Michail Rallis

Topical photodynamic therapy of murine non-melanoma skin carcinomas with aluminum phthalocyanine chloride and a diode laser: pharmacokinetics, tumor response and cosmetic outcomes

Background/purpose: Topical photodynamic therapy (PDT) is potentially useful for the treatment of non‐melanoma skin cancer and other skin diseases. We investigated the therapeutic effects of PDT using topical application of aluminum phthalocyanine chloride (AlClPc) and a diode laser emitting at 670 nm in murine non‐melanoma skin carcinomas.

Comparative characterization of the cellular uptake and photodynamic efficiency of Foscan® and Fospeg in a human prostate cancer cell line

BACKGROUND m-THPC (Foscan(®)) is one of the most potent second generation photosensitizers used in photodynamic therapy, photoactivated at higher wavelengths (652 nm). However, its strongly hydrophobic nature causes aggregation of the molecules and prevents its unbiased bioavailability in the biological media, resulting in lower accumulation in the tumor cells. Several strategies have been adopted to improve the photodynamic characteristics of the photosensitizer. Among them, very promising seems to be the encapsulation of the molecule into liposomes, due to the superior properties of liposomes as drug carriers. METHODS In this paper the photodynamic characteristics of the PEGylated liposomal formulation of m-THPC, Fospeg, using the human prostate cancer cell line LNCaP, as an in vitro model, were investigated. In addition the spectral characteristics, cellular uptake and localization, dark and light induced cytotoxicity and photodynamic efficacy of Foscan(®) and Fospeg were compared. RESULTS Fospeg, compared with Foscan, showed higher intracellular uptake at any concentration and incubation time. Regarding PDT efficacy, Fospeg produced more severe cytotoxicity than Foscan(®) at any concentration and energy dose. Using Fospeg, the lowest concentration (0.22 μM) and energy dose (180 mJ/cm(2)) was adequate to result in the death of 50% of the cells 24h post PDT while an approximately 10 times higher Foscan(®) concentration (1.8 μM) was needed to result in the same cytotoxicity. CONCLUSIONS The use of the PEGylated liposomal formulation of m-THPC resulted in the improvement of its intracellular uptake and the enhancement of its photodynamic activity. Fospeg, compared to Foscan(®), proved to be a more advantageous photosensitizer for photodynamic therapy.

In vitro percutaneous absorption of pine bark extract (Pycnogenol) in human skin

One promising class of antioxidant compounds is polyphenols, contained abundantly in pine bark extract (Pycnogenol®—pine bark extract). This medicinal extract is utilized for its anti‐inflammatory properties. Its pharmacological action in skin depends on the kinetics of its absorption. In this study the dermal bioavailability of pine bark extract was investigated. Viable human skin, adapted on continuously perfused Franz cells, was applied with 5% (w/v) pine bark solution. Samples were taken at 0.5, 1, 2, 4, 6, 8, 10, and 12‐hour intervals and analyzed for detection of pine bark extract constituents by high performance liquid chromatography (HPLC) (reversed phase column, isocratic conditions) coupled with an electrochemical detector (EC). Several constituents of pine bark extract such as gallic acid, protocatechuic acid, catechin, p‐hydroxybenzoic acid, vanillin, and one unidentified constituent were detected. These findings indicate that pine bark extract is readily absorbed by human skin and can be used for topical application.

Studies in vivo by electron spin resonance of free radical mechanisms implicated in UV-induced skin photocarcinogenesis.

Solar ultraviolet radiation (UVR) is implicated in many types of skin damage, such as photodermatoses, photoageing, erythema, pigmentation, skin cancer etc. Free radicals and reactive oxygen species are considered to play an important role in cutaneous photocarcinogenesis. But skin is endowed with photoprotective agents, namely melanins and antioxidant enzymatic and non‐enzymatic mechanisms. In this study we describe the in vivo electron spin resonance (ESR) signals of melanins after UVR exposure, using skin specimens of various types of mice, which were taken from different parts of their bodies. The ESR signals were used as a model for testing the antioxidant properties of butylated hydroxyanisole, tocopherol acetate, and octyl p‐methoxycinnamate with butyl methoxy dibenzoyl methane and superoxide dismutase (SOD). Additional UVB radiation was applied to the skin samples in situ (in the cavity of the ESR spectrometer). Suppression of ESR signals of melanins was observed in all cases.

Low-Molecular-Weight Hydrophilic and Lipophilic Antioxidants in Nonmelanoma Skin Carcinomas and Adjacent Normal-Looking Skin

Low-molecular-weight antioxidants are some of the most efficient agents of the skin defense mechanism against environmental factors, such as cosmic rays, smoke, and pollutants. The total skin concentrations of hydrophilic ascorbic and uric acids, as well as lipophilic α-tocopherol, β-carotene, and ubiquinol-10 antioxidants were determined by an HPLC-EC detector from 18 biopsies of human nonmelanoma skin carcinomas and 18 biopsies from skin areas adjacent to carcinomas. No significant differences in the concentrations of lipophilic antioxidants in both carcinomas and normal-looking skin areas adjacent to carcinomas were observed. On the contrary, ascorbic and uric acid concentrations were found to be 18 and 36% lower in carcinomas than in normal-looking skin areas, respectively. No statistical significance was observed between antioxidant concentrations and age, sex, phototype, profession, site of tumor, frequency, and time of UV light exposure either. Accordingly the antioxidant concentrations in both cancerous skin and adjacent normal-looking areas were found to be much higher than in normal skin, in contrast to literature data.

Combination of Fospeg-IPDT and a natural antioxidant compound prevents photosensitivity in a murine prostate cancer tumour model

BACKGROUND The aim of the present research was to investigate the potential use of a natural compound rich in antioxidant agents, derived from Pinus halepensis (P. halepensis), to prevent PDT induced photosensitivity. The present research progressed in two levels. The first one evolved the optimization of Fospeg-interstitial photodynamic therapy (IPDT) in a prostate cancer animal model. In the second one, P. halepensis bark extract, was evaluated for its potential use to prevent photosensitivity. METHODS Two sets of experiments were performed, IPDT only and IPDT in the presence of antioxidant. For both of them, Fospeg was administrated intravenously to SCID mice bearing prostate cancer, followed by IPDT after 6 h. For the IPDT+antioxidant experiments, P. halepensis was injected intratumourously 1 h prior the tumour illumination. Treatment outcome was monitored twice a week by an imaging system and by measuring tumour dimensions using a caliper. Photosensitivity was assessed by monitoring erythema of the tail using the imaging system. RESULTS IPDT with Fospeg and 15 J total light energy is a therapeutic scheme that can eliminate tumours in the murine model of prostate cancer. Two months after complete tumour remission no tumour recurrence was observed. Also, the cosmetic outcome of the research was excellent. The major drawback of this treatment scheme was that 90% of the animals developed photosensitivity. The addition of P. halepensis bark extract resulted in prevention of the photosensitivity, leaving PDT outcome unaffected. CONCLUSIONS The combined use of PDT and the used antioxidant agent could broaden the implementation of photodynamic therapy, by eliminating photosensitivity.

Evaluation of the PDT Effect of Foscan ® and Fospeg ® in the LNCaP Human Prostate Cancer Cell Line

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.

Diabetic skin and UV light: Protection by antioxidants

Abstract Non‐invasive biophysical methods were used to study the effect of antioxidant gels, which were prepared from Pinus halepensis bark extracts, vitamin C and water, on the skin of diabetic hairless mice irradiated with UV radiation of 1 and 2 minimal erythemal doses (MEDs). The calculated transepidermal water loss (TEWL) for diabetic mice was found to be fivefold higher on day 11 after irradiation, and in all cases, the TEWL values converged to their initial values on day 21. Both pinus and vitamin C gels inhibited the dehydration of the skin, while water gels did not show similar protection. At low dose of UV‐irradiation (1 MED), vitamin C gels showed the best hydration, while by doubling the UV dose, pinus gels induced significant skin‐protective effects. Upon irradiation, the mice treatment with pinus gel showed diminished inflammation in comparison with the other gels. Pinus also inhibited the hyperkeratosis of skin. As expected, 2 MEDs caused greater skin damage, such as inflammation, dryness, oxidative stress and rides (aging) compared to the damage induced by 1 MED. Graphical abstract Antioxidant‐gel protection of UV‐irradiated diabetic skin. Figure. No Caption available.

Topical Treatment of Skin Injury Inflicted in Mice by X-Ray Irradiation

Background/Aims: There is no treatment, without side effects, efficiently preventing or curing skin burns, caused by radiotherapy. A new experimental topical treatment protocol was assessed in mice receiving orthovoltage X-rays at an equivalent dose to that applied to human breast cancer patients in conventional radiotherapy. Methods: SKH-HR2 female hairless mice were irradiated on their dorsum with a total dose of 4,300 cGy during a 1-month period (20 fractions). The treatment group received a combination of 3 topical products, an oil-in-water cream, a gel containing Pinus halepensis bark aqueous extract, and an ointment containing olive oil extract of the marine isopod Ceratothoa oestroides. The positive control group was treated with a conventionally used commercial gel, whereas the negative control group did not receive any topical treatment. Skin alterations were evaluated by macroscopic examinations, measurements of transepidermal water loss (TEWL), melanin content, erythema intensity, hydration, and histopathology assessment. Results: Sixty days after radiation, TEWL and hydration values were abnormal and elements of acute, chronic, and granulomatous inflammation were present in all cases. The severest damage was detected in the deeper dermis. Treatment showed a comparatively beneficial effect on chronic and granulomatous inflammation while positive control was beneficial on acute inflammation. Conclusion: Skin anti-inflammatory treatment was the most effective but must be applied for several months. Further preclinical studies should be conducted, assimilating a human cancer radiation therapeutic schema with the aim of optimizing skin inflammation treatment.

Contents Vol. 29, 2016

N. Ahmad, Madison, Wis. C. Antoniou, Athens J.M. Baron, Aachen E. Benfeldt, Roskilde E. Berardesca, Rome K. De Paepe, Brussels P. Elsner, Jena A. Farkas, Zurich N. Garcia Bartels, Berlin M.W. Greaves, London R.H. Guy, Bath S. Hedtrich, Berlin E.M. Jackson, Bonney Lake, Wash. H. Kandárová, Ashland, Mass. L. Kemeny, Szeged J. Kresken, Viersen J. Krutmann, Düsseldorf B. Lange-Asschenfeldt, Klagenfurt am Wörthersee R. Neubert, Halle T. Ruzicka, Munich M. Schäfer-Korting, Berlin M. Schmuth, Innsbruck S. Seidenari, Modena P.W. Wertz, Iowa City, Iowa J. Wohlrab, Halle L. Zastrow, Monaco Journal of Pharmacological and Biophysical Research