Long-term effects of photodynamic therapy on fluorescence spectroscopy in the human esophagus.

Clinical interest in laser-induced fluorescence (LIF) spectroscopy and photodynamic therapy (PDT) is growing rapidly and may ultimately lead to close parallel use of these techniques. However, variations in LIF due to photosensitizer retention as well as tissue damage and healing processes may interfere with autofluorescence-based diagnostic methods. We have investigated the compatibility of these two techniques by quantifying PDT-induced changes in LIF in the human esophagus. Fluorescence spectra were collected endoscopically at excitation wavelengths (lambda ex) of 337, 400 and 410 nm in 32 patients. Measurements were performed immediately before and after PDT treatment with porfimer sodium and during follow-up procedures. In the months following PDT regions of reepithelialized squamous showed reduced autofluorescence in comparison with untreated squamous regions (P = 0.0007). Photosensitizer fluorescence was undetectable with lambda ex = 337 nm during follow-up procedures, whereas for lambda ex = 400 and 410 nm porfimer sodium fluorescence was noted for nearly a year after treatment. Therefore, residual photosensitizer fluorescence is likely to affect certain LIF-based diagnostic techniques during a period when patients are at high risk for tumor recurrence. Modification of LIF systems and/or the use of alternative photosensitizers may be required to optimize the detection of lesions in the post-PDT patient. Given the potential of LIF as a method for surveillance following cancer therapy, further investigation of the compatibility of specific LIF approaches with cancer pharmaceuticals may be warranted.     

The photosensitizing effect of the photoproduct of protoporphyrin IX

The photodynamic effect of a photoproduct of protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (ALA) was investigated in WiDr cells, a human adenocarcinoma cell line. The fluorescence excitation and emission spectra of PpIX and the photoproduct were measured. After 1, 3 or 5 min exposure of the ALA-incubated cells to 140 mW/cm² light at 635 nm, the photoproduct — the chlorin photoprotoporphyrin (Ppp), had an emission band around 670 nm. The Ppp excitation peak at 670 nm is well separated from the PpIX peak at 635 nm. The outcome of photodynamic therapy (PDT) was determined by measuring intracellular fluorescence intensity of propidium iodide (PI) 2 h following PDT and methylene blue (MB) staining 24 h following PDT. A significant increase in the fluorescence intensity of PI was noted when the ALA-loaded cells were exposed to 670 nm light after exposure to 635 nm, indicating enhanced cell membrane inactivation induced by the photodynamic action of the photoproduct. However, the fraction of the cells that survived following the same treatment as measured by MB staining was not significantly affected based on an analysis of variance. The fluorescence of PpIX decayed significantly during 635 nm light exposure. Exposure to light at 670 nm does not lead to any photodegradation of PpIX. The fluorescence of Ppp was bleached during 670 nm light exposure. Exposure of Ppp at 670 nm gives no PpIX back. Thus, the phototransformation of PpIX to Ppp is probably not a reversible process.     

Kinetic fluorescence studies of 5-aminolaevulinic acid-induced protoporphyrin IX accumulation in basal cell carcinomas.

Laser-induced fluorescence (LIF) investigations have been performed in connection with photodynamic therapy (PDT) of basal cell carcinomas and adjacent normal skin following topical application of 5-aminolaevulinic acid (ALA) in order to study the kinetics of the protoporphyrin IX (PpIX) build-up. Five superficial and 10 nodular lesions in 15 patients are included in the study. Fluorescence measurements are performed prior to the application of ALA, 2, 4 and 6 h post ALA application, immediately post PDT (60 J cm-2 at 635 nm), and 2 h after the treatment. Hence, the build-up, photobleaching and re-accumulation of PpIX can be followed. Superficial lesions show a maximum PpIX fluorescence 6 h post ALA application, whereas the intensity is already the highest 2-4 h after the application in nodular lesions. Immediately post PDT, the fluorescence contribution at 670 nm from the photoproducts is about 2% of the pre-PDT PpIX fluorescence at 635 nm. Two hours after the treatment, a uniform distribution of PpIX is found in the lesion and surrounding normal tissue. During the whole procedure, the autofluorescence of the lesions and the normal skin does not vary significantly from the values recorded before the application of ALA.     

Primary photodamage sites and mitochondrial events after Foscan photosensitization of MCF-7 human breast cancer cells

To determine the initial photodamage sites of Foscan-mediated photodynamic treatment, we evaluated the enzymatic activities in selected organelles immediately after light exposure of MCF-7 cells. The measurements indicated that the enzymes located in the Golgi apparatus (uridine 5'-diphosphate galactosyl transferase) and in the endoplasmic reticulum (ER) (nicotinamide adenine dinucleotide [reduced] [NADH] cytochrome c [cyt c] reductase) are inactivated by the treatment, whereas mitochondrial marker enzymes (cyt c oxidase and dehydrogenases) were unaffected. This indicates that the ER and the Golgi apparatus are the primary intracellular sites damaged by Foscan-mediated PDT in MCF-7 cells. We further investigated whether the specific mitochondria events could be associated with Foscan photoinduced cell death. The dose response profiles of mitochondrial depolarization and cytochrome c release immediately after Foscan-based PDT were very different from that of overall cell death. By 24 h post-PDT the fluence dependency was strikingly similar for both mitochondrial alterations and cell death. Therefore, although mitochondria are not directly affected by the treatment, they can be strongly implicated in Foscan-mediated MCF-7 cell death by late and indirect mechanism.     

In vivo Spectroscopic Evaluation of the Intraperitoneal Cavity in Canines

As part of a preclinical trial for the treatment of peritoneal carcinomatosis (PC) with photodynamic therapy (PDT), we have assessed changes in optical properties, tissue oxygenation and drug concentration as a result of benzoporphyrin derivative (BPD)-mediated PDT using diffuse reflectance and fluorescence measurements. PDT can effectively treat superficial disease spread, but treatment efficacy is influenced by physical properties of the treated tissue which can change over the treatment time. In this study, healthy canines were given BPD and irradiated with 690 nm light during a partial bowel resection, and spectroscopic and fluorescence measurements were made using an in-house built spectroscopic probe. Hemoglobin concentration, oxygenation and optical properties were determined to be highly heterogeneous between canines and at different anatomical locations within the same subject, so further development of PDT dosimetry systems will need to address this patient and location-specific dose optimization. Compared to other photosensitizers, we found no apparent BPD photobleaching after PDT.     

Glucose-dependent changes in NAD(P)H-related fluorescence lifetime of adipocytes and fibroblasts in vitro: potential for non-invasive glucose sensing in diabetes mellitus

The aim of this study was to test the hypothesis that glucose can be monitored non-invasively by measuring NAD(P)H-related fluorescence lifetime of cells in an in vitro cell culture model. Autofluorescence decay functions were measured in 3T3-L1 adipocytes by time-correlated single-photon counting (excitation 370nm, emission 420-480nm). Free NADH had a two-exponential decay but cell autofluorescence fitted best to a three-exponential decay. Addition of 30mM glucose caused a 29% increase in autofluorescence intensity, a significantly shortened mean lifetime (from 7.23 to 6.73ns), and an increase in the relative amplitude and fractional intensity of the short-lifetime component at the expense of the two longer-lifetime components. Similar effects were seen with rotenone, an agent that maximizes mitochondrial NADH. 3T3-L1 fibroblasts stained with the fluorescent mitochondrial marker, rhodamine 123 showed a 16% quenching of fluorescence intensity when exposed to 30mM glucose, and an increase in the relative amplitude and fractional intensity of the short lifetime at the expense of the longer lifetime component. We conclude that, though the effect size is relatively small, glucose can be measured non-invasively in cells by monitoring changes in the lifetimes of cell autofluorescence or of a dye marker of mitochondrial metabolism. Further investigation and development of fluorescence intensity and lifetime sensing is therefore indicated for possible non-invasive metabolic monitoring in human diabetes.     

Gamma-ray dosimetry by spectrofluorimetry of phenylacetic acid solution

Aqueous solutions of phenylacetic acid have been evaluated for possible use in γ-ray dosimetry. When aerated aqueous phenylacetic acid solutions are irradiated, photoflourescent species are formed and identified as hydroxyphenylacetic acid. When excited by ultraviolet light at 280 nm, the radiation-induced product shows an emission spectrum with a maximum at 307 nm. The intensity of the emission peak at 307 nm (as well as the area under the peak from 290 to 350 nm) is a linear function of absorbed dose from 0.5 to 25 Gy. This aqueous dosimeter is about ten times more sensitive than that of the conventional ferrous sulfate solution (Fricke) dosimeter. The differences in response at dose rates in the range 0.0055–67 Gy/min are negligible. Conversely, at higher dose rates (170 Gy/min), although the response is linear with dose up to 135 Gy and with proper calibration can be used up to 350 Gy, the photofluorescence signal is somewhat greater than in the lower dose rate range. The estimated random uncertainty limits (1σ) of readings of absorbed dose by the dosimeter are approximately ±2% at a dose of 10 Gy. The fluorescence signal is very much affected by the hydrogen ion concentration of the solution and the intensity of the signal is greatest in the pH range 5–9.5. The radiation chemical yield of the fluorescing species is little influenced by moderate changes in the concentration of phenylacetic acid or by deaeration of the solution. The signal is stable up to at least four weeks, if the solution after irradiation is stored at low temperature (ca. 5°C). However, when stored at room temperature, and in room light, the signal is stable only up to about four days.     

Oral aminolevulinic acid induces protoporphyrin IX fluorescence in psoriatic plaques and peripheral blood cells

Photodynamic therapy (PDT) with topical aminolevulinic acid (ALA) has been shown in previous studies to improve psoriasis. However, topical ALA-PDT may not be practical for the treatment of extensive disease. In order to overcome this limitation we have explored the potential use of oral ALA administration in psoriatic patients. Twelve patients with plaque psoriasis received a single oral ALA dose of 10, 20 or 30 mg/kg followed by measurement of protoporphyrin IX (PpIX) fluorescence in the skin and circulating blood cells. Skin PpIX levels were determined over time after ALA administration by the quantification of the 635 nm PpIX emission peak with in vivo fluorescence spectroscopy under 442 nm laser excitation. Administration of ALA at 20 and 30 mg/kg induced preferential accumulation of PpIX in psoriatic as opposed to adjacent normal skin. Peak fluorescence intensity in psoriatic and normal skin occurred between 3 and 5 h after the administration of 20 and 30 mg/kg, respectively. Ratios of up to 10 for PpIX fluorescence between psoriatic versus normal skin were obtained at the 30 mg/kg dose of ALA. Visible PpIX fluorescence was also observed on normal facial skin, and nonspecific skin photosensitivity occurred only in patients who received the 20 or 30 mg/kg doses. PpIX fluorescence intensity was measured in circulating blood cells by flow cytometry. PpIX fluorescence was higher in monocytes and neutrophils as compared to CD4+ and CD8+ T lymphocytes. PpIX levels in these cells were higher in patients who received higher ALA doses and peaked between 4 and 8 h after administration of ALA. There was only a modest increase in PpIX levels in circulating CD4+ and CD8+ T lymphocytes. In conclusion oral administration of ALA induced preferential accumulation of PpIX in psoriatic plaques as compared to adjacent normal skin suggesting that PDT with oral ALA should be further explored for the treatment of psoriasis.     

Biodistribution and Pharmacokinetic Studies of a Porphyrin Dimer Photosensitizer (Oxdime) by Fluorescence Imaging and Spectroscopy in Mice Bearing Xenograft Tumors

Herein, we present a study of the pharmacokinetics and biodistribution of a butadiyne‐linked conjugated porphyrin dimer (Oxdime) designed to have high near‐infrared (NIR) 2‐photon absorption cross‐section for photodynamic therapy (PDT). Changes in biodistribution over time were monitored in mice carrying B16‐F10 melanoma xenografts, following intravenous injection. Using fluorescence imaging of live animals and analyzing isolated organs ex vivo at different time points between 30 min and 24 h after injection, accumulation of Oxdime was measured in several organs (heart, kidney and liver) and in tumor. The concentration in the plasma was about 5–10 times higher than in other tissues. The fluorescence signal peaked at 3–12 h after injection in most tissues, including the tumor and the plasma. The change in the fluorescence emission spectrum of the sensitizer over time was also monitored and a shift in the maximum from 800 to 740 nm was observed over 24 h, showing that the Oxdime is metabolized. Significant quantities accumulated in the tumor, indicating that this PDT sensitizer may be promising for cancer treatment.     

Determination of the distribution of light, optical properties, drug concentration, and tissue oxygenation in-vivo in human prostate during motexafin lutetium-mediated photodynamic therapy

It is desirable to quantify the distribution of the light fluence rate, the optical properties, the drug concentration, and the tissue oxygenation for photodynamic therapy (PDT) of prostate cancer. We have developed an integrated system to determine these quantities before and after PDT treatment using motorized probes. The optical properties (absorption (micro(a)), transport scattering (micro(s'), and effective attenuation (micro(eff)) coefficients) of cancerous human prostate were measured in-vivo using interstitial isotropic detectors. Measurements were made at 732 nm before and after motexafin lutetium (MLu) mediated PDT at different locations along each catheter. The light fluence rate distribution was also measured along the catheters during PDT. Diffuse absorption spectroscopy measurement using a white light source allows extrapolation of the distribution of oxygen saturation StO2, total blood volume ([Hb]t), and MLu concentration. The distribution of drug concentration was also studied using fluorescence from a single optical fiber, and was found to be in good agreement with the values determined by absorption spectroscopy. This study shows significant inter- and intra-prostatic variations in the tissue optical properties and MLu drug distribution, suggesting that a real-time dosimetry measurement and feedback system for monitoring these values during treatment should be considered in future PDT studies.     

PHOTOPHYSICAL PROPERTIES OF PORPHYRIN-CHLORIN SYSTEMS IN THE PRESENCE OF SURFACTANTS

Abstract The therapeutic efficacy of PDT is related to the capability of the photosensitizer to absorb light at a wavelength that can penetrate into tissues. We have synthesized two systems, a haematoporphyrin-chlorin (HPC) and a dihaematoporphyrin ether or ester (DHE) with the terminal ring converted to a chlorin (DHEC). The presence of the chlorin moiety provides an extra band at ˜ 660 nm with a relative amplitude from 5 to 10 times larger than that of the porphyrin at 630 nm. Since both HPC and DHEC strongly aggregate in buffer, we have studied their photophysical properties in the presence of cationic surfactants at different concentrations below and above the critical micelle concentrations. Absorption spectra were measured together with emission spectra and fluorescence decays at different observation wavelengths under excitation at 364 nm. The results were compared with those obtained for DHE in the same environmental conditions. As for DHE, the presence of micelles disaggregate both compounds, resulting in a large increase in the relative emission intensity at ˜ 670 nm due to the presence of the chlorin moiety. The fluorescence decays could be fitted by two or three exponential components indicating the presence of more than one molecular species and/ or conformations. On the basis of our measurements the chlorin molecule does not seem to modify appreciably the photophysical properties of the porphyrin molecules but does superimpose its absorption and emission spectrum onto that of the porphyrin. This result may be of relevance in the possible use of these compounds in PDT.     

Fluorescence imaging during photodynamic therapy of experimental tumors in mice sensitized with disulfonated aluminum phthalocyanine

A fluorescence imaging system was used to monitor the emission of disulfonated aluminum phthalocyanine (AlS2Pc) during the photodynamic therapy (PDT) of murine tumors. Cells of the MS-2 fibrosarcoma were injected in mice in two compartments in order to cause the development of tumors in different host tissues. Two drug doses and two uptake times were considered. Moreover, the fluorescence of the AlS2Pc was excited using two wavelengths on the opposite sides of the absorption peak to detect a possible change in the absorption spectrum of the sensitizer induced by the PDT. In the tumors, the treatment induces a variation of the fluorescence intensity: in some mice a mild photobleaching takes place, in others a fluorescence enhancement occurs. Which effect predominates depends on the experimental conditions, even though a large spread of data was found amongst mice of the same group. In all mice, independently of the drug dose, uptake time or tumor compartment, a marked increase in the fluorescence signal takes place at the borders of the irradiated area. To quantify this effect we evaluated the ratio between the fluorescence intensities in the peritumoral area and in the tumor itself. This ratio increases monotonically during the PDT, showing a different behavior with the two excitation wavelengths. This indicates that the AlS2Pc absorption spectrum shifts toward shorter wavelengths as a result of the irradiation.     

Photophysical properties of Hypericum perforatum L. extracts--novel photosensitizers for PDT

We report the preparation of the methanolic extract (ME), and polar methanolic fraction (PMF) from the plant Hypericum perforatum L. The extracts contain various photosensitizing constituents such as naphthodianthrone derivatives (in 1.37% w/w), and chlorophylls (in 0.08% w/w). Upon light emission these constituents can be activated, providing photodynamic properties to the extracts, and making them a potent, new class, natural photosensitizers for use in photodynamic therapy (PDT), and photodynamic diagnosis (PDD). The absorbance spectra of the extracts are similar to the spectrum of hypericin, the main naphthodianthrone identified within, with two major bands at 548 and 590 nm. The fluorescence spectra in ethanol exhibit two main bands around 595 and 640 nm, in accordance with the spectrum of pure hypericin. The fluorescence intensity of PMF at 595 nm is only eight times less than the intensity of pure hypericin at the same wavelength, even though its hypericin concentration is only 0.57% w/w. The dependence of the PMF fluorescence signal on the pH of the medium, alone and in comparison with the signal of hypericin, has been investigated. PMF signal fades steadily, and smoothly both in acidic, and basic environment.     

Porphyrin bleaching and PDT-induced spectral changes are irradiance dependent in ALA-sensitized normal rat skin in vivo

Photobleaching kinetics of aminolevulinic acid-induced protoporphyrin IX (PpIX) were measured in the normal skin of rats in vivo using a technique in which fluorescence spectra were corrected for the effects of tissue optical properties in the emission spectral window through division by reflectance spectra acquired in the same geometry and wavelength interval and for changes in excitation wavelength optical properties using diffuse reflectance measured at the excitation wavelength. Loss of PpIX fluorescence was monitored during photodynamic therapy (PDT) performed using 514 nm irradiation. Bleaching in response to irradiances of 1, 5 and 100 mW cm-2 was evaluated. The results demonstrate an irradiance dependence to the rate of photobleaching vs irradiation fluence, with the lowest irradiance leading to the most efficient loss of fluorescence. The kinetics for the accumulation of the primary fluorescent photoproduct of PpIX also exhibit an irradiance dependence, with greater peak accumulation at higher irradiance. These findings are consistent with a predominantly oxygen-dependent photobleaching reaction mechanism in vivo, and they provide spectroscopic evidence that PDT delivered at low irradiance deposits greater photodynamic dose for a given irradiation fluence. We also observed an irradiance dependence to the appearance of a fluorescence emission peak near 620 nm, consistent with accumulation of uroporphyrin/coproporphyrin in response to mitochondrial damage.     

THE VALIDATION OF A NEW VASCULAR DAMAGE ASSAY FOR PHOTODYNAMIC THERAPY AGENTS

Abstract— The therapeutic effect of photodynamic therapy (PDT: photodynamic sensitizer + light) is partly due to vascular damage. This report describes a new vascular photodamage assay for PDT agents and a validation of the assay. The method described here quantitates changes in tissue blood perfusion based on the relative amount of injected fluorescein dye in treated and untreated tissues. A specially designed fluorometer uses chopped monochromatic light from an argon laser as a source for exciting fluorescein fluorescence. The fluorescent light emitted from the tissue is collected by a six element fiberoptic array, filtered and delivered to a photodiode detector coupled to a phase-locked amplifier for conversion to a voltage signal for recording. This arrangement permits a rather simple, inexpensive construction and allows for the simultaneous use of the argon laser by other investigators.
The routine assay for characterizing a specific photosensitizer at a standard dose consists of the sequential allocation of eight mice to a set of different light doses designed to span the dose-response range of fluorescein fluorescence exclusion (measured 8–10 min after fluorescein injection). The assay validation experiment used an anionic photosensitizer, 2-[l-hexyloxyethyl]-2-devinyl pyropheophorbide-a at a dose of 0.4 μmol/kg. The parameter estimates (n = 34 mice) from fitting the standard Hill dose-response model to the data were: median fluorescence exclusion light dose FE₅₀= 275 ± 8.3 J/cm² and Hill sigmoidicity parameter m =−3.66 ± 0.28. Subsets of the full data set randomly selected to simulate a standard eight mice experiment yielded similar parameter estimates. The new assay provides reliable estimates of PDT vascular damage with a frugal sequential experimental design.     

DNA cleavage by UVA irradiation of NADH with dioxygen via radical chain processes

Efficient DNA cleaving-activity is observed by UVA irradiation of an O(2)-saturated aqueous solution of NADH (beta-nicotinamide adenine dinucleotide, reduced form). No DNA cleavage has been observed without NADH under otherwise the same experimental conditions. In the presence of NADH, energy transfer from the triplet excited state of NADH ((3)NADH*) to O(2) occurs to produce singlet oxygen ((1)O(2)) that is detected by the phosphorescence emission at 1270 nm. No quenching of (1)O(2) by NADH was observed as indicated by no change in the intensity of phosphorescence emission of (1)O(2) at 1270 nm in the presence of various concentrations of NADH. In addition to the energy transfer, photoinduced electron transfer from (3)NADH* to O(2) occurs to produce NADH(*+) and O(2)(*-), both of which was observed by ESR. The quantum yield of the photochemical oxidation of NADH with O(2) increases linearly with increasing concentration of NADH but decreases with increasing the light intensity absorbed by NADH. Such unusual dependence of the quantum yield on concentration of NADH and the light intensity absorbed by NADH indicates that the photochemical oxidation of NADH with O(2) proceeds via radical chain processes. The O(2)(*-) produced in the photoinduced electron transfer is in the protonation equilibrium with HO(2)(*), which acts as a chain carrier for the radical chain oxidation of NADH with O(2) to produce NAD(+) and H(2)O(2), leading to the DNA cleavage.     

Protoporphyrin IX fluorescence photobleaching during ALA-mediated photodynamic therapy of UVB-induced tumors in hairless mouse skin

Fluorescence photobleaching of protoporphyrin IX (PpIX) during superficial photodynamic therapy (PDT), using 514 nm excitation, was studied in UVB-induced tumor tissue in the SKH-HR1 hairless mouse. The effects of different irradiance and light fractionation regimes upon the kinetics of photobleaching and the PDT-induced damage were examined. Results show that the rate of PpIX photobleaching (i.e., fluorescence intensity vs fluence) and the PDT damage both increase with decreasing irradiance. We have also detected the formation of fluorescent PpIX photoproducts in the tumor during PDT, although the quantity recorded is not significantly greater than generated in normal mouse skin, using the same light regime. The subsequent photobleaching of the photoproducts also occurs at a rate (vs fluence) that increases with decreasing irradiance. In the case of light fractionation, the rate of photobleaching increases upon renewed exposure after the dark period, and there is a corresponding increase in PDT damage although this increase is smaller than that observed with decreasing irradiance. The effect of fractionation is greater in UVB-induced tumor tissue than in normal tissue and the damage is enhanced when fractionation occurs at earlier time points. We observed a variation in the distribution of PDT damage over the irradiated area of the tumor: at high irradiance a ring of damage was observed around the periphery. The distribution of PDT damage became more homogeneous with both lower irradiance and the use of light fractionation. The therapeutic dose delivered during PDT, calculated from an analysis of the fluorescence photobleaching rate, shows a strong correlation with the damage induced in normal skin, with and without fractionation. The same correlation could be made with the data obtained from UVB-induced tumor tissue using a single light exposure. However, there was no such correlation when fractionation schemes were employed upon the tumor tissue.     

Pharmacokinetics and pharmacodynamics of tetra(m-hydroxyphenyl)chlorin in the hamster cheek pouch tumor model: comparison with clinical measurements

The pharmacokinetics (PK) of the photosensitizer tetra(m-hydroxyphenyl)chlorin (mTHPC) was measured by optical fiber-based light-induced fluorescence spectroscopy (LIFS) in the normal and tumoral cheek pouch mucosa of 29 Golden Syrian hamsters with chemically induced squamous cell carcinoma. Similar measurements were carried out on the normal oral cavity mucosa of five patients up to 30 days after injection. The drug doses were between 0.15 and 0.3 mg per kg of body weight (mg/kg), and the mTHPC fluorescence in the tissue was excited at 420 nm. The PK in both human and hamster exhibited similar behavior although the PK in the hamster mucosa was slightly delayed in comparison with that of its human counterpart. The mTHPC fluorescence signal of the hamster mucosa was smaller than that of the human mucosa by a factor of about 3 for the same injected drug dose. A linear correlation was found between the fluorescence signal and the mTHPC dose in the range from 0.075 to 0.5 mg/kg at times between 8 and 96 h after injection. No significant selectivity in mTHPC fluorescence between the tumoral and normal mucosa of the hamsters was found at any of the applied conditions. The sensitivity of the normal and tumoral hamster cheek pouch mucosa to mTHPC photodynamic therapy as a function of the light dose was determined by light irradiation at 650 nm and 150 mW/cm2, 4 days after the injection of a drug dose of 0.15 mg/kg. These results were compared with irradiations of the normal oral and normal and tumoral bronchial mucosa of 37 patients under the same conditions. The reaction to PDT of both types of human mucosae was considerably stronger than that of the hamster cheek pouch mucosa. The sensitivity to PDT became comparable between hamster and human mucosa when the drug dose for the hamster was increased to 0.5 mg/kg. A significant therapeutic selectivity between the normal and neoplastic hamster cheek pouch was observed. Less selectivity was found following irradiations of normal mucosa and early carcinomas in the human bronchi. The pharmacodynamic behavior of mTHPC was determined by test irradiations of the normal mucosa of hamsters and patients between 6 h and 8 days after injection of 0.5 and 0.15 mg/kg in the hamsters and the patients, respectively. The normal hamster cheek pouch showed a maximum response to irradiation 6 h after injection and then decreased continuously to no observable reaction at 8 days after injection. The reaction of the normal human oral mucosa, however, showed an increasing sensitivity to the applied light between 6 h and 4 days after mTHPC injection and then decreased again at 8 days. The hamster model with the chemically induced early squamous cell cancer in the cheek pouch thus showed some similarity to the early squamous cell cancer of the human oral mucosa considering the PK. However, a quantitative difference in fluorescence signal for identical mTHPC doses as well as a significant difference in pharmacodynamic behavior were also observed. The suitability of this animal model for the optimization of PDT parameters in the clinic is therefore limited. Hence great care must be taken in screening new dyes for PDT of early squamous cell cancer of the upper aerodigestive tract based upon observables in the hamster cheek pouch model.     

Analysis of the heterogeneity of pO2 dynamics during photodynamic therapy with verteporfin.

Photodynamic therapy (PDT) with verteporfin provides a reliable way to destroy malignant tissues. Changes in the blood flow and oxygen partial pressure (pO2) during verteporfin-PDT were studied here in the tumor tissue of the rat mammary R3230Ac carcinoma model. Oxygen microelectrodes (6-12 microns tip diameter) were used to measure the transients locally within tumors during intravenous injection of 1.0 mg/kg verteporfin followed by irradiation 15 min later with 690 nm light at 200 mW/cm2, for a cumulative dose of 144 J/cm2. The observed changes in pO2 were heterogeneous and there was a difference in the response of low-pO2 regions relative to higher-pO2 regions. The change in pO2 in hypoxic tissue regions (pO2 < 8 mmHg) had acute pO2 loss after treatment, whereas the response in regions of higher pO2 (> 8 mm Hg) was more heterogeneous with some areas maintaining their pO2 value after treatment was completed. Blood flow measurements taken on a subset of the animals indicated a significant loss in flow during the initial light delivery that remained low after treatment, indicating some vascular stasis. The results suggest that hypoxic or poorly perfused vessels may be more susceptible to acute stasis than normoxic vessels in this treatment protocol.     

Photobleaching-based dosimetry predicts deposited dose in ALA-PpIX PDT of rodent esophagus

An improved method to estimate dose to esophageal tissue was investigated in the setting of photodynamic therapy with aminolevulinic acid-induced protoporphyrin IX (PpIX) treatment. A model of treatment-induced edema in the esophagus mucosa proved to be a well controlled and useful way to test the dosimetry model, and the light from the treatment laser together with the PpIX fluorescence intensity could be quantified reliably in real time. Dosimetry calculations based upon the detected fluorescence and bleaching kinetics were used to calculate the "effective" dose to the tissue, and a correlation was shown to exist between this metric and the edema induced in the esophagus. The difference between animals with no detectable treatment effect and those with significant edema was predictable based upon the dose calculation. The underlying assumption in the interpretation of the data is that rapid photobleaching of PpIX occurs when there is ample oxygen supply, and this bleaching is not present when oxygen is limited. This leads to the prediction that integration of the light and drug dose, in intervals where appreciable photobleaching occurs, should provide a prediction of the relative dose of singlet oxygen produced. This detection system and rodent model can be used for prospective dosimetry studies that focus on optimization of esophageal PDT.