Toward understanding the high PDT efficacy of chlorin e6-polyvinylpyrrolidone formulations: photophysical and molecular aspects of photosensitizer-polymer interaction in vitro

It is recognized that chlorin e6-polyvinylpyrrolidone (Ce6-PVP) formulations are characterized by a high efficacy in photodynamic therapy of malignant tumors. Currently, a commercially available formulation of this type is Photolon((R)) (Fotolon((R))) with Ce6:PVP=1:1 (w/w) and the weight-average molecular weight of PVP is 1.2x10(4). To gain a better understanding of the role played by PVP in Ce6-PVP formulations, we carry out experiments on IR and UV-VIS absorption, steady-state and time-resolved fluorescence, time-resolved triplet-triplet absorption, octanol-water partitioning, and solubility of chlorin e6 in buffer solutions at pH 6.3, 7.4, and 8.5 in presence of PVP with Ce6:PVP ratios ranging from 1:0 to 1:1000 (w/w) for PVP samples with weight-average molecular weights of 8x10(3), 1.2x10(4), and 4.2x10(4). We show that Ce6 interacts with PVP by forming molecular complexes via hydrophobic interactions and determine the Ce6-PVP binding constant, as well as the mean number of PVP monomers per binding site. We find that complexation of Ce6 with PVP prevents Ce6 aggregation in aqueous media and leads to an enhancement of Ce6 fluorescence quantum yield, while keeping the quantum yield of the intersystem crossing essentially unchanged. Possible scenarios of how the presence of PVP can favorably affect the PDT efficacy of chlorin e6 in Ce6-PVP formulations are discussed.     

Tissue distribution and in vivo photosensitizing activity of 13,15-[N-(3-hydroxypropyl)]cycloimide chlorin p6 and 13,15-(N-methoxy)cycloimide chlorin p6 methyl ester

Photosensitizers 13,15-[N-(3-hydroxypropyl)]cycloimide chlorin p6 (HPC) and 13,15-(N-methoxy)cycloimide chlorin p6 methyl ester (MMC) absorb at 711 nm and possess high photoinduced cytotoxicity in vitro. Here we report, that photodynamic therapy with HPC and MMC provide considerable antitumor effect in mice bearing subcutaneous P338 lymphoma. The highest antitumor effect was achieved at a dose of 4 micromol/kg when 1.5 h delay between dye injection and light irradiation (drug-light interval) was used. According to the confocal spectral imaging studies of tissue sections this drug-light interval corresponds to a maximum of tumor accumulation of MMC and HPC (tumor to skin accumulation ratio is 8-10). Short (15 min) drug-light interval can be used for efficient vasculature-targeted photodynamic therapy with HPC at a dose of 1 micromol/kg, whereas MMC is ineffective at the short drug-light interval. Relationships between the features of tissue distribution and efficacy of photodynamic therapy at different drug-light intervals are discussed for HPC and MMC.     

Photodynamic therapy for malignant mesothelioma: preclinical studies for optimization of treatment protocols

Effective photodynamic therapy (PDT) depends on the optimization of factors such as drug dose, drug-light interval, fluence rate and total light dose (or fluence). In addition sufficient oxygen has to be present for the photochemical reaction to occur. Oxygen deficits may arise during PDT if the photochemical reaction consumes oxygen more rapidly than it can be replenished, and this could limit the efficacy of PDT. In this study we investigated the influence of the drug-light interval, illumination-fluence rate and total fluence on PDT efficacy for the photosensitizer meta-tetrahydroxyphenylchlorin (mTHPC). The effect of increasing the oxygenation status of tumors during PDT was also investigated. PDT response was assessed from tumor-growth delay and from cures for human malignant mesothelioma xenografts grown in nude mice. Tumor-bearing mice were injected intravenously with 0.15 or 0.3 mg.kg-1 mTHPC, and after intervals of 24-120 h, the subcutaneous tumors were illuminated with laser light (652 nm) at fluence rates of 20, 100 or 200 mW.cm-2. Tumor response was strongly dependent on the drug-light interval. Illumination at 24 h after photosensitization was always significantly more effective than illumination at 72 or 120 h. For a drug-light interval of 24 h the tumor response increased with total fluence, but for longer drug-light intervals even high total fluences failed to produce a significant delay in tumor regrowth. No fluence-rate dependence of PDT response was demonstrated in these studies. Nicotinamide injection and carbogen breathing significantly increased tumor oxygenation and increased the tumor response for PDT schedules with illumination at 24 h after photosensitizer injection.     

In Vivo Fluence Rate Effect in Photodynamic Therapy of Early Cancers with Tetra(m-hydroxyphenyl)chlorin

Abstract— Several parameters affect clinical trials in photodynamic therapy and influence the therapeutic outcome. Beside drug dose, light dose, drug-light interval and other variables, the fluence rate is a parameter that can influence the therapeutic results. In this study we have evaluated the fluence rate effect with a second-generation photosensitizer, tetra( m -hydroxyphenyl)chlorin (mTHPC) using a 7,12-dimethylbenz(a)anthracene induced early squamous cell carcinoma of the Syrian hamster cheek pouch as a tumor model. Following injection of 0.5 mg/kg of mTHPC, irradiation tests were performed at two drug-light intervals, 4 and 8 days. Wavelength and light dose were adapted from those applied routinely in clinical trials. Irradiations at 652 nm were carried out with fluences ranging from 8 to 20 J/cm² delivered at fluence rates of 15 and 150 mW/cm². Similar tests were also performed at 514 nm with a fluence of 80 J/cm² delivered at fluence rates ranging from 25 to 125 mW/cm². At both wavelengths and drug-light intervals for a given fluence, the higher fluence rates resulted in less tissue damage in tumor and healthy mucosae. However, the lower fluence rates yielded slightly less therapeutic selectivity. This study confirms that the fluence rate is of major importance in clinical PDT.     

The influence of photodynamic therapy on the ultrastructure of the normal rat bladder.

Reduced bladder capacity is a major side effect for patients receiving photodynamic therapy (PDT) for bladder cancer. A rat bladder model has been developed to address both the vascular and tissue effects of the photodynamic treatment of the urinary bladder. Bladders were exteriorized and positioned in a plexiglass tissue bath. Effects on microvasculature were assessed during PDT of the bladder by recording luminal diameter changes in arterioles and venules. Animals receiving Photofrin II (10 mg kg-1) 30 min prior to PDT scored a statistically significant reduction in the diameter of the red blood cell column in the vessels, whereas administration of Photofrin II 48 h prior to PDT was ineffective. Morphological changes included significant endothelial and vascular myocyte damage in the 30 min PDT group alone. Among the other tissue components, the mucosal lining was minimally affected and the response of the muscularis was highly variable. Smooth muscle cell changes ranged from mild contraction to frank necrosis with many of the affected cells located near the altered vascular beds. These data suggest that the clinical symptoms of reduced bladder capacity can be accounted for by vascular damage and myocyte sensitivity. Further refinements in the Photofrin II and light doses used in therapy may reduce bladder complications and allow for better management of bladder cancer.     

SKIN PHOTOSENSITIVITY AND PHOTODESTRUCTION OF SEVERAL POTENTIAL PHOTODYNAMIC SENSITIZERS

The major side effect associated with porphyrins (Photofrin II) in clinical photodynamic therapy is skin photosensitivity. In order to avoid this deleterious reaction, patients must remain out of the sunlight for approximately 1 month. A possible procedure to reduce the amount of skin photosensitivity is to photodegrade (photobleach) the compound in the skin. In this study, we report a series of experiments describing the photodegradation rates of two photosensitizers currently receiving attention due to their potential for use in PDT (mono L-aspartyl chlorin e6 and chloroaluminum sulfonated phthalocyanine). These compounds are compared to Photofrin II (PfII). Experiments consisted of determining photodegradation rates and efficiencies of the sensitizers in (i) phosphate buffered saline (PBS), (ii) PBS with fetal calf serum (to enhance absorption and simulate cellular binding or deaggregation), (iii) Chinese Hamster Ovary cells, and (iv) Balb/c mice. We performed two standardized skin sensitivity assays using the Hartely albino guinea pig irradiated with a UV blue point lamp and Balb/c mice irradiated with the therapeutic wavelength of each sensitizer. In addition, we performed a cell clonogenicity assay comparing photodegraded and fresh PfII on CHO cells. The photodegraded PfII exhibited significant phototoxicity, although the fluorescence was bleached by more than 70%. The results show that PfII causes major skin photosensitization and that the other compounds produce no substantial skin sensitivity. Our studies suggest that photodegradation of PfII with 630 nm light has little influence on the phototoxicity of the compound. In addition, skin sensitivity was not alleviated with prior photobleaching with 405 nm light.     

Encapsulation of porphyrins and chlorins in biodegradable nanoparticles: the effect of dye lipophilicity on the extravasation and the photothrombic activity. A comparative study

In the present work, we performed a preclinical inter-comparison study using several photosensitizers with the goal of optimizing photodynamic therapy (PDT) for the treatment of choroidal neovascularization (CNV) associated with age-related macular degeneration. The tested molecules were the porphyrins meso-tetraphenylporphyrin (TPP) and meso-tetra-(4-carboxyphenyl)-porphyrin (TCPP), and the chlorins pheophorbide-a (Pheo-a) and chlorin e(6) (Ce(6)). Each of these molecules was entrapped in biodegradable nanoparticles (NP) based on poly(d,l-lactic acid). The influence of the degree of lipophilicity on the incorporation efficiency of the drug in the NPs, and on the dye leakage from blood vessels as well as on the photothrombic efficiency was investigated using the chick chorioallantoic membrane (CAM) as in vivo model. NP characterization showed that the dye was more effectively entrapped in the polymeric matrix when its degree of lipophilicity increased. While less lipophilic compounds (TCPP, Ce(6)) extravasate rather easily, the more lipophilic dyes (TPP, Pheo-a) tend to remain inside the blood vessels. After injection of a drug dose of 1 mg/kg body weight and a drug-light application interval of 1 min, irradiation with light doses ranging from 5 to 20 J/cm(2) led to the highest photothrombic efficiency when using the NPs loaded with the most lipophilic molecule (TPP). The latter induced vascular damage, which was significantly higher than that observed with the other molecules tested. Thus, in addition to minimal leakage from blood vessels, the TPP in NP formulation exhibited photothrombic efficiency similar to Visudyne which was also tested in the CAM model.     

Complexes of Chlorin e6 with Pluronics and Polyvinylpyrrolidone: Structure and Photodynamic Activity in Cell Culture

Polymeric carriers are extensively used in photodynamic therapy (PDT) for increase of efficacy of photosensitizers. Here, we report the influence of nine Pluronic copolymers on phototoxicity of chlorin e6 (Ce6), in particular 5‐ to 7‐fold rise in the phototoxicity caused by hydrophilic Pluronics F127, F108, F68 and F87 and practically no influence on Ce6 of more hydrophobic polymers. The revealed value of 0.2 mg mL^?1 of Pluronic F127 concentration sufficient for half‐of‐maximal increase of Ce6 photodynamic activity proved to be close to 0.16 mg mL^?1 inherent in well‐documented carrier poly(N‐vinylpyrrolidone) (PVP). The dissociation constants of Ce6 complexes with Pluronic F127 and PVP that were estimated from UV spectra were 0.252 and 0.036 mg mL^?1, respectively, indicating higher stability of Ce6 complex with PVP. According to the results of ¹H‐NMR studies of Ce6 complexes, the porphyrin interacts not only with hydrophobic regions but also with hydrophilic sides of both polymers.     

Endobronchial phototoxicity of WST 09 (Tookad), a new fast-acting photosensitizer for photodynamic therapy: preclinical study in the pig

Photodynamic therapy (PDT) has been used for many years for both palliative and curative treatment of bronchial carcinomas. However, prolonged skin phototoxicity and reduced depth of penetration has limited the widespread use of PDT. We studied the endobronchial phototoxicity of a novel photosensitizer, WST 09 (Tookad). Fourteen pairs of Large White-Landrace male piglets were given intravenous WST 09 followed by laser light illumination of the left mainstem bronchus. Different settings for light dose (fluence), fluence rate (FR), drug dose (D) and drug-light interval (DLI) were applied to each pair. Bronchial toxicity was assessed with repeat bronchoscopic photographic evaluation as well as by pathologic examination following autopsy. Animals developed no toxicity, moderate toxicity or severe toxicity. Increased toxicity was seen with increasing D and fluence and decreasing DLI, whereas no increased toxicity was seen with higher FR. PDT-related histological changes in the normal bronchus confirm the vascular effect of WST 09. Depending on the parameter settings for fluence, D and DLI, the lesions ranged from focal intramucosal ischemia to transmural infarction with subsequent acute inflammation and fibrosis. Clinically feasible parameters for drug and light dosimetry were documented. These data will be important in determining safe starting doses for human phase I/II studies.     

Relationship between mTHPC fluorescence photobleaching and cell viability during in vitro photodynamic treatment of DP16 cells

An implicit dosimetric model has been proposed in which biological damage caused by photodynamic therapy (PDT) is monitored through the decrease in sensitizer fluorescence during treatment. To investigate this, in vitro experiments were performed in which DP16 cells were incubated in meta-tetra(hydroxyphenyl)chlorin (mTHPC) and then irradiated with 514 nm light. Photosensitizer concentration, fluence rate and oxygenation were independently controlled and monitored during the treatment. Fluorescence of mTHPC was continuously monitored via a charge-coupled device-coupled spectrometer during treatment and, at selected fluence levels, cell viability was determined using a trypan blue exclusion assay. The relationship of cell viability to normalized fluorescence was obtained for the different treatment conditions. The relationship was independent of cell medium oxygenation, treatment fluence rate and sensitizer incubation concentration except at a high mTHPC concentration (4 microg/mL). This relationship suggests that fluorescence bleaching may be used to predict mTHPC PDT damage in vitro.     

HepG2 human hepatocarcinoma cells: an experimental model for photosensitization by endogenous porphyrins

Endogenous protoporphyurin IX (PpIX) synthesis after δ-aminolaevulinic acid (ALA) administration occurs in cancer cells in vivo; PpIX, which has a short half-life, may thus constitute a good alternative to haematoporphyrin derivative (HPD) (or Photofrin). This study assesses the ability of the human hepatocarcinoma cell line HepG2 to synthesize PpIX in vitro from exogenous ALA, and compares ALA-induced toxicity and phototoxicity with the photodynamic therapy (PDT) effects of HPD on this cell line.

ALA induced a dose-dependent dark toxicity, with 79% and 66% cell survival for 50 and 100 μg ml⁻¹ ALA respectively after 3 h incubation; the same treatment, followed by laser irradiation (λ = 632 nm, 25 J cm⁻²), induced a 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 to be optimal to reach a maximum phototoxicity.

HPD induced a slight dose-dependent toxicity in HepG2 cells and a dose- and time-dependent phototoxicity ten times greater than that of ALA-PpIX PDT. After 3 h incubation of 2.5 and 5 μg ml⁻¹ HPD, followed by laser irradiation (λ = 632 nm, 25 J cm⁻²), cell survival was 59% and 24% respectively at 24 h.

Photoproducts induced by light irradiation of porphyrins absorb light in the red spectral region at longer wavelengths than the original porphyrins. The possible enhancement of PDT effects after HepG2 cell incubation with ALA or HPD was investigated by irradiating cells successively with red light (λ = 632 nm) and light (λ = 650 nm). The total fluence was kept constant at 25 J cm⁻². For both HPD and ALA-PpIX PDT, phototoxicity was lower when cells were irradiated for increased periods with λ = 650 nm light than with λ = 632 nm light alone. This suggests that any photoproducts involved either have a short life or are poorly photoreactive.

Not all cell lines can synthesize PpIX after ALA incubation. HepG2 cells, which can synthesize enzymes and precursors of endogenous porphyrin synthesis, represent a good in vitro model for experiments using ALA-PpIX PDT. In addition, ALA-PpIX PDT may represent a new, specific treatment for hepatocarcinomas.     

A Comparison of Dose Metrics to Predict Local Tumor Control for Photofrin‐mediated Photodynamic Therapy

This preclinical study examines light fluence, photodynamic therapy (PDT) dose and “apparent reacted singlet oxygen,” [¹O₂]_rx, to predict local control rate (LCR) for Photofrin‐mediated PDT of radiation‐induced fibrosarcoma (RIF) tumors. Mice bearing RIF tumors were treated with in‐air fluences (50–250 J cm^?2) and in‐air fluence rates (50–150 mW cm^?2) at Photofrin dosages of 5 and 15 mg kg^?1 and a drug‐light interval of 24 h using a 630‐nm, 1‐cm‐diameter collimated laser. A macroscopic model was used to calculate [¹O₂]_rx and PDT dose based on in vivo explicit dosimetry of the drug concentration, light fluence and tissue optical properties. PDT dose and [¹O₂]_rx were defined as a temporal integral of drug concentration and fluence rate, and singlet oxygen concentration consumed divided by the singlet oxygen lifetime, respectively. LCR was stratified for different dose metrics for 74 mice (66 + 8 control). Complete tumor control at 14 days was observed for [¹O₂]_rx ≥ 1.1 mm or PDT dose ≥1200 μm J cm^?2 but cannot be predicted with fluence alone. LCR increases with increasing [¹O₂]_rx and PDT dose but is not well correlated with fluence. Comparing dosimetric quantities, [¹O₂]_rx outperformed both PDT dose and fluence in predicting tumor response and correlating with LCR.     

DNAzyme‐Loaded Metal–Organic Frameworks (MOFs) for Self‐Sufficient Gene Therapy

DNAzymes have been recognized as potent therapeutic agents for gene therapy, while their inefficient intracellular delivery and insufficient cofactor supply precludes their practical biological applications. Metal–organic frameworks (MOFs) have emerged as promising drug carriers without in‐depth consideration of their disassembled ingredients. Herein, we report a self‐sufficient MOF‐based chlorin e6‐modified DNAzyme (Ce6‐DNAzyme) therapeutic nanosystem for combined gene therapy and photodynamic therapy (PDT). The ZIF‐8 nanoparticles (NPs) could efficiently deliver the therapeutic DNAzyme without degradation into cancer cells. The pH‐responsive ZIF‐8 NPs disassemble with the concomitant release of the guest DNAzyme payloads and the host Zn²⁺ ions that serve, respectively, as messenger RNA‐targeting agent and required DNAzyme cofactors for activating gene therapy. The auxiliary photosensitizer Ce6 could produce reactive oxygen species (ROS) and provide a fluorescence signal for the imaging‐guided gene therapy/PDT.     

A Comparative Analysis of Silicon Phthalocyanine Photosensitizers for in vivo Photodynamic Therapy of RIF-1 Tumors in C3H Mice

Photofrin® photodynamic therapy (PDT) has recently received FDA approval for the palliative treatment of to-tally and partially obstructing esophageal malignancies. However, there is a need for new PDT photosensitizers because Photofrin has a number of undesirable features. The purpose of this study was to evaluate the efficacy of four amine-bearing silicon phthalocyanines—Pc4, Pc10, Pc12 and Pc18—as potential PDT photosensitizers. Equimolar concentrations of these Pc were found to be highly effective at causing the regression of RIF-1 tumors trans-planted to C3H/HeN mice. The amount of Pc4 necessary to cause an equivalent amount of tumor regression in this model system was substantially less than the amount of Photofrin. The cutaneous phototoxicity of the silicon Pc photosensitizer was assessed by the utilization of the murine ear-swelling model. When C3H mice were exposed to 167 J/cm² of polychromatic visible light from a UVB-filtered solar simulator, which emitted UV radiation and visible light above 320 nm, the Pc produced little, if any, cutaneous photosensitivity. These results indicate that Pc4, Pc10, Pc12 and Pc18 are at least as effective as Photofrin in PDT protocols, while at the same time addressing many of the drawbacks of Photofrin.     

The Effects of Serum on Cellular Uptake and Phototoxicity of Mono-L-Aspartyl Chlorin e6 (NPe6) In Vitro

Abstract— Previous reports showed that the photosensitizer mono- l -aspartyl chlorin e6 (NPe6) binds to serum proteins. However, the influence of this binding on the cellular uptake and photodynamic therapy (PDT) phototoxicity of NPe6 is still undefined. In this paper, we studied how serum in medium affected the P388 cellular uptake and PDT phototoxicity of NPe6 in vitro. This was assessed by (1) detection of the red shift (654 nm Q band peak of absorption) induced by protein binding NPe6; (2) detection of intracellular concentration of NPe6 by HPLC and (3) measurements of the cell survival ratio after PDT by MTT assay. The 654 nm Q band peak of NPe6 shifted to 665 nm after binding of NPe6 and serum proteins. The protein-bound NPe6 cannot be uptaken by cells, thus there was no PDT phototoxicity. Nevertheless, phototoxicity recovered when the concentration of NPe6 excessed the serum protein binding ability or there was free serum protein in the medium. These data suggested that the cellular uptake of NPe6 is inhibited by serum components in the medium, and that only free NPe6 is accumulated by P388 cells even during relatively long incubations. The cytotoxicity of PDT mainly depends on the free NPe6 level in the medium.     

Optimizing meso-tetra-hydroxyphenyl-chlorin-mediated photodynamic therapy for basal cell carcinoma

Meso-tetra-hydroxyphenyl-chlorin (mTHPC)-mediated photodynamic therapy (PDT) has shown to be effective in the treatment of patients with multiple basal cell carcinoma (BCC). In the present study we further optimized the drug-light interval and examined the correlation between plasma drug levels and treatment efficacy. Thirteen patients with multiple BCC (a total of 366 lesions) were included in the study. Following intravenous administration of 0.1 mg kg(-1) mTHPC, lesions were illuminated with 10 J cm(-2) light (652 nm, 100 mW cm(-2)) at 12, 24, 48, 72 or 96 h. Plasma samples were taken prior to each illumination for determination of mTHPC levels, and tumor response was evaluated at 6 months and 1 year. Both univariable and multivariable analyses showed that optimal treatment outcome was obtained for a drug-light interval of 24 h when plasma drug levels were high. Overall, good cosmetic results with little or no scarring were obtained in 87% of the treated lesions and no serious side effects were observed. We optimized mTHPC-mediated PDT for patients suffering from multiple BCC by determining the most effective drug-light interval and showed that this treatment offers significant advantages over surgical resection.     

Hypericum perforatum L. extract - novel photosensitizer against human bladder cancer cells

The polar methanolic fraction (PMF) of the Hypericum perforatum L. extract has recently been developed and tested as a novel, natural photosensitizer for use in the photodynamic therapy (PDT), and photodynamic diagnosis (PDD). PMF has been tested on HL-60 leukemic cells and cord blood hemopoietic progenitors. In the present study, the efficacy of PMF as a phototoxic agent against urinary bladder carcinoma has been studied using the T24 (high grade metastatic cancer), and RT4 (primary low grade papillary transitional cell carcinoma) human bladder cancer cells. Following cell culture incubation, PMF was excited using 630 nm laser light. The photosensitizer exhibited significant photocytotoxicity in both cell lines at a concentration of 60microg/ml, with 4-8 J/cm(2) light dose, resulting in cell destruction from 80% to 86%. At the concentration of 20microg/ml PMF was not active in either cell line. These results were compared with the results obtained in the same cell lines, under the same conditions with a clinically approved photosensitizer, Photofrin. Photofrin was used in the maximum clinically tolerable dose of 4microg/ml, and it was also excited with 630 nm laser light. In the T24 cell Photofrin exhibited slightly less photocytotocixity, compared with PMF, resulting in 77% cell death with 8J/cm(2) light dose. However, against the RT4 cells Photofrin resulted in minimal cell death (9%) with even 8J/cm(2) light dose. Finally, the type of cell death induced by PMF photoactivation was studied using flow cytometry and DNA laddering. Cell death by PMF photodynamic action in these two bladder cell lines is caused predominently by apoptosis. The reported significant photocytotoxicity, selective localization, natural abundance, easy, and inexpensive preparation, underscore that the PMF extract hold the promise of being a novel, effective PDT photosensitizer.     

Pharmacokinetics of Tetra (m-hydroxyphenyl)chlorin in Human Plasma and Individualized Light Dosimetry in Photodynamic Therapy

The pharmacokinetics of the photosensitizer 5,10,15,20-tetra( m -hydroxyphenyl) chlorin(mTHPC) was investigated in the plasma of 20 patients by absorption and fluorescence spectroscopy. The temporal behavior was characterized by a rapid decrease in concentration during the first minutes after intravenous injection of 0.15 mg/kg mTHPC. A minimum concentration in the plasma was reached after about 45 min. The drug concentration then increased again, attaining a maximum after about 10 h, after which it decreased again with a halflife of about 30 h. Irradiation tests in the oral cavity at different time intervals after the injection revealed that the tissue re-action was only partially correlated with the mTHPC plasma level. The tissue response was stronger at later drug-light intervals (1–4 days) than during the first hours after injection even though the mTHPC plasma concentration was higher at the shorter times. Relative mTHPC concentrations were also measured in the mucosae of the oral cavity, the esophagus and the bronchi of 27 patients by light-induced fluorescence spectroscopy using an optical fiber-based spectrometer. These measurements were performed prior to photodynamic therapy (PDT), 4 days after injection of the photosensitizer. Highly significant linear correlations were found between the relative mTHPC concentrations in the mucosae of these three organs. Likewise, the plasma levels of mTHPC measured just before PDT were significantly correlated with the mTHPC concentrations in the three types of mucosae mentioned above. These results indicate that mTHPC plasma levels measured just before PDT can be used for PDT light dosimetry.     

Interaction between photodynamic therapy and BCG immunotherapy responsible for the reduced recurrence of treated mouse tumors

Subcutaneous mouse EMT6 tumors were treated by individual or combined regimens of a single Bacillus Calmette-Guérin (BCG) vaccine administration and photodynamic therapy (PDT). Six clinically relevant photosensitizers characterized by different action mechanisms were used: Photofrin, benzoporphyrin derivative, tetra(m-hydroxyphenyl)chlorin (foscan), mono-L-aspartylchlorin e6, lutetium texaphyrin or zinc phthalocyanine. Irrespective of the type of photosensitizer used, the optimized BCG protocols improved the cure rate of PDT-treated tumors. This indicates that the interaction does not take place during the early phase of tumor ablation but at later events involved in preventing tumor recurrence. Beneficial effects on tumor cure were observed even when the BCG injection was delayed to 7 days after PDT. The accumulation of activated myeloid cells that markedly increases in tumors treated by Photofrin-based PDT was not additionally affected by BCG treatment. However, the incidence of immune memory T cells in tumor-draining lymph nodes that almost doubled at 6 days after Photofrin-PDT further increased close to three-fold with adjuvant BCG. This suggests that BCG immunotherapy amplifies the T-lymphocyte-mediated immune response against PDT-treated tumors. Since both these modalities are established for the treatment of superficial bladder carcinomas, use of their combination for this condition should be clinically tested.     

Monitoring photoproduct formation and photobleaching by fluorescence spectroscopy has the potential to improve PDT dosimetry with a verteporfin-like photosensitizer

In current clinical practice, photodynamic therapy (PDT) is carried out with prescribed drug doses and light doses as well as fixed drug-light intervals and illumination fluence rates. This approach can result in undesirable treatment outcomes of either overtreatment or undertreatment because of biological variations between different lesions and patients. In this study, we explore the possibility of improving PDT dosimetry by monitoring drug photobleaching and photoproduct formation. The study involved 60 mice receiving the same drug dose of a novel verteporfin-like photosensitizer, QLT0074, at 0.3 mg/kg body weight, followed by different light doses of 5, 10, 20, 30, 40 or 50 J/cm2 at 686 nm and a fluence rate of 70 mW/cm2. Photobleaching and photoproduct formation were measured simultaneously, using fluorescence spectroscopy. A ratio technique for data processing was introduced to reliably detect the photoproduct formed by PDT on mouse skin in vivo. The study showed that the QLT0074 photoproduct is stable and can be reliably quantified. Three new parameters, photoproduct score (PPS), photobleaching score (PBS) and percentage photobleaching score (PBS%), were introduced and tested together with the conventional dosimetry parameter, light dose, for performance on predicting PDT-induced outcome, skin necrosis. The statistical analysis of experimental results was performed with an ordinal logistic regression model. We demonstrated that both PPS and PBS improved the prediction of skin necrosis dramatically compared to light dose. PPS was identified as the best single parameter for predicting the PDT outcome.