In this paper the cytotoxic effect of m-THPC, Foscan®, as well as of the liposomal formulation of m-THPC, Fospeg®,(kind offer of Biolitec) were studied post PDT in the human prostate cancer cell line LNCaP. The cells were incubated for 24h with 0.15 μg/ml and 1.2 μg/ml Foscan® and Fospeg®. Irradiation was performed with a 652nm laser and energy doses 180, 360 and 540mJ/cm2. The effect was assessed by the MTT viability test 24h after irradiation. Also the intracellular localization of Foscan® and Fospeg® was monitored by using Laser Scanning Confocal Microscopy Imaging. The results showed no dark toxicity either with Foscan® or
Fospeg® at any concentration. Also irradiation at each energy dose in the absence of any photosensitizer, did not affect cellular
viability. The cellular death caused after Photodynamic Treatment was dependent on m-THPC concentration and formulation, as well as the delivered energy dose. Fospeg® was more effective as LD50 was achieved with 0.15μg/ml at 180mJ/cm2 while for the same cytotoxic result 1.2μg/ml Foscan® was needed. Images from confocal microscopy revealed higher fluorescence intensity in the cytoplasm after incubation with Fospeg®, than upon incubation with Foscan® under the same experimental conditions.
Photodynamic therapy (PDT) has been used in the treatment of various skin diseases including non melanoma skin
carcinomas (NMSC). However, until now there are no publications concerning the efficacy of PDT after topical
application of mTHPC.
Although topical photosensitizer application presents many advantages over systemic drug administration, ALA-induced
protoporphyrin IX is the only sensitizer topically used so far.
In the present study photodynamic efficacy of the highly potent sensitizer meso-tetra(hydroxyphenyl)chlorin (mTHPC),
supplied in a novel liposome formulation is investigated after topical application in hairless SKH-HR1 mice, bearing non
melanoma skin carcinomas. The drug was applied topically for drug - light interval of 4 hours. The fluence rates were
100 and 50 mW/cm2 and two total energy doses, 10 J/cm2 and 100 J/cm2 were studied in groups of 5 animals. Three PDT
sessions were performed in each animal, once every 7 days. The final evaluation of PDT effects was performed 14 days
after the 3rd PDT treatment by measuring the geometrical characteristics of tumors.
The groups treated with 100 mW/cm2 presented a higher complete tumor remission than the group of 50 mW/cm2 but an
unusual high mortality. In the group of 50 mW/cm2 and 100 J/cm2, although the complete tumor remission percentage is
poor, the tumor growth rate was decreased. No lesion, papilloma, or tumor was observed in the treated area even six
months after tumor remission. Furthermore tumours up to 7 mm were achieved to be treated, indicating that this novel
mTHPC formulation could be used for deeper and not only superficial carcinomas or lesions.
The aim of this work is to study pharmacokinetics and photodynamic efficiency of aluminium phthalocyanine chloride
(AlClPc) in dimethylsulfoxide/Tween 80/water solution, after topical application on hairless mice bearing non-melanoma
skin carcinomas. The concentration of photosensitizer in normal skin and tumor biopsies 1-6 hours after application was
assessed by fluorescence spectroscopy of chemical extractions. The concentration of photosensitizer was 40 times higher
in tumor than in normal skin even 1 h after application.
For photodynamic therapy (PDT) AlClPc was excited by a diode laser emitting at 670 nm, 1 h after application. Seven
different combinations of therapeutic parameters were chosen. The efficiency was assessed as the percentage of complete
tumor remission, the tumor growth retardation and the cosmetic outcomes. The highest complete remission 60% was
achieved with the combination of 75 mW/cm2 with 150 J/cm2. No recurrence rate was observed in any treatment
parameters group and the cosmetic outcome in all completely treated tumors was excellent. The results show that the
effectiveness of PDT is highly dependent on fluence rate. In addition, they are promising for further investigation of this
PDT scheme in preclinical studies mainly in non-melanoma skin carcinomas up to 7mm.
Although the benefits of topical sensitizer administration have been confirmed for photodynamic therapy (PDT), ALA-induced protoporphyrin IX is the only sensitizer clinically used with this administration route. Unfortunately, ALA-PDT results in poor treatment response for thicker lesions. Here, selectivity and depth distribution of the highly potent sensitizer meso-tetra(hydroxyphenyl)chlorin (mTHPC), supplied in a novel liposome formulation was investigated following topical administration for 4 and 6 h in a murine skin tumor model. Extraction data indicated an average [± standard deviation (SD)] mTHPC concentration within lesions of 6.0(±3.1) ng/mg tissue with no significant difference (p<0.05) between 4- and 6-h application times and undetectable levels of generalized photosensitivity. Absorption spectroscopy and chemical extraction both indicated a significant selectivity between lesion and normal surrounding skin at 4 and 6 h, whereas the more sensitive fluorescence imaging setup revealed significant selectivity only for the 4-h application time. Absorption data showed a significant correlation with extraction, whereas the results from the fluorescence imaging setup did not correlate with the other methods. Our results indicate that this sensitizer formulation and administration path could be interesting for topical mTHPC-PDT, decreasing the effects of extended skin photosensitivity associated with systemic mTHPC administration.
m-THPC photodynamic therapy has been successfully studied in skin cancer, but no research effort concerning its topical application has been performed until now. Determination of the biodistribution of a special m-THPC thermogel formulation and its tumour selectivity was studied after topical application on hairless SKH-1 mice bearing non-melanoma skin carcinomas. 20 μl/cm2 of m-THPC thermogel (0.5 mg m-THPC/ml) were applied on normal and tumour area and the concentration or demarcation of tumor by mTHPC fluorescence was measured at 2, 4 and 6 hours after drug application by three methods: 1. A fluorescence imaging system capturing images at two emission wavelengths (500 and 654 nm) following 405 nm excitation. Signals from different regions of interest were averaged and the intensity ratio at 654 to 500 was calculated. 2. A fluorescence spectrometer with a fiber bundle for in vivo spectra recording after 420 nm excitation. 3 Each animal was euthanized and the photosensitizer was chemically extracted from liver, spleen, muscle, normal skin and tumour. The photosensitizer concentrations in the extracts and in plasma were determined by fluorescence spectroscopy. The in vivo methods showed a remarkable difference in the concentration of photosensitizer in normal skin and tumour. The highest concentration in tumour was observed 6h after drug application and the highest fluorescence intensity ratio of m-THPC in tumour to normal tissue was observed at 4 hours. Furthermore, no m-THPC was detected in normal tissues or plasma after drug topical application. In vivo and ex vivo results were consistent.
Measurements of concentration of sensitizers for photodynamic therapy can provide important information in the dosimetry planning and can also give input to the optimal time for treatment. There has been skepticism towards fluorescence techniques for this purpose, as the signal depends on the fluorescence yield and optical properties of the
tissue. Absorption based techniques, lack on the other hand, often the sensitivity required for many sensitizers with relative weak absorption in a wavelength region where hemoglobin absorption is dominant. A direct comparison between absorption and fluorescence techniques for measuring mTHPC concentration after topical application on hairless SKH-1 mice bearing skin carcinomas has been performed. 20 μl/cm2 of m-THPC thermogel (0.5 mg m-THPC/ml) were applied on normal and tumor area and the concentration of mTHPC was measured at 4 and 6 hours after drug application by two methods: 1. A fluorescence imaging system capturing images at two wavelengths (500 and 650 nm) following 405 nm excitation. Signals from different regions of interest were averaged and the intensity ratio at 650 to 500 was calculated. 2. A diffuse reflectance spectroscopy system with a fiber separation of 2 mm, providing the absorbance at 652 nm. Both
systems provided consistent results related to the photosensitizer concentration. The methods show a remarkable difference in the concentration of photosensitizer in normal skin and tumor. No significant difference in mTHPC concentration in tumor could be observed between the 4 and 6h groups after drug application.
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