed were subcutaneously and intramuscularly grown murine Lewis lung carcinoma, and lung micrometastases, grown on the same animal. The objective of the study was to determine whether these photosensitizers localize differently in these three tumor types. Common to all the tumors was the observation that no substantial photosensitizer fluorescence could be found in the tumor parenchyma. With Photofrin, most of the drug fluorescence was found in the periphery of the tumors, especially around blood vessels and possibly in some macrophages. On the other hand with TPPS4, most of the fluorescence was seen in the basement membrane of blood vessels and airways. The photosensitizer TPPS4 may therefore accumulate less specifically in tumor microenvironment than Photofrin. Both photosensitizers were also found to localize in necrotic areas.

Plasma samples were obtained from 4 patients at intervals after administration of Photofrin II (2 mg/kg). Total porphyrin in plasma and protein/lipoprotein fractions was estimated after hydrolysis, and levels of different Photofrin II components were measured by HPLC. Pharmacokinetic data are provided relating to rates of loss of total porphyrin from plasma and to pool size and half-lives of sensitizers bound to albumin and lipoprotein fractions. Preferential persistence of the most hydrophobic components of Photofrin II was observed.

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.

Many photosensitizers under investigation for possible use in PDT share the property of selective accumulation in malignant or abnormal tissues. However, the mechanisms by which this occurs is not understood. Although it has been established that singlet oxygen is responsible for cell killing once a photosensitizer has reached the target tissue, the cellular targets and molecular mechanisms also remain unknown. The mechanisms of selective accumulation and cytotoxicity may depend upon the photosensitizer used. In attempts to develop technology to improve selective uptake by target tissues, the authors address the question of cellular targets at a preliminary level. These studies were performed using one benzoporphyrin derivative analogue, benzoporphyrin mono-acid ring A (BPD-MA). Plasma distribution of BPD-MA shows that this lipophilic photosensitizer has a propensity to associate with plasma lipoproteins in blood. Biodistribution studies indicate that preincubation of BPD-MA with low density lipoprotein (LDL) leads to significantly greater tumor accumulation than BPD-MA in aqueous solution. Further, the technology for conjugating BPD-MA to monoclonal antibodies has been established in the laboratory. Selective photodynamic killing of a human squamous cell carcinoma cell line (A549) has been demonstrated using such a conjugate. The monoclonal antibody (MoAb) used, 5E8, has specificity for a glycoprotein detected on human squamous cell carcinoma of the lung. In this study these two delivery systems are used to compare the cytotoxicity of membrane bound and internalized photosensitizer at the cellular level. The results indicate that LDL or MoAb mediated delivery increases the selectivity and cytotoxicity of BPD-MA. Internalization of BPD-MA via either delivery mode produces significantly enhanced cell killing.

The photodynamic therapeutic properties of the oxygen containing benzophenoxazine dye Nile Blue E, NBEO, and its sulfur and selenium analogs, NBES and NBESe, were evaluated in cell culture using a murine mammary sarcoma (EMT-6) cell line. The photosensitizing efficacies of the dyes were found to correlate with their ability to generate singlet oxygen and to increase in the order: NBEO

The benzophenoxazines, including several Nile blue analogues, are a unique group of dyes that localize selectively in animal tumors. Chemical modifications of Nile blue A can yield derivatives with high 1O2 quantum yields. These derivatives represent a group of potentially effective photosensitizers for selective phototherapy of malignant tumors. In vitro evaluation of these derivatives has indicated that those with high 1O2 yields are very effective in mediating the photocytotoxicity of tumor cells. This photodynamic effect is most likely mediated through the action of 1O2, since photoirradiation under D2O enhanced and under hypoxic conditions diminished the photocytotoxic action. The subcellular localization of these photosensitizers in bladder tumor cells in culture was examined by light and fluorescence microscopies as well as by histochemical and biochemical studies. The results indicate that these dyes are localized primarily in the lysosome. The cellular uptake and retention of these dyes is energy- and pH-dependent. Agents such as nigericin, which alter the transmembrane pH gradient, reduced uptake and enhanced efflux of the dyes, while agents such as valinomycin, which reduce cellular membrane potential, had no effect on the uptake. These findings are consistent with having ion-trapping as the mechanism for the uptake of these dyes. Photoirradiation of sensitizer-treated cells obliterated lysosomes in a light-dose and drug-dose dependent fashion. Release of the hydrolytic enzymes may be the main cause for subsequent cell death since the cytolytic effect was reduced by a specific inhibitor of lysosomal proteolytic enzyme. A lysosomotropic photosensitization mechanism is therefore proposed for the photocytotoxic action of the Nile blue derivatives. This mechanism may provide an approach to the development of new photosensitizers for the effective and selective destruction of malignant tumors.

In a seminal series of papers, Meruelo and co-workers have described the potent antiretroviral effect of hypericin. Interestingly, hypericin was found to inhibit not only the production of infectious virus from chronically infected cells, but was also shown to directly inhibit reverse transcriptase activity of mature virions. The effect of hypericin on cells chronically infected with equine infectious anemia virus (EIAV), a retrovirus genetically related to HIV, is demonstrated. At concentrations of 10 (mu) g/ml, hypericin reduced production of infectious EIAV by 99.99 without causing obvious cytopathic effects. Interestingly, the results indicated that the antiretroviral activity of hypericin was wholly dependent on the presence of light. No decrease in viral infectivity was observed when hypericin and virus were incubated in the dark. Moreover, it appeared that light was an absolute requirement for the antiviral activity, as even high concentrations of hypericin (10 (mu) g/ml) were unable to reduce infectivity of as few as 100 infectious virions.

Phthalocyanines are receiving increasing attention as second-generation sensitizers for photodynamic therapy (PDT). This paper discusses some of the investigations into the mechanism of the phototoxic responses of phthalocyanine-sensitized PDT exploiting the interaction of PDT with other metabolic modulators. Among the agents which interact strongly with PDT is the K+/H+ ionophore nigericin. Under the conditions studied with chloroaluminum phthalocyanine (AlPcCl), the Na+/H+ ionophore monensin, the Ca++ ionophore A23187, and the lysosomotrophic agent chloroquine, but not the K+ ionophore valinomycin, also potentiate photodynamic cell killing. None of the latter compounds interact with PDT as strongly as does nigericin. Both nigericin and monensin partially inhibit cellular respiration; however, KCN, which inhibits respiration completely, is less effective in potentiating PDT damage than is nigericin. Nigericin treatment alone does not deplete glutathione; however, the GSH level decreases after treatment of cells with PDT and nigericin. The potentiation of the PDT response is much greater at an extracellular pH (pHe) of 6.70 than at pHe 7.30. When nigericin is present at pHe 6.70, the intracellular pH (pHi) is equilibrated with pHe. None of the other ionophores tested was able to cause the acidification of the intracellular milieu as did nigericin. The evidence to date suggests that the lowering of pHi is an important component of the mechanism by which nigericin potentiates PDT.

Sixty-three patients with malignant brain tumors were treated with intraoperative photodynamic therapy (PDT) using an argon dye pump laser and preoperatively administered hematoporphyrin derivative or dihematoporphyrin ether. In 13 cases, in addition to cavitary photo-illumination, cylindrical diffusion fibers were used to increase the amount of light energy administered to the tumor tissue intraoperatively. This interstitial photo-illumination was tolerated at light energy densities of less than 450 J/cm. In six recent cases, all of whom had large malignant gliomas and could not be illuminated adequately at a single session, cylindrical diffusion fibers were left in situ after intraoperative cavitary photo-illumination of the tumor residuum. The fibers were protected from fracturing by placing all but the exposed diffusing end in a red rubber catheter of the appropriate diameter. The fibers were externalized through a separate stab wound as would be the case for a ventricular drain. Photo-illumination was continued one or two days post-operatively. The optimal fiber couple to the argon dye pump laser was achieved by assessing the fiber side scatter with a photometer. These six patients received 585-2730 Joules during the post-operative photo-illumination. The patients tolerated the fractionated photo-illumination well. A transient scalp inflammation occurred as the consequence light transmission to skin from the implanted fibers in one case. The median survival for the whole series was 8.5 months (40 weeks) with a 1- and 2-year actuarial survival rate of 33, respectively.

Skin cancer is epidemic in the United States. A recent national cancer survey identified over 400,000 new cases of skin cancer in a single year and estimated that 40-50% of people who live to be 65 years of age will develop at least one skin cancer.1 The majority of these tumors are basal cell carcinomas. While current treatment modalities provide satisfactory cure rates for these lesions, the deformities following surgery can be devastating and provide an unsatisfactory quality of life. The author is aware of the anecdotal stories of two patients who have committed suicide following multiple surgical resections and irradiation for multifocal basal cell carcinoma. Photodynamic therapy (PDT) is a relatively new, experimental method of treatment for basal cell carcinomas that has been used by this author since 1 982. Because of its selective treatment nature, PDT has the potential to cause less deformity than surgery in certain patients by treating and preserving normal skin surrounding the areas of tumor that would necessarily be resected in the surgical margins.

Patients with endobronchial occlusion commonly experience dyspnea, cough, hemoptysis, pneumonitis, and atelectasis. If luminal patency is not re-established, obstructive symptoms may progress to sepsis and death. Although the overall survival of patients with lung cancer may not be altered by relief of airway obstruction, the prognosis for this subset of patients may be improved by eliminating the septic complications of bronchial occlusion. Techniques to treat occluded bronchi include electro-fulguration, cryotherapy, brachytherapy, laser (CO2, Nd-YAG) therapy, and photodynamic therapy (PDT). These represent local forms of treatment and are intended to be palliative. Nd-YAG and PDT are the modalities more frequently utilized in this setting. Comparison of the two treatment forms may furnish insight regarding the appropriate role for each as individual therapies and as part of the armamentarium of cancer therapies.

Eighty-eight patients with stage III endobronchial squamous cell cancer were treated with Nd:YAG laser therapy alone (49), photodynamic therapy (PDT) alone (14), or a combination of Nd:YAG and PDT therapy (25). Mean survival rates were as follows: PDT equals 4.8 months, Nd:YAG therapy equals 3.3 months, PDT followed by Nd:YAG equals 10.9 months, Nd:YAG followed by PDT equals 9.0 months, and all combination therapy equals 9.2 months. The difference between survival rates for combination therapy and Nd:YAG only was statistically significant (P equals 0.003). The authors recommend a combination of PDT and Nd:YAG laser therapies in patients who have undergone or are not amenable to conventional therapies for this tumor. Further, the authors recommend that these patients be treated early, before the tumor spreads to other bronchi or becomes large and bloody, in order to reduce the risk for perioperative complications.

Nine patients were treated with red light whole bladder photodynamic therapy (WBPDT): five had mucosal involvement (Ta) and four submucosal invasion (T1). Patients received slow intravenous injection with 2mg/kg body weight of photofrin 48-72 hours before undergoing global light treatment via a 22-French cystoscope with a 400-micron quartz fiber bulb (isotropic) tip fiber. Three months after PDT, eight of the patients had normal cystoscopy, and negative biopsy and urine cytology. Two patients who had recurrences at six and twelve months were retreated with a higher dose (20 J/cm2). They had no increased morbidity and no evidence of recurrent disease six months later. WBPDT should be considered as an important alternative treatment for patients who have recurrent or refractory superficial bladder cancer.

The treatment of non-invasive bladder carcinoma using Photofrin (sodium porfimer) and laser photodynamic therapy (PDT) is described. The patients described who were treated with this modality had positive cytology and biopsy-proven transitional cell carcinoma, no histologic evidence of muscle invasion, and negative excretory urograms.

Although clinical trials in photodynamic therapy (PDT) have been ongoing for over a decade, attempts to apply for approval of the therapy from boards of health for general use began only in 1989. The steps which are being taken to approve PDT for the treatment of endobronchial lung cancer, superficial bladder cancer and esophageal cancer are described. Technological innovations which have been suggested as increasing the ease of use of PDT as a therapeutic modality are briefly discussed.

Eleven pets with squamous cell carcinomas of the head and neck were treated with PDT using 3.0 mg/kg Photofrin II and a light dose of 300 J/cm2 at 632 nm. All tumors had measurable disease that was recurrent following at least one surgical removal. Ten of the eleven patients achieved a complete remission (CR). Of the ten animals with CR, two relapsed at 11 or 13 weeks while the other eight maintained their CR. Six animals remain disease free to the present time, with durations of 65, 56, 56, 56, 50 and 13 weeks. One died of unrelated causes at 12 weeks, and a second animal died of metastatic disease at 8 weeks. In these two cases, extensive histologic evaluation of the treated areas failed to demonstrate any evidence of residual tumor. Toxicity to the tumor bed or surrounding normal tissue was minimal. When the tumor was located adjacent to bone of the mandible or maxilla, the bone was at risk to form sequestra (in 3/5 cases). Based on these preliminary results, PDT may have applications for human patients with carcinoma of the head and neck.

The syntheses of three new cadmium(II)-containing tripyrroledimethine-derived, ''texaphyrin''-type, macrocycles, 14,15-dicyano-4,5,9,20-tetraethyl-10,19-dimethyl-13,16,21,22,23- pentazatetracyclo[16.2 .1.13,6.18,11]tricosa-1,3,5,7,9,11(23)12,14,16,18(21),19-undecaene (1), 4,5,9,28-tetraethyl-10,27-dimethyl-13,24,29,30,31- pentaazahexaacyclo$B24.2.1.13,6. 18,11.014,23.016,21]-hentriaconta-1,,5,7,9,11(31),12,14(23),15,17,19, 21,24,26(29),27-pentadecaene (2), and 4,5,9,32-tetraethyl-10,31-dimethyl-13,28,33,34,35- pentaazaheptacyclo$B28.2.1.13,6. 18,11.014,27.015,20.021,26]pentariconta-1,3,5,7,9,11(35),12,14( 27),15(20),16,18,21(26),22,24,28,30(33,31-heptadecaene (3), are discussed. These compounds, which contain extended (pi) -systems, possess strong, low-energy, Q-like absorption bands in the 650-900 nm spectral region, making them of possible interest in photodynamic applications. Time-resolved absorption and emission laser studies have been carried out and the fluorescence and triplet absorption spectra, excited state yields and lifetimes, O2-quenching rate constants, and quantum yields for formation of O2(1(Delta) g) measured and related to the structure of the macrocycle. The results of preliminary cell localization and in vitro photodynamic anti-viral, anti-bacterial, and anti-leukemic cell inactivation studies carried out with these and/or the parent (omicron) -phenylenediamine-derived parent texaphyrin system, 4, are also presented.

Syntheses and chemical characterization of a number of new porphyrin-derived sensitizers related to ''benzoporphyrin derivative'' (BPD) are described. The work discussed centers in solving the isomer problem inherent in BPD synthesis from protoporphyrin IX, with regard to the 2- and 4-vinyls and the 6- and 7-propionic acid groups (Fischer nomenclature).

Methods for the preparation of three siloxysilicon naphthalocyanines, a siloxygermanium tetraphenoxynaphthalocyanine, a siloxysilicon tetra-t-butyl-naphthalocyanine, a cofacial double-ring siloxysilicon naphthalocyanine, and a cofacial triple-ring siloxysilicon naphthalocyanine that are relevant to the search for second generation PDT sensitizers are outlined. Ultraviolet-visible spectra, NMR spectra, and other properties of these compounds are given and discussed. In addition, an attempt to make an aluminum tetradibenzobarreleno-naphthalocyanine is described.

For a number of well-defined reasons, research into the development of alternative photosensitizers to PhotofrinTM, for use in Photodynamic Therapy, continues. The efforts described in this paper cover the study of a number of sensitizers absorbing at different wavelengths, which may have selective applications in the field of photomedicine. In one approach, the use of tetraazoporphyrins (porphyrazines) as sensitizers is studied. Although related tetramacrocyclic systems such as porphyrins and phthalocyanines have been, and continue to be, well studied in this field, porphyrazines have, to-date, received little attention. In a second study, purpurins are examined as possible alternatives to PhotofrinTM. In a recent study, tissue distribution properties of tin etiopurpurin dichloride, as a function of time, were examined. Results indicate that this purpurin continues to show promise for applications in PDT.

Efficient methods for the syntheses of porphyrin and chlorin dimers joined with variable length carbon units, either at meso positions or at (beta) -pyrrolic positions, are described. The in vivo testing results for some of the dimers are also discussed. The main objective of this work is to understand the structure/activity relationships among carbon-carbon linked dimers of porphyrins and chlorins. It is anticipated that this study will assist the authors in designing photosensitizers with required chemical and photochemical properties.

Cytotoxicity of sulfonated Chlor-.Aluminium Phthalocyanine (C1A1SPc) was investigated for 2 bladder tumor cell lines and for normal bladder wall cells as control. The particular interest has been focussed on the correlation of the sensitizer's uptake with the incubation time and on the subcellular morphologic changes following PDT. Cells were cultivated in monolayer cultures using DMEM + 15 % FCS. For the assessment of the cellular uptake, the cells were incubated with 40 micro-g CIAISPc for 2, 4, 6 and 24 hours respectively. The sensitizer was then extracted from the cells using 3ml 0.1 m NaOH for 1 hour at 37 °C. Concentration of the sensitizer was then calculated measuring absorption of the extracted substance at 670 nm with a Zeiss photometer. To evaluate the subcellular changes, an incubation time of 4 hours was chosen. Following PDT (100 mW/cm2 for 2 minutes) the cells were assessed under the electron microscope in transmission technique. To identify and assess the mitochondria in particular, cells were stained with DAB. As expected, the uptake of CIA1SPc in vitro depends on the incubation time. Rapid increase in concentration can be demonstrated from 2 to 4 hours incubation time, whereas following 6 and 24 hours the increase is less marked. Nevertheless, a short incubation of 2 or 4 hours, which would be realistic for a topic application of the sensitizer under clinical conditions (bladder tumors!), already yields a significant and probably sufficient intracellular concentration for subsequent PDT. Following PDT, most of the investigated cells show significant changes of the mitochondria which is confirmed by DAB staining. The number of intact mitochondria in treated cells is significantly smaller than in non-treated cells.

Red blood cells from BALB/c mice were labeled with short-lived (gamma) -emitting 99mTc and injected in EMT-6 tumor-bearing animals as a probe for the blood circulation. 99mTc-concentrations in light-exposed versus non-treated tumors were established for different photosensitizers at various dye doses and time intervals post-photodynamic therapy. At minimal dye doses for tumor cure, Photofrin IITM induced a doubling of the tumor radioactivity within the first hour post-PDT, followed by a marked decrease in the 99mTc-concentration at 24 hours post PDT. This pattern is characteristic of extensive hemorrhage followed by occlusion of the blood vessels. Under similar conditions the mono- and tetrasulfonated zinc phthalocyanines induced tumor response without prolonged effects on the tumor radioactivity levels. Accordingly, with the latter sensitizers, indirect cell kill via obstruction of the blood supply does not appear to constitute a major factor in eliciting a tumor response.

The photosensitization mechanism and kinetics of hematoporphyrin derivative (HPD), photofrin II (PF-II) and Zn2+-phthalocyanine tetrahydroxyl (ZnPC) are compared with the newly introduced sensitizers Zn2+ and Mg2+-tetrabenzoporphyrin (ZnTBP and MgTBP), in lipid vesicles. 9,10-dimethyl anthracene was used as a fluorescent target for singlet oxygen-mediated photosensitization. From the kinetics the quantum yield of singlet oxygen generation was calculated. MgTBP is compared with hematoporphyrin for the ability to photodamage bacterial cells and human cancer cells. MgTBP was found to have a comparable capacity for such a biological effect.

The photodvnmtc prooerttes o+ ch1or.ne e trtmetnvi ester(Eá methyl pheophorbid a (MPh) and pheophvtin a (Ph) in Ehrllch carcinoma cells were investigatea ooth in vitro ana in vivo. Mu above mentioned ohotoseneitizere(PS) induced the hotoirradiated death o4 neoolaem cciii. There was an inverse relationshlo between the hvdroDhobictty of the PS and ite ohotodvnamic activity Uess hvbroahobic Eó was shown to be most eF4ective PSI'. ccoraina to our data irradiation of Ehrlici, carcinoma cefle in the oresence of E. causea a aramatic aecrease in ATP level. The latter could be connected with suporession of the intensity of anaerobic glvcolvsis ana conEumDtion of oxygen. It could be supposeb that a decrease in AlP level in Ehrlich carcinoma cells is mainly due to th irncouplinç of OxIdiitive phophory1aition. At the same time. upp1ementrv addition of doxorubicin a chemical decreaging thea functional activity of mitochondria) to E increased both in vitro and in vivo of phototoc i ci ty of Eo. The key rol e of an enerceti c status of tumour cells in their resistance to the photodynamic action is proposed.

The authors have theoretically designed and analyzed the optical components and mechanical structures for an imaging x-ray microscope which will be configured to operate in the water window. This instrument affords new and noninvasive strategies for examining living tumor cells without the use of dyes, stains or other exogenous chemicals, which can produce limiting artifacts. The Water Window Imaging X-Ray Microscope is based on doubly reflecting, normal incidence multilayer optical technology, such as has been previously employed in telescopes for high resolution x-ray imaging of the sun. Multilayer coatings have now been fabricated with near theoretical reflectivities and perfect bandpass matching for the new rocket-borne solar observatory, the Multi-Spectral Solar Telescope Array (MSSTA). These telescopes employ multilayer mirror substrates with sub-angstrom (rms) smoothness which are made possible by Advanced Flow Polishing. Recent developments in multilayer coating technology and Advanced Flow Polishing methods used with Zerodur and Hemlite grade sapphire have paved the way for the development of the Water Window Imaging X-Ray Microscope. In this narrow water window wavelength regime of the x-ray spectrum, which lies between the K absorption edges of oxygen (23.3 $ANS) and of carbon (43.62 $ANS), water is relatively highly transmissive and carbon is highly absorptive. This principle allows the Water Window Imaging X-Ray Microscope to delineate, with high resolution and high contrast, carbon-based structures in the aqueous physiological environments found within living cells. The theoretical design and analysis of the microscope optical and mechanical components are described and the fabrication effort underway for the development of this new optical instrument, which should improve diagnosis and greatly benefit experimental studies of tumor cell biology, is discussed.

The prognosis for cancer patients becomes much better if a tumor is diagnosed, localized and treated early, in a precancerous stage. The difficulty lies in the localization of cancerous tumors. Carcinoma in situ (CIS) refers to a tumor which is approximately 100 microns thick and one which has not penetrated through the epithelium wall or become invasive (2). A tumor of this size cannot be detected by existing techniques such as x-ray, computer tomography, magnetic resonance imaging, nuclear medicine or conventional endoscopy under white-light illumination. However, these tumors can be localized and destroyed by photodynamic diagnosis and therapy. This research shows that digital image processing and the technique of digital image ratioing contribute to photodynamic diagnosis and the early localization of cancer. A software package has been developed as a result of this research. The software package quantifies the usefulness of digital image processing for tumor localization and detectability. System parameters such as the endoscope distance and angle variations, tumor size and tumor concentration, sensitivity and specificity of the system have been tested and quantified.

The goal of this study was to investigate laser induced fluorescence spectroscopy (LIFS) as a method of localization of atherosclerotic lesions not visible by angiography using Photofrin IIr enhanced fluorescence. Twenty-four New Zealand White rabbits divided into six groups varying in type of arterial wall lesion and Photofrin IIr administration time (i.v.) were used. Aortic wall fluorescence signals were acquired from the aortic arch to iliac bifurcation. The output of a He-Cd laser (442 nm, 17 mW) was directed at the arterial wall through a 400 micron fiber. The fluorescence signal created in the arterial wall was collected via the same fiber and analyzed by an optical multi-channel analyzer (OMA). The ratio of fluorescence intensities at 630 nm (Photofrin IIr) and 540 nm (autofluorescence of artery wall) was analyzed (I630nm/I540nm). Intensity ratio values 24 hours after administration of Photofrin IIr were found to be as follows: in normal artery wall of 0.30 +/- 0.14 (n equals 3), in mechanically damaged wall of 0.91 +/- 0.65 (n equals 2) and, in atheromatous tissue, 0.88 +/- 0.54 (n equals 4). The intensity ratio of atheromatous tissue without Photofrin IIr was 0.23 +/- 0.04 (n equals 7). These results suggest that the use of Photofrin IIr allows in vivo atheroma detection by LIFS because of its ability to accumulate in atheroma. In addition, accumulation of Photofrin IIr was found in artery walls traumatized by balloon catheter intervention. Using this method, a catheter-based LIFS system may be developed for atheroma detection.

Results on tunable diode laser spectroscopy applications in analysis of human expiration are presented. Carbon monoxide concentrations in exhaled air of several non-smoking adults were measured with high sensitivity. Obtained CO contents slightly exceed atmosphere level and correlate with it. Simultaneous measurements of carbon monoxide and carbon dioxide were carried out using single diode laser generated at 4.7 microns. Dependences of their concentration in exhaled air on breath-holding time were investigated.