Hemoptysis (sometimes life threatening) is a frequent symptom of patients with endobronchial tumors. We recorded the amount of hemoptysis of 140 patients before treatment of the tumors with Photodynamic Therapy (PDT) and at subsequent follow up examinations. Follow up was 100. A hemoptysis scale of: 0 equals none; 1 equals streaks; 2 equals drops and clots; 3 equals large clots life threatening; 4 equals massive (life threatening requiring transfusions) was recorded.

Photodynamic therapy (PDT) utilizing Photofrin has proven to be an effective modality used in the treatment of solid tumors. In particular, it can be applied via endoscopy to lesions developing in luminal organs. A phase II study was conducted for submission to the Japanese Ministry of Health and Welfare. In this protocol an excimer dye laser was used to deliver 630 nm light via a quartz fiber passed through an endoscopic working channel two days subsequent to i.v. injection of photosensitizer. In this study, 98 patients with superficial cancer of various organs were treated. Of these, 88 patients could be evaluated, including 33 with roentgenographically occult lung cancer, 10 with esophageal cancer, 24 with gastric cancer, 18 with cervical cancer and three with bladder cancer. Complete remission as evaluated endoscopically, pathologically, and cytologically was obtained in 83 out of 98 (84.7). There was no serious complication except mild skin photosensitivity, which was seen in four patients. It was concluded that PDT can be efficacious in the treatment of superficial cancers and that complete remission can be achieved when suitable photoradiation conditions are met.

In this study, the subcellular targets and cellular mechanisms of the photodynamic action of (delta) -aminolevulinic acid (5ALA) were investigated in rat mammary carcinoma cells (MTF7) in vitro. A constant dose of 5ALA (200 (mu) g/ml) was given and the light dose was varied (20-480 J/cm²). The effects of PDT were examined at 0 hr, 3 hr, and 24 hr after the treatment using the following assays: (1) Trypan Blue Dye Exclusion Assay for cell membrane damage; (2) MTT Assay for mitochondrial damage; (3) ³H-Thymidine Incorporation Assay for DNA synthesis; (4) Cell Colony Formation Assay for cell survival. The MTT and colony formation assays are the best predictors of cell survival early and late after treatment. Cell membrane damage and almost complete disruption of mitochondrial function are early mechanisms of 5ALA PDT cytopathic effect. Reduction of DNA synthesis is a later, and possibly, a secondary effect of disruption of mitochondrial function although this conjecture has to be explored further. Reduction of DNA synthesis and cell membrane damage show complicated relationships with light dose while loss of mitochondrial function and long term survival are essentially complete with the lowest light dose used in this study.

We have found that an esophageal centering balloon improves light delivery to esophageal mucosa during photodynamic therapy (PDT). However, balloon pressure on esophageal mucosa could possibly reduce mucosal blood flow and oxygenation, therefore reducing the photodynamic therapy effect. Studies were performed in the canine esophagus of 10 animals to investigate whether increasing the size of the centering balloon and hence the pressure on esophageal mucosa would alter the tissue effect of PDT. It was concluded that increasing balloon size resulted in reduced tissue damage when mucosal equivalent light dose was administered during photodynamic therapy. Proper sizing of centering balloons will be necessary for balloon PDT of esophageal mucosal dysplasia or cancer.

Barrett's esophagus is a common premalignant disease. The aim of this study is to determine if low dose photodynamic therapy is capable of ablating Barrett's epithelium allowing regrowth of normal squamous mucosa. Methods: Patients with specialized Barrett's esophagus of at least 3 cm length were given 1.5-1.75 mg/kg of hematoporphyrin derivative intravenously. After 48 hours, esophagoscopy and videotaping of the Barrett's was performed. A 1.0-1.5 cm cylindrical diffusing fiber delivered light of 630 nm in a dose of 150-200 J/cm using a tunable dye laser at a power of 400 mW/cm. Following entry, patients were placed on omeprazole 20 mg/day for six months. Results: Twelve patients (8 men) mean age of 62 +/- 4 have had repeat endoscopy performed at 25 +/- 2 weeks following entry. The Barrett's segment length decreased from 8+/- 1 to 5+/- 1 cm after PDT (p < 0.01) with a corresponding change in the appearance and location of the squamocolumnar junction. Six (50%) of the patients had a 4 cm or more decrease in the length of their Barrett's esophagus with normal squamous mucosa on biopsy. Adverse events included transient odynophagia, minor sunburn, and transient chest pain. Conclusions: Photodynamic therapy of Barrett's esophagus can cause a regression in length of the Barrett's segment with replacement by normal squamous epithelium.

Forty-nine patients with neoplastic diseases of the larynx, oral cavity, pharynx and tracheobronchial tree have been treated with photodynamic therapy with follow-up to 40 months. Those patients with primary recurrent leukoplakia, carcinoma-in-situ, and T1 carcinomas obtained a complete response after one photodynamic therapy (PDT) treatment and remain free of disease. Eight patients with T2 and T3 carcinomas treated with PDT obtained a complete or partial response, but in all cases, the carcinomas recurred locally, many times with overlying normal mucosa. This is due to the inability to adequately deliver laser light to the depths of the tumor bed, despite aggressive use of interstitial implantation. PDT is highly effective for the curative treatment of early carcinomas (CIS, T1) of the head and neck. Further development of devices to measure and deliver light into the depths of a tumor bed are required prior to the use of PDT to effectively treat larger solid tumors of the head and neck.

Delta-aminolevulinic acid ((delta) -ALA) has been recently proposed as a tumor photosensitizer precursor with increased selectivity and decreased toxicity for the treatment of neoplasms. We investigated the conversion and cytotoxicity of (delta) -ALA in a human hepatoma cell line to determine its clinical potential. SK-HEP-1 (ATCC) cells were plated on 35 mm coverslips in media for use in a digital fluorescence microscopic imaging system. (delta) -ALA was added to achieve final concentrations between 0-5 mM. Cells were excited with 450-490 nm light while a 610 nm long pass filter was used to assess fluorescence from conversion to protoporphyrin IX, the putative photosensitizer. After maximal fluorescence was obtained at each initial concentration of (delta) -ALA, cells were radiated with 10 J/cm2 of light from a xenon lamp fitted with a 515 nm band pass filter. After photoradiation, cell death was assessed by flow cytometry using propidium iodide labeling. Protoporphyrin IX accumulation was constant at K_sequals0.001 until a plateau was achieved 2 hours after the addition of (delta -ALA. Photoradiation with 10 J/cm2 at a concentration of 1 mM (delta ALA resulted in a linear increase in cell death over time with 5% cell death at 2 hours and 12% at 5 hours compared to controls. Interestingly, controls with (delta) -ALA alone demonstrated a cytoprotective effect with a logarithmic relationship between increasing cell survival and increasing dose of drug.

The use of aminolevulinic acid (ALA), a precursor of protoporphyrin IX (PPIX), is a new concept in PDT. The goal of our study was to compare two different incubation times of ALA in two cell lines and to evaluate phototoxic potential in correlation with extraction data of PP IX. For PDT a concentration of 1, 5, and 10 mM of ALA for Waf bladder cancer cells and 0.1, 1, and 1.5 mM for Mefa fibroblasts was used. The incubation time was 3.5 or 5.5 h. Irradiation was performed with an argon-dye laser emitting light at a wavelength of 630 nm. After irradiation, cells were incubated for two days and compared with control cells. For extraction of PP IX, cells were incubated with ALA at the same concentration as for PDT experiments for 3.5, 5.5, and 7.5 h. In both cell lines the survival rate was decreased after an incubation time of 5.5 h as compared to 3.5 h independent of the ALA-concentration or the fluence. Light and ALA alone had no effects on cells. The extraction data showed a good correlation with these results. In both cell lines the highest fluorescence of PP IX was measured after an incubation time of 5.5 h.

The photobiology of a group of iminium salts was examined. The nonfluorescent copper derivative (CDS1) had an almost undetectable triplet yield, but could catalyze phototoxic effects in cell culture and experimental animal tumors, apparently without the involvement of singlet oxygen. The Zn analog and the metal-free iminium salt both exhibited fluorescence, and were somewhat more efficacious that CDS1, perhaps because both type I and type II processes were available. The nonfluorescent Ni analog was inactive as a photosensitizer. Fluorescent probes indicated that CDS1 and its zinc analog catalyzed photodamage at mitochondrial loci, the metal-free derivative at membrane loci. Because of its very low fluorescence yield, the metal-free iminium salt showed only faint intracellular fluorescence, but the Zn analog was unusual in this regard, with irradiation leading to a photoproduct with very intense intracellular fluorescence which was not readily photobleached.

As a new treatment model for endometriosis, photodynamic therapy was applied to endometriotic and endometrial cultures. It could be demonstrated that both endometrial components (epithelium and stroma) were present in the cultures, proved by immunocytology and electron microscopy. No major differences were seen between endometriotic and endometrial cells. The cultures were treated by HpD-sensitized PDT. Incubation time was 24 h and concentrations of 5 and 10 (mu) g/ml were used. Irradiation was performed by an argon-pumped dye laser at 630 nm with a power density of 80 mW/cm2. Evaluation both morphologically and by trypan blue exclusion test, was effected 24 h after irradiation. Toxicity in endometriotic and endometrial cultures was practically identical. Stroma cells were more sensitive to photodynamic treatment than epithelial cells. Complete stromal cell destruction was reached at 15 J/cm2, whereas epithelial cells showed 100 lethality at 40 J/cm2 (10(mu) g/ml HpD). These and subsequent results demonstrate that the sensitivity of stromal cells was about seven times higher than that of epithelial cells.

We compared the photodynamic efficiency of pulsed to cw laser irradiation in a cell culture experiment with a NIH ovarian cancer cell line (NIH-OVCAR3). Our photosensitizer was a cationic Zn(II)-phthalocyanine with an absorption maximum near 675 nm. The laser systems we use are an excimer laser pumped DCM-dye laser ((tau) equals 15 ns) and a cw Ti:sapphire laser. The photodynamic activity of the photosensitizer strongly depends on the pulse fluence and decreases with increasing fluence due to saturation of the sensitizer. In another experiment no changes in the light penetration depth into the tissue for pulsed irradiation could be detected up to a pulse intensity of 3 MW/cm².

The purpose of this study was to determine whether phospholipid liposomes that are bound by the human bladder carcinoma cells can improve the delivery of the photosensitizer and the effectiveness of photodynamic therapy.

This in vivo study was carried out to determine to what extent meso-tetra(hydroxyphenyl)chlorin (mTHPC), a chemically very stable second generation photosensitizer, can be 'photobleached'. Experiments were designed to determine if the degradation was dependent on the light dose and if clearance of drug could be achieved in an exposed skin area after a single exposure. Rabbits were injected with 0.3 mg/kg mTHPC and, utilizing injection to exposure intervals of 2 and 6 days, multiple skin areas were exposed to nonhyperthermic light at the absorption maximum (652 mn) for this drug. The exposed areas and control areas were excised immediately (or after an interval) and analyzed for drug content by chemical extraction and spectrofluorometry. Blood was taken prior to and again immediately after light exposure (or after 4 hours) to determine the drug levels circulating in the plasma. The drug content in skin could be reduced by half using light doses of 10 to 320 J/cm² independently of the fluence. Our findings indicate that 'bleaching' is dependent on the drug concentration in plasma. The concept of intentional 'bleaching' designed to reduce skin photosensitivity either prior to or after treatment is discussed with respect to these findings.

The treatment of solid human tumors of volume up to 50 cm3 will require significant gains in photosensitizing effect over that obtained with Photofrin and 630 nm light. Some techniques of interstitial radioisotope brachytherapy can be exploited for the uniform delivery of laser light to solid tumor volumes. Our dosimetry planning system (T-PIPET) was used to design 7- and 9- fiber illuminators for the treatment of R3327-AT rat prostate tumors by interstitial PDT. Relatively uniform light fields within encompassed tumor volumes could be achieved with needle spacings of 0.9 and 1.2 cm for light of 670 and 750 nm, respectively. Novel photosensitizers derived from pheophorbide and bacteriopheophorbide and activated by 673 and 753 nm light, respectively, were at least 1000X more potent than Photofrin in photokilling EMT-6 tumor cells in vitro. Tumor response in vivo resulted from perfusion shutdown and secondary ischemic cell death. Complete tumor response and some cures were demonstrated when R3327-AT rat prostate tumors of 3.5-4.5 cm3 volume were treated with 400 J of 673 nm light delivered 1 hour after the i.v. administration of 2mg/kg Ph4-OH. Current studies will optimize the vehicle for drug delivery, the time between drug and light administration and the light dose uniformity required throughout a treatment volume for maximizing tumor cures. A two-fold gain in tissue penetrance by longer wavelength light combined with at least a 100X gain in in vivo effectiveness by these novel photosensitizers makes feasible the treatment of solid human tumors by interstitial PDT with current laser systems.

A photodosimetry computer program, T-PIPET, was developed to rapidly compute maps of relative light intensity within tumors illuminated by implanted optical fibers. Light attenuation was measured along radial tracks from laterally-diffusing fibers implanted into both the R3327-AT and R3327-H Dunning rat prostate carcinomas and input to the computer program. The calculations assumed (1) uniform optical property of tissue through the tumor, (2) uniform and equal illuminance from the length of optical diffuser and between fibers and (3) precise needle implantation. Values of relative light intensity were computed, color-coded and imaged for a 5 X 5 cm tumor cross section (a 150 X 150 pixel array). Illuminators consisting of seven laterally-diffusing fibers implanted as six adjacent equilateral triangles were tested. The uniformity of light fields within encompassed tumor volumes was determined as a function of fiber spacing. Measures of light intensity along specific tracks within tumors agreed well (+/- 10%) with values of relative light intensity predicted by T-PIPET. The rapid falloff of light dose beyond the illuminator will assist in minimizing normal tissue damage outside the tumor volume. To cure slid tumors, a specific light dose must be delivered throughout the tumor volume and recurrence might be expected from tumor zones which are underdosed. A 9-fiber illuminator has been constructed to deliver light more uniformly to the tumor periphery. These illuminators have been tested with R3327-AT tumors illuminated with 673 nm light after i.v. administration of a pheophorbide-base photosensitizer.

This paper considers rapid calculations of light distributions in a tissue for photodynamic therapy. The delivery system considered is a single optical fiber, although other configurations are easily modeled. The influence of optical heterogeneities in the tissue is considered. The optical distributions surrounding an optical fiber within a tissue can be well approximated by a point source of light placed one 'reduced mean free path' (MFP) in front of the fiber tip. One MFP equals 1/((mu) _a + (mu) _s(1-g)). Then the simple diffusion theory expression for 3D diffusion from a point source predicts the light distribution in the tissue. The influence of optical heterogeneities in the tissue can also be well approximated. A method is described for representing regions of increased absorption (such as a region with increased vascularity) as virtual sources of 'negative radiant power' such that linear superposition of the true primary source and the virtual sources yields the net light distribution in the heterogeneous tissue. The method is rapid, flexible, and generally accurate to within about 15% error. The method yields light distributions in optically complex issues.

The Swiss nude mouse model is currently used as an animal model to investigate the efficacy of photodynamic therapy in ovarian cancer treatment. The disease requires treatment illumination of the entire abdominal cavity. The close proximity of the internal organs in the abdomen of a mouse and the vastly different optical properties of these organs present a challenge to light delivery and dosimetry. In this study the efficacy of different internal and transcutaneous light delivery geometries was investigated by scanning a transverse plane of the peritoneum with optical fiber fluence-rate detectors. The placement of the implanted optical fibers in the abdominal cavity was verified post mortem in selected animals by high resolution CT imaging. Preliminary experiments were performed to correlate the biological response with actual total fluence delivered to the abdominal cavity. Optical fiber fluence rate detectors were implanted in the peritoneum and abdominal cavity and the animal was treated with PDT. Cell survival of one hour post light treatment harvested cells from the peritoneum was used as a biological response quantifier.

A spectrofluorometry system has been developed for the collection of laser induced fluorescense spectra of tissue during endoscopy. In this system, a catheter with seven optical fibers was used to deliver the excitation light and collect the emitted fluorescence. The system enables one to switch from regular endoscopy into fluorescence measurement in 50 ms using a computerized shutter system. The fluorescence spectra can be recorded in 100 ms. This spectrofluorometry system has been used to obtain spectra from bronchial, larynx and nasopharyngeal tissues when employed with the appropriate endoscopes. The results demonstrate that laser induced fluorescence can be used to differentiate abnormal tissue from normal tissue. The illumination and fluorescence collection patterns of this system have been modeled using a Monte Carlo simulation. The Monte Carlo simulation data shows that the spectra recorded by our collection pattern is very close to the intrinsic spectra of tissue. The experimental results and the Monte Carlo simulation suggest that changes in fluorescence intensity are more robust for the detection of early cancers than the differences in spectral characteristics.

Recently, a new photosensitizer, 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-1 (HPPH) was developed for PDT which possesses more rapid clearance from skin and greater cytotoxicity per drug dose than Photofrin. The spectral characteristics of HPPH shows an absorption band at 665 nm (50,000 M-1cm(superscript -1 and peak emission at 680 nm. The aid of this study was to examine HPPH as a fluorescent diagnostic compound for the detection of transformed tissues using the in vivo fluorescence photodetector. The model of tissue transformation was the carcinogen (DMBA [9, 10-dimethyl-1, 2- benzanthracene])-induced premalignant and malignant lesions of the hamster buccal cheek pouch. The results demonstrated significant correlations between fluorescence levels and histological developments at all time points after injection. Time course studies of HPPH showed highest fluorescence readings at 48 hours after injection of 0.5 mg/kg HPPH (mild-moderate dysplasia, 0.35 +/- 0.17 volts; papillary disease with severe dysplasia, 0.58 +/- 0.33 volts; and squamous cell carcinoma, 1.04 $OM 0.32 volts). Therefore, it appears that HPPH may be a promising fluorophore for the detection of transformed tissues.

Lasers have traditionally been the preferred light source for activation of the photosensitizing agents used in photodynamic therapy (PDT). Their monochromaticity, high power, and the ability to efficiently couple that power into optical fibers have dictated their use. Dye lasers, metal vapor lasers, or ion gas lasers have been used in the past as the excitation source for PDT, largely because they provided the only available alternatives. These laser systems are very large and complex, and are very expensive to operate. The introduction of high power visible red laser diodes have provided a cost effective alternative to existing lasers for use in PDT. This paper will describe the features of a prototype preclinical red laser diode source for photodynamic therapy, and will present the results of an animal study conducted with this device. The study, using the photosensitizer SnET2, compared the efficacy of PDT performed with the diode laser system with the results obtained from a traditional dye laser system. Future plans for a clinical version of the system will also be discussed.

Laser-induced fluorescence spectroscopy was employed to measure fluorescence emission of normal and malignant tissue during endoscopy in patients with esophageal adenocarcinoma. A nitrogen/dye laser tuned at 410 nm was used for excitation source. The fluorescence lineshape of each spectrum was determined and sampled at 15 nm intervals from 430 nm to 716 nm. A calibration set from normal and malignant spectra were selected. Using stepwise discriminate analysis, significant wavelengths that separated normal and malignant spectra were selected. The intensities at these wavelengths were used to formulate a classification model using linear discriminate analysis. The model was used to classify additional tissue spectra from 26 malignant and 108 normal sites into either normal or malignant spectra with a sensitivity of 100 percent and specificity of 98 percent.

We have previously reported results of experiments in which EMT6/Ro spheroids were subjected to Photofrin-PDT consisting of a fixed incident fluence (60 J-cm^-2) delivered at 200, 50, and 25 mW-cm^-2. Surviving fractions from treated spheroids decreased as the incident fluence rate was lowered over this range. We have interpreted these data using a model wherein the cells compromising the surviving fractions are assumed to originate from within a therapy-induced anoxic volume resulting from Type-II photochemistry. In this paper, we demonstrate direct measurements of the phenomenon in individual photosensitized EMT6 spheroids. Steady-state measurements of ³O₂ gradients in and around metabolizing spheroids allow determination of the ³O₂ diffusion constant and the rate of metabolic ³O₂ consumption within a spheroid. Time-dependent measurements obtained at a single spatial location during laser irradiation are fit to numerical solutions of a pair of time-dependent diffusion with consumption equations. Fits allow a determination of the rate of PDT-induced ³O₂ consumption. Based on these fits, it is possible to calculate the spatial and temporal distributions of oxygen within a spheroid undergoing PDT.

Photofrin and 5-aminolevulinic acid are photosensitizers that show promise in the photodynamic treatment of cancer, port-wine stains, atherosclerosis and viral lesions. Photofrin is a mixture of porphyrins which, upon the absorption of light, become temporarily cytotoxic. One side-effect associated with the use of Photofrin is long-term cutaneous photosensitivity. It is possible that topical application of this photosensitizing dye will ameliorate such a side-effect. Another way to avoid the cutaneous photosensitivity in photodynamic therapy is to use 5- aminolevulinic acid, which is a porphyrin precursor that causes an increase in the synthesis and concentration of the photosensitizer protoporphyrin IX. 5-aminolevulinic acid is usually applied topically, and so minimizes cutaneous photosensitivity while maximizing the local protoporphyrin concentration. There are a host of disorders in oral mucosa that are potentially treatable by photodynamic therapy. However, since stratum corneum presents an impermeable barrier to many pharmaceuticals, it is not clear that topical application of the photosensitizer will result in a clinically relevant tissue concentration. We have therefore studied the permeation behavior of Photofrin and 5-aminolevulinic acid by applying them to the surface of ex vivo oral mucosa tissue positioned by a Franz diffusion cell. In order to increase the permeability of the photosensitizer across the stratum corneum, we studied the effects of four different drug carriers: phosphate buffered saline, dimethylsulfoxide, ethanol and Azone with isopropyl alcohol.

This paper reports further studies of Radiation Induced Fibrosarcoma (RIF-1) tumor cells which have been made resistant to Photodynamic Therapy by multiple treatment and regrowth in vitro using the hematoporphyrin derivative photosensitizer Photofrin. Previous work has shown both structural and functional changes in the mitochondria of the resistant (RIF-8A) cells. Colocalization of Photofrin and the mitochondrial localizer Rhodamine-123 was assessed by double-label confocal fluorescence microscopy (CFM). At 18h Photofrin incubation, there was strong correlation in discrete subcellular sites between Photofrin and Rhodamine fluorescence. However, in RIF-8A cells there were also discrete regions of Rhodamine localization which showed weak or no Photofrin fluorescence. This was not observed in RIF-1 cells. CFM measurements also showed that the Photofrin fluorescence after 18h incubation was reduced by increasing concentration of Rhodamine (30 min. incubation), and that this dependence was different for the two cell types. The RIF-8A cells were also shown to be cross-resistant to cisplatin and to have an associated reduced level of Pt-DNA adducts, suggesting the possibility of increased repair capacity. Cross-resistance was not observed, however, with a ruthenium phthalocyanine photosensitizer nor, as previously reported, with other chemotherapeutic agents such as Adriamcyin. Thus, there is a complex pattern of cross-resistance with these cells. Preliminary observations of the effects of a respiratory chain inhibitor (oligomycin) and an uncoupler of oxidative phosphorylation (FCCP) indicate differences between RIF-8A and RIF-1 which may be related to the condensed mitochondrial structure of the RIF-8A cells.

In the context of PDT, photobleaching refers to the permanent degradation of the photosensitizer due to chemical modification or to the transient reduction of absorption caused by ground state depletion during pulsed irradiation. This paper consists of a theoretical study of the influence of both of these phenomena on the depth of necrosis achieved by PDT. Calculations were based on the assumptions that (1) the tissue is a homogeneous semi-infinite medium, (2) irradiation is a cw or pulsed broad external beam, (3) necrosis occurs when the number of photons absorbed by the photosensitizer per unit tissue volume exceeds a threshold. A six-flux model was used to calculate the fluence distribution and to include the effects due to temporal and spatial variations in photosensitizer concentration caused by the bleaching process. Results are presented as response surfaces showing contours of equal necrosis depth in the space defined by total delivered light fluence and initial photosensitizer concentration. Photobleaching modifies the response surface by introducing a steep 'cliff' and by partially eliminating the effects of photosensitizer self-shielding. Differences between transient and permanent photobleaching are illustrated and discussed.

Laser blood flow measurements were performed on the tumor surface of 45 oral cancers growing on the shank of nude mice to detect changes in microcirculation (mc) during and up to 6 hours after PDT. PDT was performed in 27 animals (9.9 mg HPD/kg b.w. 24 hours pre radiation; 150 mW/cm², 200 J/cm²), 17 mice received only the photosensitizer without laser radiation. Simultaneously skin mc in the contralateral leg was measured to detect systemically caused changes in mc. Specimens of the tumor regions were taken two days later and analyzed histologically. With regard to our tumor model and the PDT conditions our results favor a mainly direct effect of PDT on the tumor cells rather than tumor necrosis caused by decreased mc. Although the method of laser doppler flowmetry (LDF) is very sensitive to all kinds of faults and blood flow can't be quantified in absolute units, changes of mc on the tumor surface are sufficient to be detected.

Light excitation of photosensitizing porphyrins leads to cytotoxic reactions. In addition, photobleaching and photoproduct formation occur indicating photosensitizer destruction. Photoproducts from hematoporphyrin (HP) fluoresce in aqueous solution at 642 nm, whereas photoproducts from protoporphyrin (PP) in hydrophobic environment emit around 670 nm and exhibit pronounced absorption at 665 nm. Photoproduct formation depends on singlet oxygen. The photoproducts exhibit faster fluorescence decay kinetics compared with nonirradiated porphyrins, as shown by time-grated spectroscopy and fluorescence decay measurements. Photoproduct fluorescence was observed during light exposure of cells and of tumor-bearing, nude mice, following administration of Hematoporphyrin Derivative (HpD), tetramethyl-HP, and PP. Photoconversion was also detected with naturally-occurring porphyrins (PP-producing bacteria) and ALA-simulated biosynthesis of PP in tumor tissue and in skin lesions of patients (psoriasis, mycosis fungoides). The efficiency of PDT with porphyrin photoproducts was found to be low in spite of the strong electronic transitions in the red spectral region.

Various microscopy techniques were employed to measure subcellular porphyrin distribution in ALA-incubated tumor cells and tumor spheroids. In addition, photodynamically-induced intracellular changes in morphology and fluorescence were detected. Studies were carried out by confocal laser scanning microscopy, polarization microscopy, video-intensified microscopy, video-enhanced contrast microscopy, time-resolved and time-gated spectroscopy, and spectrally-resolved fluorescence imaging using a tunable liquid crystal filter. It was found that ALA-incubation results in the biosynthesis of protoporphyrin IX (PP IX) in the mitochondria. Long ALA incubation times (>6 h) results in PP IX release to the cytoplasm. PP IX fluorescence has a maximum at 635 nm and a fluorescence decay time of 16 ns. No evidence for the existence of short-lived dimers, aggregates of PP IX, or other porphyrins was found. Cell irradiation resulted in cytotoxic reactions which, in turn, led to mitochondrial swelling followed by destruction of the cell membrane. In addition, PP IX photodestruction and the formation of short-lived chlorin-type photoproducts were observed.

In this study, the effect of PDT on tumor microvasculature was studied by light and electron microscope. Walker-256 tumors, implanted subcutaneously in rats, were exposed to 200J/cm² 630nm light, 24h after injection of PsD-007 at dose of 15mg/kg of body weight. Animals were killed and tumors were removed at time 0, 1/2, 1, 2, 6, 12 and 24h after treatment. The tumors with PDT displayed progressive injury to both microvasculature and tumor cells. Pathological changes such as swelling or vacuolization of mitochondria in endothelial cells could be seen immediately following PDT. At 6h post PDT, the injury to endothelial cells became more severe. Microthrombosis, diffuse hemorrhage and perivascular tumor cell mecrosis are also noted. By 12-24h after PDT, complete destruction of microvessels and obvious tumor cell mecrosis were observed. Untreated tumors or light only or drug only didn't show these changes. The results indicated that disruption of tumor microvasculature may be an important mechanism of action of PDT.

Highly phosphorescent photosensitizer Pd-tetra (o-methoxy-p-sulfo) phenyl porphyrin (Pd-MSPP) was used to follow the primary events of photodynamic action - quenching of triplet states by free oxygen in different systems: water solutions of proteins, cells and tissues in vivo and in vitro. The photosensitizer forms complexes with proteins in solutions and biosystems showing remarkable hypsochromic shifts of band and an increase of the quantum yield and lifetime of phosphorescence at the binding to proteins. In absence of oxygen the lifetime of phosphorescence is almost single exponential, and depends on the energy of lowest triplet state of the sensitizer. The photochemical quenching of the triplets by cell components is negligible. In presence of free oxygen the quenching of the sensitizer triplets takes place. The emission spectrum of singlet oxygen with maximum 1271 nm was recorded in water protein solutions and quantum yield of sensitized luminescence was measured. In the systems studied, oxygen consumption was detected and oxygen concentration was estimated in the course of photodynamics by an increase in photosensitizer phosphorescence lifetime, using laser flash photolysis technique. At least two exponential kinetics of the phosphorescence decay shows that the distribution of the free oxygen is not uniform in tissues.

Despite aggressive surgical and radiotherapy, recurrence rates for patients with recurrent head and neck carcinomas remain high. Photodynamic therapy has been successfully used to treat patients with early carcinomas. This is due to the ability of the activating laser light to penetrate up to one centimeter into tissue resulting in destruction of microscopic tumor with preservation of normal tissues. Employing this principle, PDT was used as an adjuvant intraoperative therapy following resection of tumor in four patients with recurrent infiltrating carcinomas of the head and neck. All patients tolerated the treatment without complications and remain free of disease six months post-treatment. Adjuvant intraoperative PDT may improve cure rates of recurrent head and neck malignancies by providing for larger tumor-free margins of resection while preserving normal structures.