This presentation does not attempt to review the vast field of technology adapted for photodynamic therapy (PDT). The goal, which is rather more modest although not less ambitious, is to try to highlight different aspects of clinically relevant technological applications. We hope in this attempt to bridge the gap separating basic scientists, engineers, various high tech manufacturers, and clinicians and thus bring the benefits of photodynamic therapy closer to the patients. Arbitrarily then, we concentrate on three major aspects: (1) Energy sources and appropriate therapeutic wavelengths. (2) Delivery devices which include superficial, intercavitary, and intravascular applicators. (3) Dosimetry and more specifically the dosimetry of energy delivered, absorbed, and distributed in the tissues; the dosimetry of the photosensitizers in vivo as well as the relationship between the dosimetry of the delivered stimulating energy and the rate of photobleaching. We also briefly discuss the diagnostic fluorescence.

Photodynamic therapy using 5-aminolevulinic acid-induced photosensitization (ALA PDT) via endogenous protoporphyrin IX (PpIX) synthesis has been reported as efficacious, using topical formulations, in the treatment of a variety of dermatologic diseases including superficial basal cell carcinoma, Bowen's disease, and actinic (solar) keratoses. Application of ALA PDT to the detection and treatment of both malignant and non-malignant diseases of internal organs has recently been reported. Local internal application of ALA has been used for the detection, via PpIX fluorescence, of pathological conditions of the human urinary bladder and for selective endometrial ablation in animal model systems. Systemic, oral administration of ALA has been used for ALA PDT of superficial head and neck cancer and of colorectal cancer. This paper reviews the current clinical status of ALA PDT.

The chemical and biological properties of 5,10,15,20-tetrakis(m-hydroxyphenyl)chlorin as a PDT photosensitizer are discussed, and its promise in clinical applications is outlined.

Aminolaevulinic acid (ALA) induced porphyrin biosynthesis and the resulting in vitro phototoxicity have been determined in both SV40 transformed Swiss mouse 3T3 fibroblasts and PtK₂ epithelial cells. Both cell lines respond to the addition of exogenous ALA, producing porphyrin linearly with ALA concentrations up to 0.3 mM. Notably the only accumulating porphyrin detected by HPLC was PpIX. Although the levels of PpIX are both dependent on the time and concentration used, the final intracellular porphyrin concentration is dictated by the presence of serum. When ALA is added in medium containing 10% new born calf serum, 90 - 95% of the induced porphyrin appears in the incubation medium. In the absence of serum, the intracellular PpIX levels are maintained and only under these conditions can successful in vitro PDT be performed. Gel permeation chromatography has indicated that the afflux of PpIX is promoted by the low density and high density lipoproteins, with an unknown protein (mw < 66000) contributing significantly to the effect seen. It appears that this protein is present at very low concentrations in both foetal and new born calf serum.

Mesotetrametahydroxyphenylchlorin (mTHPC) is compared to mTHPC derivatized with one or more polyethyleneglycol (PEG) chains of about 6000 amu. The latter compound is called mTHPC-PEG hereafter. In particular the uptake and removal of these two compounds is studied in tumor and other tissues after injection as a function of the time. In a first model this is done with nude mice bearing human colorectal xenografts (LS174T); in a second model it is studied in a Chinese hamster with a 7,12 dimethylbenzanthracene (DMBA) induced early squamous cell carcinoma in the cheekpouch mucosa. In the nude mice with tumors grown to between 70 and 130 mg, tumor to muscle dye ratios are found to attain a maximum value of 4.8, eight hours after injection of mTHPC. With mTHPC-PEG the tumor to muscle ratio attained a value as high as 18.6, forty-eight hours after injection. In the hamster model the maximum tumor to normal mucosa ratio for mTHPC was about 3.5 at times up to 2 hours after injection. For the mTHPC-PEG this ratio is always near unity in this model.

Golden Syrian hamsters are evaluated as an animal model for light induced fluorescence (LIF) photodetection and phototherapy of early squamous cell carcinomas of the upper aerodigestive tract, the esophagus, and the traecheo-bronchial tree. Carcinomas of this type are induced on the hamster cheek pouch mucosa by the application of the carcinogen 7,12-DMBA. For phototherapeutic experiments on the animals we utilized meso-(tetrahydoxyphenyl) chlorin (mTHPC). This drug is currently in phase I and II clinical trials for ENT patients presenting superficial `early' squamous cell carcinomas. By means of LIF we measured in vivo the kinetics of the uptake and removal of mTHPC in the normal and tumoral cheek mucosa and in the skin. The photodynamic therapy (PDT) reaction of the tissue after excitation of the photosensitizer with laser light at 652 nm was studied. Both pharmacokinetics and PDT efficacy are compared between animal model and clinical results with special emphasis on selectivity between normal and tumoral mucosa. These first experiments show that this tumor model in the hamster cheek pouch seems to be suitable for testing new photosensitizers preceding their clinical application as well as for optimization of the multiple parameters of clinical PDT.

In this study the effect of PDT on the antitumoral specific immunologic response was evaluated. We compared the specific cytolytic activity (CLA) by a chromium release assay of primed mouse spleen T lymphocytes sensitized against syngeneic mastocytoma P511 cells. P511 cells, or lymphocytes, or both cells were treated or not with photofrin and/or light (514 nm). Photofrin II alone (1 (mu) g/ml, 2 hours) reduced CLA 59% when P511 were treated. Photofrin II (1 (mu) g/ml) followed by light (25 Joules/sq cm) also reduced CLA 35%. Photofrin II alone (0.5 (mu) g/ml, 2 hours) reduced CLA 8% when only lymphocytes were treated. And Photofrin II (0.5 (mu) g/ml) followed by light (25 Joules/sq cm) also reduced CLA 45%. When both cells were treated with Photofrin II alone or followed by light (25 Joules/sq cm) the CLA was also reduced respectively 19, 41%.

Laser-induced fluorescence spectroscopy is a promising technique for detecting colonic dysplasia in vivo but, at present, the biological basis for the success of the method is poorly understood, and little information is provided as to the morphological/molecular origin of tissue fluorescence. We present a methodology for establishing this, taking as a starting point a recently completed prospective LIF clinical study of colonic polyps. The method is based on a morphological model of tissue fluorescence with three components: the intrinsic lineshapes of the fluorophores, the spatial distributions of their intensities, and the optical parameters of tissue. We measure these using fluorescence microspectroscopy, microscopic imaging and tissue optics, respectively. The model predicts the features of the clinical spectra, and quantifies the respective intrinsic and architectural contributions. The results can be inverted to extract biological parameters from the spectra, and used to select optimal excitation wavelength(s) and guide probe design. Implications for the detection of non-polypoid dysplasia are discussed.

Helicobacter pylori is associated with a wide range of pathologies in the upper gastrointestinal tract. Current treatments employing antibiotics are disappointing, and an endoscopic PDT might offer a better alternative. Methylene blue is a widely known histological dye and has been in use for photodynamic therapy experimentally for some years. A prospective application of MB is photosensitization of Helicobacter pylori, but little is known about its effect with light on normal mucosa of the stomach. We studied the fluorescence microscopy of the stomachs of 3 ferrets which had been sensitized by oral route with three different concentrations of MB 1 hour prior to sacrifice. MB at all doses was seen to concentrate on the surface of the mucosa and shows little deeper penetration. As Helicobacter lie on the superficial mucosa, this study suggests that oral dosing with MB should sensitize these bacteria. These findings are an important preliminary to an in vivo trial of PDT for the treatment of H pylori.

From 1/1985 to 7/1993, 18 patients underwent endoscopic photodynamic therapy (PDT) for early stage esophageal squamous cell carcinoma -- as two patients had two synchronous esophageal cancers, 20 lesions were treated. Tumors were staged as Tis in 7 cases and T1 in 13. The average light energy delivered was 50 J/cm² and 70 J/cm² for the treatment of Tis and T1, respectively. To obtain a more uniform distribution of laser light in 12 cases the irradiation was performed through the wall of a transparent tube previously placed over the endoscope and advanced into the stomach. The overall results show a complete response in 14/20 (70%) tumors. Three patients developed a local recurrence, 6, 12, and 14 months after therapy. After a follow-up of 5 to 75 months, there was no evidence of disease in 10/18 patients (56%). The actuarial survival rate was 95%, 79%, and 26% at 1, 3, and 5 years, respectively. Complications were skin reaction in one patient and esophageal stenosis at the treatment site, that gradually responded to endoscopic bougienage, in 2 patients. Endoscopic PDT proved to be safe and effective in the treatment of superficial carcinoma of the esophagus.

The authors developed a new high power red laser diode system for photodynamic therapy (PDT) with Mono-L-aspartyl Chlorin e6 (NPe6). The laser beam has a low wavelength (664 nm) and relatively high power. Our results suggest that a certain increase in the tumor tissue level of NPe6 and a certain increase of laser light dose reaching a deeper layer of tumor caused an increase in percent cure. In addition, the effectiveness of PDT and also tumor recurrence following PDT depend to some extent on the total laser dose reaching deeper layers of tumors. Furthermore, the effectiveness of PDT tends to correlate with the amount of photobleaching by PDT.

Mouse lymphoma L5178Y-R cells respond to photodynamic therapy (PDT) by undergoing rapid apoptosis, which is induced by PDT-activated signal transduction initiating in the damaged cellular membranes. To relate the level of PDT damage and photosensitizer to the mechanism of cell death, apoptosis has been detected by agarose gel electrophoresis of fragmented DNA and quantified by flow cytometry of cells after staining with Hoechst33342 and propidium iodide, a technique which can distinguish between live, apoptotic, and necrotic cells. When the silicon phthalocyanine Pc 4 or Pc 12 served as photosensitizer, lethal doses (as defined by clonogenic assay) of PDT induced apoptosis in essentially all cells, whereas supralethal doses prevented the characteristic degradation of DNA into oligonucleosomal fragments. In contrast with aluminum phthalocyanine (AlPc) cells died by apoptosis after all doses studied. It appears that high PDT doses with Pc 4 or Pc 12 damage enzymes needed to carry out the program of apoptosis; the absence of this effect with AlPc suggests either a different intracellular location or different photocytotoxic mechanism for the two photosensitizers.

Hypocrellins are naturally occurring compounds with photosensitizing properties in biological systems. We have prepared synthetic derivatives of hypocrellin B, which have promise as photosensitizers in the clinical application of photodynamic therapy. The inherent fluorescence of four selected compounds has provided a means of determining their uptake and distribution in monolayer cultures of EMT6/Ed murine tumor cells, via fluorescence confocal microscopy.

A method is presented of monitoring the low level fluorescence emitted by the photosensitizing agent protoporphyrin IX during superficial photodynamic therapy of skin carcinomas, using 630 nm illumination. A fiber optic probe samples the light field which is filtered and recorded by an optical spectrum analyzer. The technique is minimally invasive and can proceed concurrently with light dosimetry measurements. This paper presents in vitro data that define the sensitivity and selectivity of the technique, along with preliminary in vivo measurements. These indicate that it is the rate of phototransformation of the photosensitizer, rather than the total light dose, that determines the optimum treatment duration. Clinically effective treatment therefore depends upon achieving a threshold concentration of drug throughout the volume of the lesion. In this way the effect of phototransformation does not inactivate the drug before complete tumor necrosis occurs.

Clinical results obtained endoscopically with an imaging fluorescence photodetection apparatus based on the use of one excitation wavelength and detection of the fluorescence emission performed in two separate spectral windows are presented. Early cancer photodetection of micro-invasive and in situ carcinoma of the bronchial mucosa after intravenous injection of meso-tetrahydroxyphenylchlorin (m-THPC) was performed during sessions involving photodynamic therapy. Preliminary results are also presented in the context of detecting transitional cell carcinoma of the bladder performed by fluorescence imaging photodetection of protoporphyrin IX (PPIX) following topical application of 5-delta aminolevulinic acid (ALA).

Lysosomes and intracellular acidic compartments seem to play an important role in the context of PDT. Some photosensitizers are localized in the lysosomes of tumor-associated macrophages. Liposomes, which are lysosomotropic drug carriers, are used to deliver photosensitizers in tumors. Liposomes are taken up by the liver cells after intravenous injection. Intracellular pathway and liposomes localization in the different liver cells require sacrifice of the animals, cell separation, and observation by electronic microscopy. Little is known about liposomes kinetic uptake by the acidic intracellular compartments in vivo. We propose in this study a new method to follow liposomes uptake in the liver in vivo using a fluorescent pH-sensitive probe. We have already demonstrated the ability of fluorescence spectroscopy and imaging using a pH-dependent probe to monitor pH in living tissues. As pH of lysosome is very low, the kinetic of liposome uptake in this intracellular acidic compartment is followed by monitoring the pH of the whole liver in vivo and ex vivo. Liposomes-encapsulated carboxyfluorescein are prepared by the sonication procedure. Carboxyfluorescein is used at high concentration (100 mM) in order to quench its fluorescence. Liposomes are injected to Wistar rats into the peinil vein. After laparotomy, fluorescence spectra and images are recorded during two hours. Results show a rapid fluorescence increase followed by a slow phase of fluorescence decrease. pH decreases from physiological value to 6.0. After sacrifice and flush with cold saline solution, pH of liver ex vivo is found to be 5.0 - 5.5. These data show a rapid clearance of released dye and an uptake of liposomes by the liver cells and, as liposomes penetrate in the acidic compartment, dye is released from liposomes and is delivered in lysosomes leading to the decrease of pH.

For a given light and photosensitizer dose the degree of reaction of a tissue after photodynamic therapy (PDT) with tetra(meta-hydroxyphenyl)chlorin (mTHPC) shows large differences from one patient to another. This can be explained by variation of the photosensitizer (PS) concentration in the tissue of interest and can lead to undertreatment of the lesion and overtreatment of the surrounding normal tissue. A better prediction of the degree of reaction can be obtained by taking into account the drug concentration measured by light induced fluorescence (LIF) and by adjusting the light dose consequently.

Since 1984, 58 patients bearing malignant brain tumors were treated 70 times with photodynamic treatment (PDT). The patient population consisted of 11 primary glioblastoma WHO grade IV, 39 recurrent glioblastomas, 3 malignant meningiomas, 3 recurrent melanomas, and 2 metastasis of carcinomas. The patients were sensitized with hematoporphyrin derivative (HPD) 2.5 mg/bodyweight 24 - 48 hours prior to craniotomy and tumor resection. The light-irradiation was performed by an Argon pumped dye laser (Aurora M) superficially and/or interstitially at a dose ranging up to 250 J/cm². The median survival of primary glioblastomas was 19 months and for recurrent glioblastomas 7 months, respectively. Malignant meningiomas, as well as melanomas, did not benefit from PDT, whereas one patient with a metastasis of an adenocarcinoma is still recurrence free since 18 months, the other recurred after 6 months. HPD extractions of the tumor revealed significantly different concentrations among the various tumors, but also between identical histologies. The survival, however, did not correlate with the HPD concentration in the tumor. PDT prolongs median survival of primary glioblastomas significantly, and doubles the survival of recurrent high grade gliomas. Furthermore the treatment of recurrent low grade gliomas and metastasis to the brain are promising indications for PDT.

Tissue fluorescence measurements utilizing a point monitoring fluorosensor and a multi-color fluorescence imaging system were performed in patients with various malignant tumors in the head and neck region. Also precancerous lesions were investigated and characterized by means of laser-induced fluorescence. Oral administration of low-dose (delta) -amino levulinic acid was used for the induction of protoporphyrin IX, which exhibits a strong fluorescence in the red wavelength region. The blue-green fluorescence emitted from the tissue natural chromophores was also utilized for tumor demarcation. By incorporating both Protoporphyrin IX and natural fluorescence in a tumor demarcation criterion a tumor demarcation with a tumor-to-normal-surrounding-tissue of about 5 - 10:1 contrast was achieved. Hypertrophic tissue did not exhibit tumor specific signals.

The response of a human glioma cell line (U-87MG) and normal rat brain to photodynamic therapy (PDT) was investigated in athymic nude rats. U-87MG was implanted in the right hemisphere, and allowed to grow for 17 days. The animals were injected IV with Photofrin^R (12.5 mg/kg) 48 h prior to PDT of both normal and tumored brain (35 to 280 J/cm², 100 mW/cm²). The results were compared with that obtained under identical conditions with 9L gliosarcoma implanted in Fisher 344 rats. No tumor or normal tissue response was evident in the Nude rats at 35 J/cm². Tumor and normal tissue response was evident at incident optical energy dose levels of 140 and 280 J/cm². The response of the U-87MG tumor is consistent with that observed with the 9L gliosarcoma, however, the response in the nude rat normal brain was less than that observed in the Fisher 344 animals. These data indicate: (1) a possible contribution of the cellular immune system to the PDT reaction, (2) the human glioma xenograft model may contribute important insights into tumor and normal brain PDT responses.

Treatment of a solid tumor with photodynamic therapy (PDT) requires interstitial illumination, since the penetration depth of visible light in tissue is limited. Accurate light dosimetry in solid tumors requires the use of a mathematical model describing light propagation in tissue. By comparing the modeled fluence with measurements of fluence in tissue, the optical coefficients of the tissue can be found. We prefer to use a flat cleaved probe for measurements of the fluence in tissue instead of a spherical probe. In comparing the results of the model to the experimental data, we must deal with the fact that the model gives us the radiant fluence, while the experimental data is the power measured by the flat cleaved fiber with non-isotropic response. Measurements of the angular fluence in a tissue phantom show that the degree of anisotropy in angular fluence decreases with increasing distance from the source. The same result is found in chicken muscle. A calibration factor which modifies the measurements of fluence with a flat cleaved probe to account for the change in anisotropy in the angular fluence is presented.

In order to develop tumor-localizing fluorescent diagnostic agents without phototoxicity, various heterodimers linked by some spacers between a chlorine derivative and its Mn or Cu complex were synthesized. The representative agent of them was named HAT-DO1 and has a molecular formula of m-phthalyl-{[13,17-bispropanoic acid-3-ethenyl-8-formylethylidene-7- hydroxy-2,7,12,18-tetramethyl-porphyrinate]-manganese (III)]}-(3'-ethenyl-8'- formylethylidene-7'-hydroxy-2',7',12',18'-tetramethyl-porphine-13',17'- bispropanoyl aspartic acid)-bishydrazone.

The illumination of a mixture of the photosensitizer methylene blue and the reagent tetraphenylcyclopentadienone by 630 nm light results in a change in the absorption spectrum of the mixture dependent on the light energy absorbed. The absorption peak of the reagent at 500 nm is used to quantify the photodynamic response by placement of small capillary tubes containing the mixture within an optical phantom. The light distribution within the phantom was measured simultaneously. The fluence rate predicted by diffusion theory is correlated to that of the photodynamic response. Methylene blue is commonly used as a standard photosensitizer and this study also allows comparison of the relative photodynamic effects of different photosensitizers.

Nine patients with malignant pleural mesothelioma underwent extensive surgery followed by intra-operative photodynamic therapy. Two mg/kg Photofrin was given 48 hours prior to surgery. The thoracic cavity and eventual remaining lung were exposed to 15 - 30 Joules/cm² of 630 nm laser light. Tumor tissue was analyzed by microscopic photometrical techniques. Five patients with mixed or epithelioid tumors with fluorescence intensity > 100 gray level/pixel seemed to benefit from the given therapy. One patient was free of disease 18 months after treatment. Two patients were treated for metastasis after 12 months with no sign of intrathoracic recurrence. Both are still alive, one without further sign of disease 32 months after initial treatment. Two patients presented generalized disease after 9 and 13 months and intrathoracic recurrence several months later. Two patients with poorly differentiated tumors and 2 patients with moderate to highly differentiated tumors, but with fluorescence intensity < 100 gray level/pixel, presented recurrences after 4 months. PDT-efficiency seems to be predicted by the intensity and distribution of drug-induced fluorescence in tumor tissue. PDT may enhance the possibility to achieve complete local tumor control after excision. Multimodal therapeutic approach of local and systemic disease seems mandatory to further improve survival.

The absorption spectrum of disulphonated aluminum phthalocyanine (AlS₂Pc) between 650 nm and 695 nm was measured in vivo by means of time-resolved reflectance. The experiments were performed on mice bearing the L1210 leukemia 1, 4, and 7 hr after the i.p. administration of 2.5 mg/kg body weight (b.w.) of AlS₂Pc. The absorption peak is centered at 685 nm, red-shifted of 10 - 15 nm with respect to the spectra obtained in solution in various environments. Measurements performed in vitro confirm the results in vivo and seem to suggest that the extracellular environment can cause the shift in the absorption line shape.

The decay of the surviving cell fraction, F, during photodynamic treatment of a cell population, F₀, vs. energy dose obeys an exponential law. The integrated logarithmic form yields a straight line with intercept 0 and slope -k. The reciprocal slope yields LD₉₀, i.e., the energy fluence (J/cm²) necessary to kill 90% of the cell population F₀. In reality there are deviations from this simple set of equations. At low power densities most photosensitizers are unable to kill cells. This leads to an `optical threshold' without cell destruction but frequently with cell stimulating effects, so called `overshots.' This general phenomenon occurs in a very pronounced form with melanotic melanoma cells.

Liposomal formulations of the photosensitizer zinc(II)-phthalocyanine (Zn-Pc) readily interact with plasma lipoproteins in vitro and in vivo, leading to a redistribution of the photosensitizer amongst the major lipoprotein classes. As lipoprotein binding may facilitate the uptake of lipophilic photosensitizers by proliferating tissues, we have examined the fate of intravenously administered liposomal Zn-Pc in normolipaemic rats and rats rendered hyperlipaemic through dietary intervention. Differences were found in the plasma decay profiles of Zn-Pc and liposomal phospholipid between the two groups of animals.

The liposomal zinc(II) phthalocyanine (Zn-Pc) formulation CGP55847 was administered intravenously (0.5 mg Zn-Pc/kg) to C57/BL6 mice bearing subcutaneously implanted B16 melanomas or to Swiss mice bearing intramuscularly implanted Ehrlich carcinomas. Tumors were removed 3 or 24 h after dosing, and the Zn-Pc content and intratumoral distribution determined by extraction and quantitative fluorescence microscopy. Localization of the photosensitizer within the tumor mass occurred more rapidly in the highly vascularized Ehrlich carcinoma compared to the less highly vascularized B16 melanoma. Zn-Pc was evident in and around blood vessels 3 but not 24 h after dosing. More Zn-Pc was found in necrotic areas compared to viable tumor tissue; little or no Zn-Pc was detected in the muscle tissue invaded by the Ehrlich carcinoma. At the cellular level, Zn-Pc was associated with membranes and the cytosol but not the nucleus.

The EAhy 926 cell is a hybridoma line derived from human endothelium and A549/8 cells. They display stable endothelial characteristics and may provide an indication of how endothelial cells respond to photodynamic therapy. Cells were grown as monolayers, seeded at a density of 10⁴ cells/35 mm dish, and then incubated with zinc (II) tetrasubstituted phthalocyanines (carboxylated, sulphonated, pyridinium or diethanolamine sulphonamide). After 24 hours, the cells were illuminated with laser light at 680 nm or 692 nm as appropriate. The response to each photosensitizer was evaluated using cell proliferation, clonogenicity, and release of tissue factor.

Five-aminolevulinic acid (ALA) is being used to induce formation and accumulation of endogenous protoporphyrin IX both in preclinical and clinical studies on photodynamic therapy and diagnosis. At the high concentrations needed for clinical application, ALA is not stable in aqueous solutions in the neutral to basic pH range. This could be of critical importance for the clinical use of ALA. The chemical instability of ALA was studied by using UV-Vis absorption spectroscopy. Our results show that ALA undergoes a chemical reaction, which is a nonreversible condensation process yielding two different products -- a hydropyrazine and a pyrazine, the latter formed by oxidation of the hydropyrazine. Preliminary kinetic studies of this reaction showed a complex pH-, concentration and temperature-dependency of the reaction rate. On the basis of these studies we optimized conditions for the preparation of ALA solutions to be used in clinical trials, leading to the expected accumulation of protoporphyrin IX in tumor cells.

To investigate pharmacokinetics of ALA-induced porphyrins the time course of fluorescence in neoplastic tissue and normal tissue following i.v. administration of ALA (100 mg kg^-1, 500 mg kg^-1 or 1000 mg kg^-1 b.w.) was studied by means of intravital fluorescence microscopy. As a model the amelanotic melanoma (A-Mel- 3) growing in a transparent skin fold chamber preparation was used (n equals 18). Maximal fluorescence intensity was measured after 2.5 h in tumor and after 6 h in surrounding host tissue, highest tumor/host tissue ratio at 90 min (500 mg kg^-1 b.w.). Heterogeneity of fluorescence intensity between different tumors ranged from 24% to 361% following injection of 500 mg/kg b.w. Twenty-four hours after systemic administration of ALA fluorescence intensity was decreased to baseline. Based upon these results a three- compartment model was calculated to simulate fluorescence kinetics in solid, well vascularized tumors assuming first-order kinetics. This compartment model supports the experimental findings, that the transient selectivity of ALA-induced fluorescence in tumors is caused by earlier uptake of ALA in tumors due to increased microvascular permeability and diffusion in neoplastic tissue. The heterogeneity might represent the different metabolic capacity of tumors to form porphyrins following exogenous administration of ALA.

The carboxylation state of porphyrin metabolites causes their hydrophilic or lipophilic properties and subsequently their distribution in tissues, cells, and subcellular compartments. The profile of porphyrin metabolites either in normal skin or in malignant skin tumors after administration of (delta) -aminolevulinic acid has been studied in detail, yet. Explant cultures of normal skin and neoplastic tissues, e.g., keratoakanthoma and basal cell carcinoma, were incubated with 1 mM ALA for 36 h. Total porphyrin concentration and percentage of porphyrin metabolites were determined quantitatively in tissues and corresponding supernatants. Seventy - ninety percent of total porphyrins could be detected in the supernatants of all samples. The highly carboxylated porphyrins were the prevailing metabolites in the supernatants as well as in the tissues. The basal cell carcinoma produced significantly more protoporphyrin and the keratoakanthoma significantly more coproporphyrin as compared to normal skin. The results show that explant cultures offer an easy approach to examine the enzymatic capacity in porphyrin biosynthesis of various tissues. Benign and malignant human tissues produce different porphyrin metabolites, which may be useful for selective and more effective photodynamic diagnosis or therapy.

A case of a 62-year-old woman suffering from psoriasis who was treated orally with arsenic 25 years ago is reported. The cumulative dose of arsenic trioxide was 800 mg. Since 10 years ago arsenic keratoses, basal cell carcinomas, Bowen's disease and invasive squamous cell carcinomas mainly on her hands and feet have developed, skin changes were clearly a sequence of arsenic therapy. Control of disease was poor, her right little finger had to be amputated. Topical photodynamic therapy with 5-aminolevulinic acid was performed on her right hand. Clinical and histological examinations 6 months after treatment showed an excellent cosmetic result with no signs of tumor residue.

Seven hundred sixty three basal cell carcinomas (BCCs) in 122 patients were treated by photodynamic therapy by 5-aminolevulinic acid (ALA) in cream topically applied, either alone, in combination with dimethyl sulphoxide (DMSO) and ethylenediaminetetraacetic acid disodium salt (EDTA), or with DMSO as a pretreatment. After 3 hours cream exposure 40 - 200 Joules/cm² of 630 nm laser light was given. Fluorescence imaging of biopsies showed highly improved ALA penetration depth and doubled ALA-induced porphyrin production using DMSO/EDTA. Treatment response was recorded after 3 months. After a single treatment 90% of 393 superficial lesions responded completely, independent of using DMSO/EDTA. In 363 nodulo-ulcerative lesions the complete response rate increased from 67% to above 90% with DMSO/EDTA for lesions less than 2 mm thickness and from 34% to about 50% for lesions thicker than 2 mm. Recurrence rate observed during a follow-up period longer than 12 months was 2 - 5%. PDT of superficial thin BCCs with ALA-induced porphyrins and DMSO/EDTA equals surgery and radiotherapy with respect to cure rate and recurrence. Cosmetic results of ALA-based PDT seemed to be better than those after other therapies. In patients with the nevoid BCC syndrome the complete response rate after PDT was far lower.

A series of landmark contributions were made at our laboratories in PDT. Indeed, we were the first to show biodistribution and selective in vivo tumor localization of endogenous porphyrins induced and stimulated by 5-ALA as a newly developed technique. Several other reports showed novel approach by using it clinically with excellent cosmetic results on skin cancer patients. Our present report would assess the effectiveness of this modality on Egyptian patients with various skin cancers. An argon pumped-dye laser was used to emit non-thermal laser light doses. We also present promising clinical trial from using m-THPC on Egyptian patients with squamous carcinoma of the lower lip. Preliminary observations reveal that (1) m- THPC-PDT presents a welcome therapeutic option in treatment of squamous carcinoma of the lips without loss of their function with excellent cosmetic results. (2) Topical PDT with endogenous porphyrins by ALA is very effective in treatment of selected superficial basal cell carcinomas in situ and early invasive squamous cell carcinomas in the skin.

Photodynamic therapy (PDT) using topical application of the Protoporphyrin IX (PpIX) precursor (delta) -amino levulinic acid (ALA) in various malignant skin tumors is a new promising treatment modality. We have treated an extensive number of non-melanoma malignancies of the skin over the past three years with very satisfying initial results. For superficial, shallow lesions one treatment session is sufficient. In thicker lesions, such as nodular basal cell carcinomas, complete treatment response is achieved after two or three treatment sessions. In conjunction with the treatment procedure the tissue fluorescence and the superficial blood flow have been investigated during and after the treatment procedures. The PpIX build-up has been detected in vivo and the degree of tumor selectivity has been evaluated using laser-induced fluorescence. Also changes in the bloodflow in tumors compared to normal skin before, during, and after the treatment procedure has been followed using a laser- Doppler perfusion imaging system. Results from the measurements in basal cell carcinomas (BCCs), Mb. Bowen lesions (squamous cell carcinoma in situ) and cutaneous T-cell lymphoma lesions are presented.

Seventeen patients with oral cancer and precancer were treated with photodynamic therapy using systemic 5-aminolaevulinic acid. A brisk PDT response was noted in all patients but damage was confined to the epithelium on histological evaluation.

Over the past 14 months we have treated a wide variety of head and neck cancer patients with mTHPC mediated photodynamic therapy. This drug is a very powerful second generation photosensitizer with significant advantages over earlier drugs. Patients treated range from palliative to primary (curative) to adjunctive (intraoperative) cases. Following promising results we are now setting up a multicenter European study to treat early head and neck cancer with this drug/light combination, along with other collaborative studies to look further at the role of adjunctive PDT.

For the application of photodynamic therapy and diagnosis many different photosensitizers have been developed. It is important to compare these photosensitizers for their activity. It is generally accepted that the most important mechanism of cell killing is via the production of singlet oxygen. We therefore performed basic studies to detect singlet oxygen using a luminol reaction. The relative singlet oxygen yields from 4 photosensitizers (Photofrin, ATX-S10, mTHPC and NPe6) were measured by the detection of luminol chemiluminescence at 445 nm wavelength in Menzel's buffer solution at pH 10.5. NPe6, ATX-S10 and mTHPC all showed singlet oxygen productive abilities. These photosensitizers are new promising photosensitizers. These results show a possibility of comparison of each photosensitizer.

Experimental study has been carried out using metatetrahydroxyphenylchlorin (m-THPC) in female Syrian golden hamsters. For quantitative fluorescence study, using a CCD camera, the dose of 1.0 mg/kg bodyweight of mTHPC was given intracaval and animals were sacrificed 1 and 4 hours and 1, 2, 3, 4, 5, 6 days afterwards and specimens from the pancreas, stomach, duodenum, gallbladder, aorta and vena cava were examined. A sensitizing dose of 0.3 mg/kg and 0.1 mg/kg respectively was given for PDT, which followed after 2 and 4 days and animals were sacrificed after 1, 2, 3, 4 and 7 days. The treated areas included the pancreas, antral part of the stomach, duodenum and wedge of lesser omentum. In the treated pancreas necrosis up to 3 - 4 mm, marked ulceration in treated duodenum up to 3 - 4.5 mm and in the stomach up to 2 - 2.5 mm in diameter were observed. No macroscopic and histological signs of necrosis were seen in bile duct and major blood vessels. Sealed duodenal perforation occurred in all treated animals with 0.3 mg/kg sensitizing dose and was not observed in 0.1 mg/kg dose with protected duodenum during PDT. Using breaks during PDT (3 X 1; 3 X 3 minutes, respectively) approximately 30% increase of pancreatic necrosis has been observed. In this model, only the duodenum is at risk of irreversible damage. The risk to normal organs in this region is similar to that of photofrin and sulphonated aluminum phtalocyanine. mTHPC is a promising photosensitizer for tumors in the region of the pancreas, although care is required in the region of the duodenum.

(delta) -Aminolaevulinic acid (ALA) is a photodynamic therapy (PDT) agent that utilizes the haem biosynthetic pathway to create therapeutic levels of photoactive agents within tissues. Photosensitizer dosimetry and drug concentrations in target tissues are areas of uncertainty within PDT research. A program is described that uses numerical methods to model mathematically the haem biosynthetic pathway from ALA to haem as a set of partial differential rate equations. The data generated allow analysis and correlation with functions describing the kinetic behavior governing the reactions. This analysis provides insight into the production of protoporphyrin IX and other photoactive agents from exogenous ALA and provides a method for optimizing parameters, and for highlighting metabolic steps to which the product formation is most sensitive.

We assessed the ability of the human hepatocarcinoma cell line HepG2 to synthesize PpIX in vitro from exogenous ALA and analyzed ALA-induced toxicity and phototoxicity on this cell line. ALA induced a slight dose-dependent dark toxicity, with 79 and 66% cell survival respectively for ALA 50 and 100 mg/ml after 3-h incubation. Whereas the same treatment followed by laser irradiation (l equals 632 nm, 25 J/sq cm) induced dose-dependent phototoxicity, with 54 and 19% cell survival 24 h after PDT. Whatever the incubation time with ALA, a 3-h delay before light exposure was found optimal to reach a maximal phototoxicity. Photoproducts induced by porphyrin light irradiation absorbed light in the red spectral region at longer wavelengths than did the original porphyrins. The possible enhancement of PDT effects after ALA HepG2 cell incubation was investigated by irradiating cells successively with red light (l equals 632 nm) and light (l equals 650 nm). Total fluence was kept constant at 25 J/sq cm. Phototoxicity was lower when cells were irradiated for increased periods of l equals 650 nm light than with l equals 632 nm light alone. Any photoproducts involved had either a short life or were poorly photoreactive. HepG2 cells, synthesizing enzymes and precursors of endogenous porphyrin synthesis, represent a good in vitro model for experiments using ALA-PpIX-PDT.

Preferential conversion of 5-aminolevulinic acid (5-ALA) to protoporphyrin-IX (Pp-IX) occurs in malignant tissue, with accumulation to diagnostic and therapeutic levels. Recent studies have suggested selective conversion in epithelial tissue following oral or intravenous administration. Topical application avoids systemic photosensitization. However, the glycosaminoglycan (GAG) layer lining the urinary bladder is believed to be a protective barrier generally limiting mucosal absorption. Our objective was to evaluate uptake and conversion of 5-ALA following intravesical or oral administration. Using a rat model, Pp-IX content within epithelial and muscularis layers was quantitated by fluorescence confocal microscopy. Following intravesical administration, Pp-IX accumulated predominantly in the urothelium; whereas following oral administration, Pp-IX accumulated in both the urothelium and muscularis. Intravesical 5-ALA administration is feasible and may afford selective photosensitization of the urothelium for treatment of carcinoma in situ.

Photodynamic therapy (PDT) has considerable potential for the treatment of superficial bladder neoplasia. Complications such as scarring of the detrusor muscle and prolonged cutaneous photosensitivity may be reduced by using the new photosensitizer precursor, 5- aminolaevulinic acid (ALA). After instillation of ALA, the concentration, pH, and time of bladder retention of ALA solution were found to be the key factors to a satisfactory PpIX buildup in the mucosa. The optimum PpIX fluorescence intensity ratio between mucosa and muscle layer is 10 to 1 with a pH 5.5, 1% ALA solution retained for 5 hours. Higher concentration resulted in more mucosal PpIX formation, but less selectivity. Unbuffered ALA was unsuitable for bladder instillation. Two days after laser treatment with 25 J/cm² at 630 nm with optimal sensitization, typical histological findings were urothelial sloughing and lamina propria edema without obvious muscle damage. After 7 days, recovery of the urothelium was almost complete and fibroblast infiltration was minimal. ALA induced PpIX after bladder instillation may be an appropriate photosensitizer for future management of superficial bladder cancer.

The objective of this study is to investigate tumor responses when treated with the combination of photodynamic therapy and hyperthermia. Intra-tumoral oxygen levels (pO₂) and 30 day tumor regrowth were investigated. C3H murine mammary leg tumors were implanted and grown to a diameter of 6 mm. The combined treatment was designed based on the data from our previous studies of tumor response and tumor tissue oxygen levels after various doses of hyperthermia and PDT alone. Photofrin (12.5 mg/kg, i.p.) was administered to the animals 24 hours before light treatment (630 nm, 200 J/cm², 300 mW/cm²). Hyperthermia (43.5 degree(s)C, 30 to 90 minutes) was delivered 4 hours after the PDT treatment. TCD₆₀ and TCD₈₀ were observed for the combined therapy with 60 and 90 min hyperthermia, in comparison to the TCD₁₀ and TCD₂₀ for the hyperthermia alone, respectively. The results indicate that improved therapeutic results can be achieved with the combined therapy designed based on the tissue PO₂ profiles after single modality treatment.

Selectivity is the most emphasized advantage of photodynamic therapy (PDT). However, at drug and light doses used for clinical applications, response from normal tissue surrounding the tumor reduces the real selectivity of the drug-light system and increases the surface of the area responding to the treatment. It is now evident that light irradiation of a sensitized patient produces damage at a various degree not only in the tumor but also in non-neoplastic tissues included in the field of irradiation. We report our experience in endoscopic PDT of early stage tumors in tracheobronchial, gastrointestinal and urinary tracts, describing early and late local complications caused by the damage of normal tissues adjacent to the tumors and included in the field of light irradiation. Among 44 patients treated, local complications, attributable to a poor selectivity of the modality, occurred in 6 patients (14%). In particular, the rate of local complications was 9% in patients treated for esophageal tumors, 14% in patients with gastric tumors, 9% in patients with tracheobronchial tumors, and 67% in bladder cancer patients. Clinical pictures as well as endoscopic findings at various intervals from treatment showed that mucositis is a common event following endoscopic PDT. It causes exudation and significant tissue inflammatory response, whose consequences are different in the various organs treated. Photoradiation must be, as much as possible, limited to the malignant area.

To improve our knowledge on PDT of human early squamous cell carcinomas of the upper aero-digestive tract and to evaluate new photosensitizers, we have set up the Syrian hamster as an animal model. A 0.5% oily solution of DMBA was applied topically to the left buccal pouch mucosa 3 times weekly. The contralateral buccal pouch served as control. Groups of 5 animals were sacrificed at 6, 8, 10 and 12 weeks from the first applications. Tissue samples of the buccal mucosa were analyzed by histopathologic and immunohistochemical techniques and compared with preneoplastic and neoplastic changes which are seen in the human carcinogenesis of the upper aero-digestive tract. After 6 to 9 weeks from the beginning of the application, we observed different degrees of epithelial dysplasia and after 10 weeks microinvasive carcinomas. The sequence of dysplastic changes to early carcinoma was reproducible in different groups of animals, and they were closely comparable with the human forms of `early' squamous cell cancer. Hyper- and dyskeratosis were present at all stages of tumor development. We are particularly interested in (mu) -invasive tumor forms appearing 10 weeks after the first application because they are potentially amenable to photodynamic therapy.

A series of experiments was undertaken to determine the effects of the combination of light and the tissue photosensitizer, tin etiopurpurin, on the canine prostate. Mongrel dogs were injected intravenously with 1.0 mg/kg of photosensitizer twenty-four hours prior to light delivery. Laser light, 660 nm, was administered either transurethrally or interstitially and tissue effects were assessed by histopathologic examination. Both techniques of light delivery resulted in hemorrhagic necrosis of the surrounding tissue. Photodynamic therapy may offer a novel approach to the treatment of both benign and malignant diseases of the prostate.

Experiments were undertaken to determine whether 5-aminoleuvinic acid in combination with light could be used as an adjunct to intestinal bladder augmentation with the aim of removing intestinal mucosa with subsequent re-epithelialization of the treated segment with urothelium. Histopathologic studies of so-treated intestinal segments used in bladder augmentation demonstrate the feasibility of this approach.

Five-aminolaevulinic acid (ALA) induced protoporphyrin IX (PpIX) has proven to be a useful photosensitizer for photodynamic therapy (PDT). In living cells, the conversion of PpIX to photoinactive haem is catalyzed by ferrochelatase in the presence of tissue iron and inhibition of this final committed step results in increased accumulation of PpIX. The in vivo effect of a new iron chelator, 1,2-diethyl-3-hydroxypyridin-4-one (CP94), on the buildup of PpIX in different bladder layers was evaluated. In CP94 treated rats, 5 - 7 hours after intravesical instillation of ALA solution, the fluorescence intensity of PpIX in the urothelium was doubled whilst in the muscle layer it remained low at a similar level to those seen without the iron chelator. With CP94, further reduction of skin photosensitization is possible as a similar photodynamic effect on the bladder could be achieved at lower ALA concentration. The addition of CP94 seems an effective and convenient way to potentiate ALA induced PpIX tissue selectivity.

A new cancer-treatment model, photodynamic therapy (PDT) combined with a type I topoisomerase inhibitor, camptothecin derivative (CPT-11), against HeLa cell tumors in BALB/c nude mice has been developed using a wide-band tunable coherent light source operated on optical parametric oscillation (OPO parametric tunable laser). The Photosan-3 PDT and CPT-11 combined therapy was remarkably effective, that is the inhibition rate (I.R.) 40 - 80%, as compared to PDT only in vivo. The analysis of HpD (Photosan-3) and CPT-11 effects on cultured HeLa cells in vitro has been studied by a video-enhanced contrast differential interference contrast microscope (VEC-DIC). Photosan-3 with 600 nm light killed cells by mitochondrial damage within 50 min, but not with 700 nm light. CPT-11 with 700 - 400 nm light killed cells within 50 min after nucleolus damage appeared after around 30 min. The localization of CPT-11 in cells was observed as fluorescence images in the nucleus, particularly the nucleoral area produced clear images using an Argus 100.

We characterized sites of photodamage catalyzed by two cationic photosensitizers tetrabromo- rhodamine 123 (TBR), which is recognized by the multidrug transporter, and a monocationic porphyrin (MCP), which is not. The transporter is an outward transport system associated with examples of drug resistance. Irradiation of multidrug-resistant cells treated with TBR resulted in highly selective photodamage to the transporter site, while MCP catalyzed non- specific membrane damage to cells regardless of transporter expression.

In photodynamic therapy (PDT), red light is administered 24 - 72 hours post intravenous (i.v.) injection of hematoporphyrin derivatives (HpD). In an earlier animal model study, more effective therapeutic response was obtained when red light irradiation was carried out 15 mins after the injection of HpD. The effectiveness of this immediate PDT protocol has been correlated to the high serum level of HpD immediately after administration and the destruction of the microcirculation system as the dominant tumor destruction mechanism. This study examines the pharmacokinetics and the serum levels of HpD in rats and also in human patients. Such data can assist in defining the optimum time delay for light irradiation in the PDT of cancer.

The distribution of Photofrin and several other photosensitizers among major cellular populations contained in solid mouse tumors was examined using flow cytometry. Seven tumor models were included in the analysis: sarcomas EMT6, KHT, RIF, FsaR and FsaN, Lewis lung carcinoma and squamous cell carcinoma SCCVII. In all these tumors, the highest photosensitizer levels were found in a subpopulation of tumor associated macrophages consisting of activated cells (as suggested by their increased size, granularity, and the number of interleukin 2 receptors). There was no evidence of selective photosensitizer accumulation in malignant tumor cells. Results consistent with these observations were also obtained with the carcinogen induced squamous cell carcinoma growing in hamster cheek pouch.

The murine squamous carcinoma cell line (SCCVII) was genetically engineered to produce high levels of granulocyte-macrophage colony stimulating factor (GM-CSF). Lethally irradiated GM-CSF producing cells were injected under the subcutaneously growing parental SCCVII tumor at various times before and/or after PDT. Even a single treatment with GM- CSF producing cells injected two days before PDT markedly enhanced the tumor cure rate when compared to the PDT treatment alone. Effective potentiation was observed with PDT mediated either by Photofrin or by benzoporphyrin derivative.

The thresholds in sensitizer concentration and in light dose rate for PDT effects have been examined on the basis of a chemical reaction. The examination revealed that there exists basically no thresholds of drug and light dose-rate effects in PDT, but apparently there does. Also it was suggested that PDT effects depend largely on peak power of laser and not so largely on the total joule. And for PDT the use of pulse laser with relatively high peak power is better than the use of the lower one, especially cw laser.

Stochastic properties of backscattered light are used for tissue discrimination. Tissue penetrated red light provided by a low power semiconductor laser is picked up by glass fibers and converted to electrical signals. The time varying signal parts are analyzed by power spectra and coherency. Both signal curves and results of analysis demonstrate the validity of the method for discrimination between physiological and malignant tissue.

Since coherence of laser light is not necessary for PDT of skin tumors, attempts have been made to construct incoherent lamps. A recent development is PDT 1200 (Waldmann Medizintechnik/FRG), a light source based on a 1200 watt metal halogen lamp. Emission of 580 to 740 nm radiation is achieved by using cut off filters. Power density can be varied from 30 mW/cm² to 200 mW/cm² in an area from 100 to 300 cm². Biologic effectiveness was proved by comparison with the radiation of an argon-pumped dye laser emitting light at 630 nm. Three human cell lines were incubated with photofrin (5 (mu) g/ml and 10 (mu) g/ml) for 24 hours. After irradiation a MTT-test was performed to assess cell viability. Results clearly proved biological effectiveness of the light source PDT 1200. No significant difference in cell viability was detected using either concentration of sensitizer. Therefore, we believe that PDT 1200 is a promising new light source for photodynamic therapy of skin lesion.

Several types of non-coherent light sources can be used for dermatological applications of photodynamic therapy (PDT). Such sources are relatively inexpensive, stable, and easy to operate, requiring very little maintenance. A further advantage is that large lesions can be irradiated in a single treatment. In order to assess the matching of the spectral output of the light source with the photochemo-therapeutic agent chromophore, a measurement of spectral irradiance with a monochromator must first be carried out. Also, in order to assure flatness of irradiation field over the entire bandwidth, an irradiance measurement as a function of position in the beam must be performed. Data of this type obtained from different light sources is presented and discussed in terms of predicted PDT efficacy.

Five aminolaevulinic acid (ALA) is a promising agent for PDT sensitization as it can be given orally and only causes skin photosensitivity for 1 - 2 days. In fluorescence and photodynamic studies 26 patients with benign and malignant gastrointestinal tumors (M 17, F 9; mean age 79) were given 30 - 60 mg ALA orally (single or divided doses) and biopsies taken of tumor and normal tissue at 1 - 24 hours for fluorescence microscopy. With 30 mg/kg, highest protoporphyrin IX (PPIX) levels were seen in oesophagus, duodenum and less in colon, but without tumor selectivity. Better tumor selectivity was seen in the colon after 60 mg/kg (5:1). Six patients had transient rises in transaminases and five mild nausea. Sixteen patients were later treated (after further ALA) with red light (628 nm, bare fiber or diffuser, 50 - 100 J at 50 mW at each site). All but two showed subsequent necrosis, but only 0.5 - 1.5 mm depth. PDT with ALA is simple, safe, and promising for tumors in the GI tract. Modification of treatment parameters may make it suitable for larger lesions.

Fifteen patients with Barrett's esophagus and dysplasia were treated with photodynamic therapy. Four patients also had early, superficial esophageal cancers and 5 had esophageal polyps. Light was delivered via a standard diffuser or a centering esophageal balloon. Eight patients maintained on omeprazole and followed for 6 - 54 months are the subject of this report. Photodynamic therapy ablated dysplastic or malignant mucosa in patients with superficial cancer. Healing and partial replacement of Barrett's mucosa with normal squamous epithelium occurred in all patients and complete replacement with squamous epithelium was found in two. Side effects included photosensitivity and mild-moderate chest pain and dysphagia for 5 - 7 days. In three patients with extensive circumferential mucosal ablation in the proximal esophagus, healing was associated with esophageal strictures which were treated successfully by esophageal dilation. Strictures were not found in the distal esophagus. Photodynamic therapy combined with long-term acid inhibition provides effective endoscopic therapy of Barrett's mucosal dysplasia and superficial (Tis-T1) esophageal cancer. The windowed centering balloon improves delivery of photodynamic therapy to diffusely abnormal esophageal mucosa.

A new sensitizer, hematoporphyrin monomethyl ether (HMME), purified by the Second Military Medical University was used with a gold vapor laser for PDT in 8 cases of alimentary cancers. The results showed that 2 cases of early stage gastric cancer and 1 case of rectal polyps with malignancy revealed CR, SR was obtained in 1 case of esophageal cancer and 4 others were MR. No sunburn occurred within the 8 patients who received 12 doses of HMME at 5 mg/kg 2 - 3 hours prior to laser treatment and who were kept away from sun light only 6 hours. Hematoporphyrin monomethyl ether is an effective single compound and safer for PDT.

A non-invasive probe has been devised and clinically used to perform in vivo measurements of the optical properties of the esophageal wall and oral cavity. The absorption and reduced scattering coefficients are determined from the observation of the spatial distribution of the diffuse reflectance at the tissue surface under a narrow beam illumination of the tissue. The determination of these two coefficients enables us to evaluate the value of the effective attenuation coefficient which is of major interest in the field of light dosimetry for photodynamic therapy (PDT). An invasive isotropic micro-probe has also been designed and clinically used to directly measure in vivo the value of the fluence rate in tissues. The principle of this probe is based on the fluorescence generated in a ruby sphere by the light which propagates in the tissue. This fluorescence which can be excited between 350 and 680 nm is isotropically emitted and in part collected by an optical fiber glued against the ruby sphere. Results, obtained with both probes at 514 and 630 nm, i.e., wavelengths of interest in photodynamic therapy, with actual clinically used photosensitizers are summarized and compared. The agreement obtained between these two techniques validates the principle of these measurements.

Five-aminolaevulinic acid (ALA) was applied orally and by aerosol inhalation to one patient in order to check the feasibility of photodynamic therapy (PDT) and photodynamic fluorescence diagnosis (PDD) of lung cancer. For PDD, ALA was given by inhalation using a conventional jet nebulizer. Protoporphyrin IX (PP IX)-fluorescence in the bronchial mucosa and the tumor was assessed visually and by spectroscopy using an optical multichannel analyzer. At a second session ALA was given orally and PDD as well as PDT were performed. The therapeutic effect on the tumor was controlled by bronchoscopy 5 and 12 weeks after PDT. Inhalative and oral application of ALA were both well tolerated. No adverse effects were observed. PP IX- fluorescence could be easily detected 3 h after ALA administered by inhalation or 5 h after ALA orally. Fluorescence ratio between tumor and normal tissues was better after the oral administration of ALA. Five and twelve weeks after PDT, marked reduction of tumor volume and recanalization of the left upper lobe were found.

From 1983 to 1994 Phase II and III clinical studies at Henry Ford Hospital demonstrated complete or partial responses in 46 of 47 patients treated with hematoporphyrin-derivative photodynamic therapy (HPD-PDT) for a variety of benign and malignant upper aerodigestive tract disease: (1) superficial `condemned mucosa' or `field cancerization' of the oral cavity; (2) stage III/IV head and neck cancer; (3) mucocutaneous AIDS-related Kaposi's sarcoma of the upper aerodigestive tract; (4) recurrent laryngotracheal papillomatosis; (5) severe dysplasia/adenocarcinoma in situ in Barrett's esophagus; (6) partial or completely obstructing terminal esophageal cancer. HPD-PDT produced complete responses in 19 patients (follow up 6 months to 8 years) with `field cancerization' (CIS, T₁) of the oral cavity and larynx (6), adenocarcinoma in situ in Barrett's esophagus (2), mucocutaneous Kaposi's sarcoma (9), obstructing esophageal carcinoma (1), and stage IV squamous cell carcinoma of the nasopharynx (1). PDT treatment protocols, results, complications, and application as adjunct or primary oncologic therapy for head and neck disease are reviewed.

In this study the metabolism in tumors and various tissues of intravenously administered (delta) -aminolevulinic acid was investigated. Amelanotic melanoma (A-Mel-3) were implanted in the dorsal skin of Syrian golden hamsters. Distribution and metabolism of i.v. injected (delta) -aminolevulinic acid in blood was studied by determination of (delta) - aminolevulinic acid and protoporphyrin concentration in red blood cells. In addition extraction of various tissues, e.g. tumor, liver, kidney, and normal skin was performed, to verify fluorescence kinetic studies by determination of total porphyrin concentration by photometry and of distribution of the porphyrin metabolites by HPLC. In untreated animals the total porphyrin concentration in all tissues examined were comparably low. In red blood cells the maximal concentration of (delta) -aminolevulinic acid as well as protoporphyrin was detected 45 min after i.v. injection of (delta) -aminolevulinic acid. Porphyrins accumulated in melanoma reaching a maximum tumor:skin tissue ratio of 6.9:1 at 45 min after i.v. injection of (delta) -aminolevulinic acid. A second high tumor:skin tissue ratio of 5.7:1 could be measured at 24 h after injection, but at this point in time the protoporphyrin content in normal skin was higher than 45 min after injection. The kidney may not be strongly affected by i.v. administration of (delta) -aminolevulinic acid, whereas the liver reveals an accumulation of porphyrins, e.g. protoporphyrin. Concluding from these results in this experimental tumor model, i.v. administration of (delta) -aminolevulinic acid seems to be a promising modality to perform photodynamic therapy more effectively and more selectively by irradiation 45 - 180 min after injection of (delta) -aminolevulinic acid.

To provide basic scientific information for utilizing photodynamic therapy to treat prostatic carcinoma, PDT dosimetry and effective treatment volume were investigated using canine prostate as an in vivo animal model. The prostate glands were surgically exposed. Isotropic tip fibers were inserted into the gland for either light irradiation or detection. PDT doses of 400 J and 1, 3 or 5 mg/kg Photofrin were used for single fiber irradiation treatments. All animals were terminated at 1 week and the prostates evaluated histopathologically. The results show that the lesion size was dependent on the treatment dose. The maximum lesion dimension was approximately 1.6 cm. The data acquired from the single fiber irradiation study were then used to plan for multi-fiber interstitial PDT treatments with a goal of achieving a larger treatment volume for practical purpose. The data indicate that the lesion size from the multiple fiber treatment could be predicted based on the single fiber treatment with the light pattern summed from all sources.

By rotating the optical axis of a nonlinear optical crystal ((beta) -BaB₂O₄), a tunable laser beam could be obtained from an optical parametric oscillator (OPO) laser. When the crystal was optically pumped by the third harmonics of the 1064 nm Nd:YAG laser, we had a coherent beam from 410 nm through 2550 nm continuously without changing the optical cavity. We compared photodynamic therapy (PDT) effects of two photosensitizers, phenophorbide a(Phd) and Photosan-3(Ph-3, hematoporphyrin-polyester), on Wistar rat liver. Twenty-four hours after sensitization (5 mg/kg i.v.), 670 nm and 630 nm light (75 mW/cm²) was irradiated for Phd and Ph-3 respectively at energy doses of 25, 50, and 100 J/cm². The rats were sacrificed 24 hours after laser irradiation and analyzed pathologically. Phd produced more severe necrosis than Ph-3. Twenty-five J/cm² of Phd was identical with 100 J/cm² of Ph-3. Next, we treated HeLa cell tumors of nude mice by Phd 670 nm PDT and Ph-3 630 nm PDT. The PDT effects of the two photosensitizers on HeLa cell tumors were similar to those on normal liver tissue. In conclusion the OPO laser could make it possible to compare PDT effects of photosensitizers by activating them with their matched wavelengths.

An informative computer program containing laser therapy and puncture methods has been developed. It was used successfully in connection with the compact Russian medical laser apparatus HELIOS-O1M in laser treatment and the education process.

At present lasers are chosen in photodynamic therapy because they produce light of superior beam and spectral qualities and of more penetrating longer wavelengths. However there is stronger absorption by porphyrins at less penetrating shorter wavelengths. The response of a rat intradermal fibrosarcoma to topical 5-aminolaevulinic acid photodynamic therapy was compared using 630 nm light from a copper vapor/due laser or broad spectrum red or white light from a modified slide projector. When the tumors were between 2 - 3 mm thick they were randomized to treatment with one light source at a range of fluences all at the same sub- hyperthermic fluence rate. Laser light and white light produced a greater tumor growth delay than broad spectrum red light, and at higher fluences white light was comparable to laser light. For the treatment of thin, superficial tumors using topical aminolaevulinic acid photodynamic therapy an inexpensive, portable, white light source may be an effective alternative to a laser.

Locally recurrent breast carcinoma and skin metastasisses on the chest wall can be difficult to treat. Conventional treatments like radiation-, chemo- and hormonal therapy have shown poor results in these patients. In comparison to this, PDT has some advantages and less side effects. We can observe a tumor accumulation of a systemic applied photosensitizer (PS). The PS can be stimulated by light of a wavelength of 630 nm and a phototoxic effect in the tumor occurs. We treated 7 patients with locally recurrent breast carcinoma 15 times with PDT. The intravenous application of the PS (Photofrin II, 1.5 mg/kg BW) was done 24 - 96 hours before local laser light radiation. The light source was an Ar-Dye laser with a wavelength of 630 nm. Due to a local tumor necrosis we observed a tumor reduction in each case. In 5 patients we saw a complete local remission with a good cosmetic result. Side effects were rare. All patients suffered from pain in the treated area. No major phototoxicity effects were seen. PDT can induce complete local tumor remissions in patients with cutaneous metastasisses after locally recurrent breast carcinoma. In absence of other metastasisses PDT is possibly a curative treatment. One of the major advantages of this treatment are the rare side effects, rare complications and the possible repetition of the PDT.

Photodynamic therapy has been proposed as an intra-operative adjunct to surgical resection of tumors invading bone. To assess this, we studied the effects of PDT in normal bone. Forty- four rabbits were sensitized with Photofrin 3 mg/kg, 5-aminolaevulinic acid (ALA) 400 mg/kg, or meso-tetrahydroxyphenylchlorin (mTHPC) 0.3 mg/kg. A mandibular incisor was removed and the socket irradiated with a cylindrical diffusion fiber (630 nm Photofrin and ALA, 650 nm mTHPC, 100 J per treatment). Irradiation was given 1 or 48 hours after Photofrin, 72 hours after mTHPC, whilst 2 doses were given 2.5 and 4 hours after the first fractionated dose of ALA. The socket of the ipsilateral maxillary incisor was used as a nonirradiated control to assess healing without PDT. Other controls assessed healing after irradiation of unsensitized animals. Rabbits were killed 3, 10, and 21 days after treatment. Tooth socket healing appeared to be the same in all groups of animals with evidence of woven bone formation by 10 days. We conclude that PDT is unlikely to have any effect on healing in normal bone, which makes it suitable for treating tumors invading bone.

Photodynamic therapy (PDT) using benzoporphyrin derivative (BPD, Verteporfin) and whole body irradiation, can affect the course of adoptively transferred experimental allergic (autoimmune) encephalomyelitis (EAE) in PL mice. Murine EAE is a T cell-mediated autoimmune disease which serves as a model for human multiple sclerosis. Using a novel disease induction protocol, we found that mice characteristically developed EAE within 3 weeks of receipt of myelin basic protein (MBP)-sensitized, in vitro-cultured spleen or lymph node cells. However, if animals were treated with PDT (1 mg BPD/kg bodyweight and exposed to whole body 15 Joules cm² of LED light) 24 hours after receiving these cells, disease onset time was significantly delayed. PDT-treated mice developed disease symptoms 45 +/- 3 days following cell administration whereas untreated controls were affected within 23 +/- 2 days. In contrast, application of PDT 48 or 120 hours following injection of the pathogenic cells had no significant effect upon the development of EAE. Experiments are in progress to account for the protective effect of PDT in this animal model. These studies should provide evidence on the feasibility of PDT as a treatment for human autoimmune disease.

Leukocyte mediated tissue damage is proposed to contribute to the tumoricidal effect of photodynamic therapy (PDT). To elucidate this hypothesis leukocyte-endothelium interaction and red blood cell velocity in the microvasculature of an amelanotic melanoma and normal tissue in a skin fold chamber preparation were investigated before and after PDT (5 mg/kg Photofrin^R; 630 nm; 10 or 100 J/cm²). One hour after PDT (10 and 100 J/cm²) the number of adhering leukocytes increased in subcutaneous but not in tumor microvessels. At this time red blood cell velocity remained unaltered in microvessels of subcutaneous tissue but decreased (10 J/cm²) and even ceased (100 J/cm²) in tumor microvessels. These results indicate that tumor destruction induced by PDT is not mediated by leukocyte-endothelium interaction.

Usually cell viability (CV) after PDT is observed in methods based on metabolic parameters (MTT assay) or dye exclusion tests [trypan-blue (TBT)]. Although these tests are accurate in the determination of cell death, functional impairment can not be easily detected. We examined the effects of PDT on human dermal fibroblasts (DF) to determine whether collagen gel contraction (CGC) can be inhibited and whether it correlates well with CV as measured by the TBT. Five-aminolevulinic acid (5-ALA) (1, 5, 10 (mu) g/ml) was added to DF cultured in dishes. Twenty-four hours later, a collagen type I solution was added and irradiation with a tunable dye laser (630 nm, 100 mW/cm², 80 J/cm²) was performed. After 24 h, diameters of contracted gels and TBT were measured. As a result, PDT with 5-ALA inhibited CGC and affected CV as a function of dose level. Consequently, CGC data allows a better estimation of the threshold 5-ALA dose affecting the cells.

Photodynamic therapy (PDT) of tumors, as a primary treatment or as an adjunctive intra- operative therapy, may expose major vascular structures to injury. PDT has also been proposed to prevent neointimal hyperplasia following angioplasty of stenotic arteries. This study aimed to determine the effect of PDT on the normal rabbit carotid artery, and to determine whether this injury resulted in weakening of the vessel wall. PDT of the carotid arteries of NZW rabbits, using either disulphonated aluminum phthalocyanine or 5- aminolaevulinic acid induced protoporphyrin IX as photosensitizers, was performed using a light dose of 100 J/cm². Histological examination of the carotids treated with both drugs demonstrated full thickness loss of cellularity 3 days following photodynamic therapy. Treated vessels all remained patent and no inflammatory infiltrate was observed. Elastin van Gieson staining showed preservation of inner and medial elastic laminae and medial and adventitial collagen. Further rabbits were similarly treated with PDT to 1 cm segments of both common carotids and sacrificed at 3, 7, and 21 days. The carotids were exposed and control and treated segments subjected to intraluminal hydrostatic distension until the vessels ruptured. No reduction in the pressure required to rupture the vessels was evident in treated vessels compared with controls. It is concluded that in spite of full thickness cell death, PDT treated arteries are not at risk of thrombotic occlusion or hemorrhage.

While laser beams are essential for endoscopic photodynamic therapy (PDT), many types of superficial tumors can be treated effectively using incoherent light sources which are appropriately filtered. This paper reports on clinical trials using simple and effective red light sources modified from an overhead projector and a surgical headlamp with fiber optic output. More than 1 W of effective red light can be obtained and used effectively in the palliative control of large superficial lesions in initial trial cases of three types of malignancies. The use of simple light sources for the treatment of superficial tumors avoids the technical complexities associated with laser sources and enables ready observations of treatment response. Such clinical trials should be conducted extensively in the development of PDT as a viable cancer treatment modality prior to the development of endoscopic procedures.

We describe a general purpose instrument capable of a wide range of noninvasive measurements potentially useful for PDT dosimetry. Examples of applications are presented along with some limitations of the approach.

Advances in short arc technology and optical filter coatings led to the design and construction of a table-top light source in 1989; the first viable and cost-effective alternative to a laser. The device can deliver over 3 W within a 30 nm band centered at any wavelength from 200 nm to 1200 nm at fluence rates of over 1 W cm^-2. Its relative biological effectiveness (RBE) in vitro has been proven alongside an argon pumped dye laser and a copper vapor pumped dye laser. These in vitro tests showed an efficiency of hematoporphyrin derivative (HPD) induced cellular photoinactivation close to that of the argon/dye laser (RBE 100%), with a mean RBE for the lamp of 87 +/- 3% (p < 0.05). The lamp proved to be superior to that of the copper/dye laser system with an RBE of up to 150% at fluence rates above 50 mWcm^-2. In vivo tests show that the extent and depth of tumor necrosis are comparable to that of an argon/dye laser. An in situ bioassay using tumor regrowth delay is currently underway. Early clinical trials show clearance of Bowen's disease and actinic keratosis using the same light fluences as costly PDT lasers.

We have used ultra-low light level fluorescence microscopy to examine the suggestion that the relatively poor response of human basal cell carcinomas (BCC) to topical 5-aminolaevulinic acid (ALA)-based photodynamic therapy (PDT) arises from limited drug penetration into the lesion. The distribution of ALA-induced protoporphyrin IX (PpIX) in human BCC and Bowen's disease was examined and, in almost all cases, was found to be most intense in those regions of tumor immediately adjacent to the dermis. This distribution was independent of tumor type, and did not appear to be affected by tumor depth in the skin. It is suggested that ALA penetration may not limit the efficacy of ALA-PDT in the treatment of BCC. Failure of superficial ALA-based PDT in basal cell carcinoma may, instead, be related to the histological structure of this type of lesion.

We have synthesized approximately 700 kinds of porphyrin derivatives and studied their side chain structures and affinities for tumor tissues. On the basis of these studies, a tumor localizing photo-chlorine photosensitizer named ATX-S10 has been synthesized for PDT and fluorescent diagnosis of malignant lesions. A non-photosensitive fluorescent diagnostic agent named HAT-D01 has also been synthesized. Both derivatives have the fluorescence near 680 nm that is far from the auto-fluorescence of biological tissue. For detection of tumor tissue, we have developed a new device that can pick up 670 - 680 nm fluorescence selectively and convert the intensity of fluorescence to the sound. By using this simple new device after ATX- S10, HAT-D01 administration we could detect well the malignant lesions.

Analysis of sensitizer kinetics is essential for the performance of light irradiation when tumor concentration and tumor-to-normal tissue ratios are optimal. In our study, nude mice were grafted with human adenocarcinoma 15 days before m-THPC IP injection. Fluorescence was recorded through an optic fiber spectrofluorometer at 650, the most intense, and 714 nm, and intensity was proportional to the injected dose. In tumor, skin and muscle, maximum fluorescence was obtained with 1.6 mg/kg 72 h after injection (44 counts per second). Tumor- to-skin and -muscle ratios obtained by HPLC analysis and spectrofluorometric measurements decreased between 12 and 72 h (15 to 1.5), indicating that tumor selectivity decreases with time.

Fluorescence after photosensitization has the potential to identify lesions not visible on conventional endoscopy. We assessed 12 patients at high risk of or with established GI cancers (u ulcerative colitis, 1 colon polyp, 2 familial polyposis with duodenal polyps, 2 early oesophageal cancers). Fluorescence images (excitation 390 nm) were recorded with endoscopic equipment and additional spot measurements (optical multichannel analyzer). Patients were given 10 - 60 mg/kg ALA orally or 2 mg/kg Photofrin i.v. 60 mg/kg ALA gave high levels of PP IX (proto-porphyrin IX) in all areas, but 10 - 15 mg/kg resulted in selectivity in macroscopically inflamed colon. Photofrin gave oesophageal tumors selectivity at 4 and 48 hours. Photofrin patients subsequently had PDT. Photobleaching was documented in 3. We conclude that these techniques have potential as `optical biopsy tools' and for screening for early neoplastic changes.

We investigated the in vivo fluorescence detection of premalignant and malignant lesions in carcinogen-induced tumors of the hamster buccal cheek pouch and micrometastases in rat lymph nodes using hexylpyropheophorbide-a (HPPH) at 0.5 mg/kg and He-Ne laser-induced fluorescence photometry. Time course studies of HPPH in hamster tumor model showed maximal relative fluorescence readings at 48 hours after i.p. injection in each stage (dysplasia: 0.35, papilloma: 0.58, squamous cell carcinoma: 1.04). Squamous cell carcinoma showed significantly greater fluorescence than papillomas and dysplasias at all time points (p < 0.01). Metastatic lymph nodes were significantly greater than normal lymph nodes at all time points (p < 0.01). Maximal fluorescence levels of normal lymph nodes were observed at 6 hours after i.v. injection. Metastatic lymph nodes still showed high fluorescence levels at 72 hours. Micrometastases showed fluorescence levels between the levels of metastatic and normal lymph nodes (normal: 0.40 < micrometastasis: 0.62 < metastasis: 0.75 at 48 hours after i.v. injection). The results demonstrated good correlation between fluorescence levels and histopathological developments at all time points. Therefore, HPPH may be a promising fluorophore for the detection of developing malignancies and occult disease.

An ordinary fluorescence spectrometer equipped with a dichroic mirror and fiber optics can be used to measure sensitizer fluorescence in tumors and normal tissues. Our findings with such an instrument showed that after oral administration of 60 mg/kg 5-aminolevulinic acid (ALA) mucosas and rectal adenomas fluoresced stronger than normal skin. Also basal cell carcinomas (BCCs) showed a stronger porphyrin fluorescence than skin after topical application of ALA. The porphyrin fluorescence of BCCs decayed rapidly during standard laser irradiation at 630 nm. The degradation rate of porphyrins in the tumor was approximately constant down to at least 0.3 mm below the tumor surface, in agreement with an optical penetration depth of more than 1 mm.

For the application of photodynamic therapy (PDT) for wider lesions of early gastric cancer we employed a new model of an excimer dye laser (EDL), because it is necessary to increase the average output for the irradiation of enough energy to the wider lesion within a limited time, in addition to protecting the single quartz fiber from destruction. The characteristics of the new laser are as follows: wavelength, 630 nm; pulse energy, 4 mJ; peak power, 400 kW; pulse width, 10 nsec; frequency of repetition, 80 Hz; average output, 320 mW. The PDT can be applicable for wider lesions of early gastric cancer by employing the new model of EDL, that can produce the average output of 320 mW with repetition of 4 mJ in 80 times per second.

Arthritic synovitis is best cured by total removal of the inflamed synovia (equals synovectomy). This can be performed by open chirurgy, by arthroscopy, or by radiosynoviothesis, i.e., injection of (beta) -radiating rare earth metals into the joint cavity. All these procedures are more or less non-quantitative and may lead to a recidive. The idea was to destroy the inflamed synovia by PDT without destruction of the underlying tissue (cartilage and bone). So, the sensitivity of the cartilage-building cells, which can be grown in cell culture, has to be studied.

Protoporphyrin (Pp IX) is the final intermediate product before haem and can be stimulated to a phototoxic reaction with light. The presence of 5-aminolevulinic acid can increase the intracellular biosynthesis of Pp IX in certain types of tumor cells. The photosensitizing concentrations of Pp IX make laser light induced fluorescence diagnostics (LIFD) and photodynamic therapy possible. A topical application of a 5-aminolevulinic acid solution requires a waterproof occlusive dressing for several hours. We developed a simple technique for a practical preparation for PDT using a hydrocolloid dressing. The normal surrounding skin can be spared. We present our first therapeutic experience with a case of cutaneous breast cancer in a 65-year-old female patient. Six hours after topical application of 10% isotonic 5- aminolevulinic acid under the hydrocolloid dressing PDT was performed (Ar-Dye Laser, 630 nm wavelength). Twenty four hours after PDT a superficial tumor necrosis could be observed with a maximum depth of tumor necrosis of 2 - 3 mm. The surrounding normal skin was without any inflammation.

HPV associated bowenoid papulosis of the anogenital region are classified as carcinoma in situ. The treatment can be difficult and recurrence rates are high. Extended surgical resections may have complications such as anal sphincter insufficiency. PDT does have some advantages and less side effects in the treatment of these tumors. We treated one female patient with an extended perianal bowenoid papulosis. Previous surgical resection led to local recurrence and partial sphincter insufficiency. Twenty-four hours before local laser light radiation (Ar-Dye laser, 630 nm wavelength), a systemic photosensitizer was applied (Photofrin II, 1.5 mg/kg BW). Four courses of PDT were performed within one year. We observed a total tumor necrosis in every radiation area. The previous sphincter insufficiency improved during the sessions. Side effects were rare. Pain in the radiation was stopped within 2 - 3 days under pain medication. PDT can induce a total local tumor necrosis in perianal bowenoid papulosis. Concerning local expansion, PDT can be a curable treatment.

Early clinical results of PDT on colon tumors using up to 60 mg/kg ALA given orally (comparable to 30 mg/kg if given i.v.) with light doses of 50 J showed only superficial necrosis. We investigated whether this could be due to subthreshold tissue concentrations of Protoporphyrin IX (PP IX) or suboptimal light treatment schedules. Normal Wistar rats were sensitized with 25 - 400 mg/kg ALA given i.v. and irradiated with 100 mW red light at 630 nm (copper vapor pumped dye laser) 10 - 200 J delivered via a fiber placed touching the colon mucosa at laparotomy at the time of peak PP IX concentration (1 - 4 hours depending on the dose used). Animals were killed 3 days later and the diameter of mucosal necrosis measured. No lesion was seen with 25 mg/kg. Increasing the ALA dose from 50 - 400 mg/kg, the lowest light dose to produce a lesion of > 5 mm fell from 100 J to 25 J. If ALA doses for rats and humans are comparable (which is likely) these results suggest that the drug and light doses used clinically are close to the thresholds levels, so better clinical results may be achieved by increasing the value of one or both.

A system of testing of potential photosensitizing agents for PDT by the illumination of cells in monolayer is described. The system allows tunability of the excitation wavelength, uniform illumination over a 35 mm cell dish, variation of the irradiance and monitoring of the power delivered throughout the duration of illumination. Knowledge of these parameters allows rapid processing, precise light dosimetry, limitation of possible heating effects as well as optimization of the excitation efficiency. The system is applied to the illumination of an endothelial hybridoma cell line using 680 nm and 692 nm light in the presence of phthalocyanine sensitizers.

Hydrophilic silicon phthalocyanines derivatives with diaxial polyethyleneglycolmonomethylether ligand with different chain lengths were synthesized. Water soluble SiPc(OmPEG 5000)₂ shows a completely monomeric UV/VIS spectrum in water. No influence of the ligand's chain length on photophysical properties, e.g., singlet oxygen quantum yield was found. First aspects of the photosensitizing properties were gained by photohemolyse experiments with human erythrocytes. SiPc(OmPEG 5000)₂ is far more active than other water soluble phthalocyanines which are aggregated in water.

The phthalocyanines form a continuously growing family of potential photosensitizers. Their chemical and photophysical properties, and therefore their photodynamic activities, are influenced by the type of the central metal ion as well as the type and localization of substituents. Plasma and tissue distribution kinetics are also dependent on the chemical features of a dye molecule. We therefore investigated the pharmacokinetics of newly synthesized octa-(alpha) -butyloxy-zincphthalocyanine (8-(alpha) -bo-ZnPc) on the Lewis lung carcinoma in order to evaluate the application possibilities of this compound in experimental PDT treatments. The zinc complex was chosen on the basis of the known effect of this metal on the photochemical and photophysical properties of phthalocyanines. The pharmacokinetics of octa-(alpha) -butyloxy-zincphthalocyanine were studied on the Lewis lung carcinoma by absorption spectroscopy in mice. Afterwards a regression analysis was performed using the absorption spectroscopic data.

Structural changes after photodynamic therapy of atherosclerotic lesions of the thoracic aorta were analyzed by scanning electron microscopy. Cholesterol fed atherosclerotic rabbits were injected intravenously with 5 mg/kg of NPe6. At 6 hours after NPe6 loading, a diode laser irradiated angioscopically on the surface of atheroma with the total energy of 200 mJ/cm². Scanning electron microscopy showed degeneration of atherosclerotic plaques of the thoracic aorta examined at one week after photodynamic therapy. NPe6 could be a potent photosensitizer for photodynamic therapy of atheroma.

A new photosensitizer, PH-1126, was administered intravenously into the ear vein of cholesterol-fed atherosclerotic rabbits at a dose of 1 mg/kg of body weight. At 24 hours after PH-1126 administration, the atherosclerotic lesions of the abdominal aorta were irradiated by krypton ion laser at 647 nm of wavelength with 100 J/cm². The abdominal aorta irradiated by laser beam was excised for histological analysis using the scanning electron microscopy and transmission electron microscopy. In the atherosclerotic plaque irradiated by krypton ion laser, migratory cells were observed and some of these cells were migrated into the lumen of the vessel. These findings suggested that these migratory cells from atherosclerotic plaque were foam cells destroyed by photodynamic therapy.

We have designed and constructed a simple, cheap and effective diffuser/applicator for intraluminal photodynamic therapy in oesophageal cancer. A cylindrical diffusing optical fiber can be easily located in the center of the oesophageal lumen with the use of a modified naso- gastric Ryles tube. This allows more uniform illumination of the luminal circumference. Measurements are presented of the light field generated by this delivery system in an optical phantom. These demonstrate that the presence of the Ryles tube imposes only a small modification on the output of the bare diffuser. The light doses received adjacent to the diffusing section are identical, within the accuracy of measurement, both with and without the tube. This ensures adequate illumination of a circumferential oesophageal tumor using a contained fiber, without adjustment of the established treatment parameters.

The fluorescence bleaching and photoproduct formation of PPIX was investigated in Ehrlich ascite carcinoma (EAC) cells (ALA induced and exogenous applied) as well as in micelles (Triton X-100) and DMSO. In all the cases fluorescence bleaching of PPIX occurs but the photoproduct formation is quite different. Besides the rapid fluorescence bleaching of the ALA induced PPIX in EAC cells the formation of a water soluble porphyrin with fluorescence maxima at 616 nm and 678 nm (coproporphyrin) was observed. In the presence of the triplet- and oxygen-quencher (beta) -carotene the fluorescence bleaching of PPIX can be reduced.

PDT kills smooth muscle cells (SMC) in vivo and thus prevents intimal hyperplasia after angioplasty. It causes little inflammation and structural integrity of the artery is not compromised. We have studied the process of the SMC death in vitro. Cultured rat SMC (cell line sv40 ATCC) were sensitized with aluminum disulphonated phthalocyanine (AlS₂Pc), and then irradiated with 675 nm laser light (2.5 J/cm²). Controls were studied using only sensitizer or laser for treatment. The cells were incubated and the dying process observed with a time lapse video and microscope system. PDT caused a characteristic pattern of death. Cells lost contact with neighbors, shrank, and showed hyperactivity and membrane ruffling. The cells imploded into active and condensed membrane bound vesicles which were terminally reduced to residual bodies. These are the morphological changes of apoptosis. The control cells which were given AlS₂Pc alone or laser alone showed no death. PDT induced cultured arterial SMC death by apoptosis rather than necrosis. An apoptotic mechanism of cell death in vivo would explain the relative lack of inflammation and local tissue destruction in the face of massive death.

Photodynamic therapy (PDT) with Photofrin^R, nonsulfonated aluminum phthalocyanine (AlClPc), and tin ethyl etiopurpurin I (SnET2) was investigated on normal and tumored (9L gliosarcoma) rat brain. Rats were injected 48 h prior to PDT (3 - 280 J/cm², 100 mW/cm²) with Photofrin^R (12.5 mg/kg) and AlClPc (2.0 mg/kg) and 24 h prior with SnET2 (1.0 mg/kg). Substantial lesions were present in normal brain with Photofrin^R and with AlClPc at all energy levels. No lesions were present with SnET2. Tumor response for Photofrin^R and AlClPc did not occur with doses less than 140 J/cm². Drug concentrations for tumored/contralateral hemisphere for Photofrin^R and AlClPc were 1.86 and 7.86, respectively. These data indicate: (1) normal brain is more sensitive than the tumored brain with Photofrin^R and AlClPc, and (2) normal brain sensitivity to SnET2 is less than that with Photofrin^R and AlClPc.

To determine the optimum wavelength for irradiation with coherent light after 5-ALA-induced protoporphyrin IX-sensitization, HaCaT cells were allowed to take up 5-ALA (30 (mu) g/ml) for 24 h. A tunable dye laser was used for PDT (40 mW/cm², 30 J/cm², wavelengths between 622 - 649 nm in steps of 3 nm). Afterwards, an MTT assay was performed to check cell viability (CV). Four groups (n equals 8) were formed: A equals controls, B equals irradiation only, C equals 5-ALA only, D equals 5-ALA and irradiation. CV of groups B, C, D were compared to those of group A (100%). Group B showed no significant difference (92% range at different wavelengths between 72% and 100%). Group D showed a significant decrease of CV (61%, range at different wavelengths between 35% and 86%). Lowest values for CV were detected at 634 and 637 nm. We believe therefore, that PDT with 5-ALA as sensitizer shows better cytotoxicity when wavelengths at 634 - 637 nm are used.

Eighty-three patients suffering from moderate or advanced malignant tumors were treated by combined chemotherapy and photodynamic therapy (PDT) in our hospital. The short term result of such management is very promising, the effectiveness seems to be nearly 100% and the general responsive rate is 79.5% (CR + PR). If compared with another group of 84 similar patients whom were treated with PDT alone, the short term efficacy is 85.7% while the general response rate is 54.7% (P < 0.01), there is a significant statistic. The better result of the combined approach is probably due to the action of the chemotherapeutic agent, potentially blocking the mitosis of the cellular cycle at certain phases of the cancer cells, making the cell membrane become more permeable to the photochemical agent, HPD, and eliciting a better cancerocidal effect.

The only early therapy of rheumatoid arthritis in orthopedic surgery is a synovectomy, which is restricted to more or less big joints. A laser-synovectomy of small joints is ineffective yet. An alternative method may be photodynamic therapy. In our study we describe the photodynamic effect of Photosan 3 in a cell culture study.

Photodynamic therapy (PDT) using benzoporphyrin derivative, monoacid ring A (BPD), and transdermal light was able to significantly treat symptoms of adjuvant-enhanced arthritis in MRL-lpr mice. Clinical and histological evaluation showed that PDT was able to modify the progression of adjuvant-enhanced arthritis up to 10 days after induction. When PDT was used on arthritic joints displaying swelling, it prevented further deterioration of clinical symptoms (76%, 16/21). However, it did not significantly effect the histopathologic parameters. As we have previously reported that mitogen activated MRL-lpr splenocytes were shown to be more susceptible to in vitro PDT we postulate that our findings reflect a selective destruction of adjuvant activated lymphocytes in the circulation and/or joints. The application of PDT to eliminate activated cells responsible for the inflammatory reaction at the arthritic site may have significant clinical implications for the treatment of rheumatoid arthritis.

Endometrium was destroyed by Nd-YAG laser under B ultrasonic monitoring through direct application of a fibrous catheter specially designed by Dong-lin Lee (an optical engineer). Of the 12 patients 11 cases had dysfunctional uterine bleeding and one case had postmenopausal uterine bleeding. The mean time of laser treatment was 5' 45', the mean volume of irrigating solution was 850 ml, and the mean estimated blood loss was less than 9 ml. All patients showed neither evidence of infection nor uterine perforation. Follow-up examination for 3 - 16 months showed amenorrhea in 4 cases, spotting in 4 cases, and obviously reduced menstrual flow in 3 cases. One patient, partly treated, had normal menstruation.

The photodynamic therapy of cancer has been improved by the developments of laser technology and photosensitizers the last 15 years. The relative yield of active oxygen species generated in the photosensitizing reaction was obtained in vitro by photochemical techniques and ESR measurements with spin trapping reagents in order to find an effective photosensitizer. Cethyltrimethyl ammonium bromide (CTAB) micellar aqueous solutions and human melanoma cultivated (HMF) cell suspensions were used for the in vitro experiments. Photosensitizer was incorporated into the CTAB micelles or HMF cells, and 5,5'-dimethyl-1- pyroline-N-oxide (DMPO) and 2,2,6,6-tetramethyl-4-pyperidone hydrochloride (TMPD) were used for the detection of OH, C (type I reaction) and ¹O₂^* (type II reaction), respectively. After irradiation by laser light or by monochromatic light, the ESR spectra were analyzed. It was found that type I reaction was predominant for the aggregate form of the photosensitizer and type II for the monomer form in CTAB micellar solutions. It was suggested that the predominant type II reaction in HMF cell suspension indicated the monomerization of the photosensitizer by the cells also in the case of hematoporphyrin oligomer (HpO).

Fluorescence offers a non-invasive means to assess the structure and status of a tissue. In the clinic, two basic tasks are required: (1) surveillance of large tracts of tissue, best done with video endoscopy in order to efficiently find sites of abnormality; and (2) spectroscopy of local sites, best done with an optical fiber fluorescence spectrometer in order to specifically characterize a tissue site and eliminate false positives. The role of tissue optics in distorting fluorescence measurements is presented.