Giuseppe Roberti

Photodynamic therapy with topical δ-aminolaevulinic acid for the treatment of plantar warts

Treatments currently employed for plantar warts are often painful (electrosurgery, cryotherapy) and not always effective (keratolytic agents). In this paper we investigate the effect of photodynamic therapy (PDT) with topical delta-aminolaevulinic acid (ALA) on plantar warts. In order to remove the superficial hyperkeratotic layer of the warts an ointment containing 10% urea and 10% salicylic acid was applied for 7 days. After gentle curettage, a cream containing 20% ALA was applied under an occlusive dressing for 5 h on 64 warts, while 57 warts (controls) received only the vehicle. Both the ALA-treated warts and the controls were irradiated using a visible light lamp (with a range of 400-700 nm, peaking at 630 nm). The light dose was 50 J/cm(2). Patients were followed-up for 22 months. Two months after the last irradiation session 48 (75.0%) out of 64 ALA-PDT treated warts had resolved. By contrast only 13 (22.8%) of the 57 control warts had done so. During the treatment a few patients complained of a mild burning sensation. The absorption of ALA by the verrucous tissue was demonstrated by in vivo fluorescence spectroscopy. This study shows that topical ALA-PDT can be an alternative treatment for plantar warts. Further studies will be necessary in order to optimize the concentration of ALA and duration of treatment.

LASER PHOTOSENSITIZATION OF CELLS BY HYPERICIN

Abstract— Administering a light dose of 90 J/cm2 at 599 nm during incubation with hypericin to a highly differentiated normal epithelial cell line(FRTL–5), derived from Fisher rat thyroid, and to a neoplastic cell line(MPTK–6), derived from the lung metastases of a thyroid carcinoma induced in Fisher rats, produces cell kill at drug doses 1000 times lower than those necessary to cause the same mortality in the dark. The photocytocidal activity of this polycyclic quinone drug on neoplastic cells is superior to that of antitumor anthraquinone drugs, such as daunomycin and mitoxanthrone, and to the photosensitized antiviral activity previously reported for hypericin.

Hypericin photosensitization of tumor and metastatic cell lines of human prostate

We have investigated the photoactivating effect of hypericin on two cancer cell lines: PC-3, a prostatic adenocarcinoma non-responsive to androgen therapy and LNCaP, a lymphonodal metastasis of prostate carcinoma responsive to androgen therapy. The two cell lines are incubated for 24 h with hypericin at concentrations ranging from 0.001 to 0.3 microg/ml in cell culture medium. The cells are irradiated at 599 nm (fluence = 11 J/cm2) using a dye laser pumped by an argon laser. Hypericin exerts phototoxic effects on both cell lines, while it does not produce toxic effects in the absence of irradiation. These results suggest that photodynamic therapy (PDT) with hypericin could be an alternative approach to the treatment of prostatic tumors, and could be beneficial in tumors that are non-responsive to androgen therapy.

Preliminary tests of a prototype system for optical and radionuclide imaging in small animals

We have assembled a prototype system for multimodal (radionuclide and optical) in vivo planar imaging of small animals (mice) using single photon emission radiotracers (Tc-99m) and a fluorescent marker (hematoporphyrin). Preliminary tests of the separate (optical and radionuclide) prototype imaging systems are presented, aimed at assessing their features and at determining the experimental protocol for in vivo imaging. Tests were performed on anesthetized healthy or tumor bearing mice. The gamma radiation detector is a small area (11 /spl times/ 11 mm/sup 2/) hybrid pixel detector based on the Medipix1 ASIC readout technology (64 /spl times/ 64 square pixels of 170 /spl mu/m by side), bump-bonded to a 300 /spl mu/m thick silicon detector. High spatial resolution in radioimaging (in the order of 1 mm) is achieved in vivo with a pinhole tungsten collimator (0.35 mm diameter, 90/spl deg/ acceptance angle, field of view of over 20 mm at 10 mm source distance). A future setup will use the Medipix2 hybrid detector (256 /spl times/ 256 square pixels, 55 /spl mu/m by side) bump-bonded to a 1 mm thick CdTe pixel detector. The laser-induced in vivo fluorescence imaging system comprises a pulsed light source (Nd:YAG laser, /spl lambda/=532 nm, energy/pulse = 30 mJ, pulse width = 50 ps, repetition rate = 10 Hz) used to excite the fluorescence emission (600-760 nm) of injected hematoporphyrin compound, a low sensitivity CCD camera and a commercial image analysis system. Images of normal and tumor regions are acquired by using a cut-on filter (/spl lambda/>600 nm). Digital image subtraction then enhances the tumor contrast with respect to the background. The final experimental protocol, only partly implemented here, includes independent and then combined optical/radio imaging of control mice and of a solid tumoral area (human thyroid derived anaplastic carcinoma) after injection of the radiotracer and/or of the fluorophore. In this work, the accumulation of the radionuclide in selected organs and of the fluorophore in the tumor provides the signal contrast in the two imaging modalities. Fluorescence spectroscopy of excised tissue samples is also performed to help the interpretation of fluorescence images. Results of in vivo combined imaging on tumor in mice will be shown in a next paper.

Multiple processor version of a Monte Carlo code for photon transport in turbid media

Abstract Although Monte Carlo (MC) simulations represent an accurate and flexible tool to study the photon transport in strongly scattering media with complex geometrical topologies, they are very often infeasible because of their very high computation times. Parallel computing, in principle very suitable for MC approach because it consists in the repeated application of the same calculations to unrelated and superposing events, offers a possible approach to overcome this problem. It was developed an MC multiple processor code for optical and IR photon transport which was run on the parallel processor computer CRAY-T3E (128 DEC Alpha EV5 nodes, 600 Mflops) at CINECA (Bologna, Italy). The comparison between single processor and multiple processor runs for the same tissue models shows that the parallelization reduces the computation time by a factor of about N , where N is the number of used processors. This means a computation time reduction by a factor ranging from about 10 2 (as in our case where 128 processors are available) up to about 10 3 (with the most powerful parallel computers with 1024 processors). This reduction could make feasible MC simulations till now impracticable. The scaling of the execution time of the parallel code, as a function of the values of the main input parameters, is also evaluated.

High-Resolution

We report on tests of a radionuclide imaging system for in vivo investigations in small animals with low-energy photons as from 125I (27-35 keV). Imaging optics features a high-resolution coded aperture mask and a fine pitch hybrid pixel detector (silicon 300-mum or 700-mum thick, or CdTe 1 mm thick) of the Medipix2 series (55 mum pitch, 256 x 256 pixels). The coded aperture had 480 70-mum holes in 100-mum-thick tungsten. Laboratory tests with a 109Cd 22 keV source and a microfocus X-ray tube (35 kVp, Mo anode) show a system resolution of about 110 mum at magnification m = 2.12 and a sensitivity improvement of 30:1 as compared to a 300-mum pinhole collimator. The field of view also depends on magnification: in the experiments presented, it varied from 6 mm (m = 2.12) to 21 mm (m = 0.66). 125I in vivo mouse thyroid imaging with the 70 mum coded aperture, a 300 mum pinhole and a 100 mum parallel hole collimator was also performed to obtain a qualitative comparison. This low energy, semiconductor-based, compact gamma-ray imaging system can be used as a gamma-ray sub-millimeter resolution imager for energies below about 35 keV and it is the basic imaging unit of a small animal Single Photon Emission Computed Tomography system (MediSPECT) built at University of Napoli Federico II and Istituto Nazionale Fisica Nucleare (INFN), Napoli.

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Abstract— —We investigate the efficacy of daunomycin, some imino‐ and amino‐substituted daunomycin analogues and the disubstituted aminoanthracenedione, mitoxantrone, in photosensitizing short‐term cell kill upon irradiation in the long wavelength visible range, during incubation of Fisher rat thyroid cells with the drugs. While all compounds exhibit similar cytocidal effects on our cell line, in the absence of irradiation, administering 86 J/cm2 at wavelengths either coincident or close to drug absorption peaks causes greater enhancement in cell mortality for the 4‐demethoxydaunomycin analogues than either the parent drug or its 5‐imino‐derivative. A lower enhancement is observed with mitoxantrone. In particular, C50 doses (i.e. concentrations that would kill 50% cells) as low as ∼10−9 M are found for both 6‐ and 11‐amino 4‐demethoxydaunomycin, compared with the values obtained in the absence of light, which are 2.59 × 10−4 and 0.43 × 10−4 M, respectively. Our previous studies of the photophysical and photochemical properties of the excited states of these drugs, and ESR and spin trapping studies of photosensitized generation of singlet oxygen, which were extended in this work to include mitoxantrone, indicate that the cytocidal effects proceed via type I rather than type II mechanisms.

I Small Animal Imaging With a Coded Aperture and a Hybrid Pixel Detector

We report on tests of a combined fluorescence and radionuclide planar imaging system for in vivo investigation on small animals. Combined images of anaesthetized mice bearing a surface solid tumor are presented. The fluorescent marker is a hematoporphyrin compound laser-excited with green light and imaged in the red fluorescence emission with a standard monochrome charge coupled device (CCD) camera. The gamma-ray (/sup 99m/Tc) pinhole imaging system uses a semiconductor pixel detector obtained by hybridizing a Silicon (300-/spl mu/m thick) or a CdTe (1-mm thick) pixel detector to the Medipix2 (55-/spl mu/m pitch) readout integrated circuit for single photon counting. The acquisition of combined images of the tumor area (fluorescence: animal top view; radionuclide: bottom view) shows that the tumor area can be imaged in a few minutes, with a few millimeter resolution (1-mm pinhole diameter), radioactively (/sup 99m/Tc MIBI, 74 MBq), and with the optical system. Combined imaging revealed also a different uptake of the two types of tumors studied (one grown from anaplastic human thyroid carcinoma-derived cells, the other from human papillary carcinoma-derived cells). Future progress will be toward a more compact optical setup and the use of a thicker CdTe detector.

ENHANCEMENT OF ANTITUMOR DRUG CYTOTOXICITY via LASER PHOTOACTIVATION

UNLABELLED Photodynamic therapy (PDT) with 5-aminolaevulinic acid (ALA) is an alternative tool for the treatment of superficial non-melanoma skin cancers. Recently ALA-PDT has been employed with encouraging results also for warts, condylomata and psoriasis. In this study the effects of topical ALA plus irradiation with visible light on intact human skin have been evaluated. Five skin areas (A, B, C, D, and E) on the inner upper part of the arms of five healthy volunteers (skin types III and IV) were treated with (A) ALA 20% in base cream without irradiation, (B) only the vehicle (base cream) without ALA, (C, D and E) ALA cream at the concentrations of 5, 10 and 20%, respectively; all treatments were applied with an occlusive dressing. Four hours after ALA or vehicle application areas B, C, D and E were irradiated with a fixed dose of 40 J/cm(2). ALA penetration through the intact skin was evaluated by in vivo fluorescence determination. The effects on healthy skin were evaluated by clinical and chromometric examinations, light microscopy, immunohistochemistry and electron microscopy. RESULTS (1) in vivo fluorescence demonstrated that ALA is able to penetrate through the intact skin, when applied with occlusive dressing and induces a classical fluorescence peak due to Protoporphyrin IX (PpIX) formation, which is the active photosensitiser. (2) Skin areas receiving ALA plus irradiation showed erythema and swelling just after the irradiative session and hyperpigmentation 48-72 h later. (3) Colourimetric data confirmed significant skin colour changes: values a* (representing the erythematous changes) increased only on the skin areas where ALA+irradiation were applied and during the 48 h after irradiation, thereafter a* began to decrease; values L* (pigmentation) increased during the 2 weeks following treatment. (4) Histopathological, immunohistochemical (S100, HMB-45) and electron microscopic findings showed an absolute increment of the number of melanocytes, which appeared clearly activated. In conclusion the application of ALA cream followed by irradiation is able to induce a pigmentation response in healthy human skin, at least in skin types III and IV. This melanocytic activation could have a potential for the treatment of skin disorders characterised by hypopigmentation.

Experimental study on in vivo optical and radionuclide imaging in small animals

Abstract— We have compared the cytotoxicity of daunomycin in vitro to highly differentiated normal epithelial cells (Fisher rat thyroid cells, FRTL‐5) and to two neoplastic cell lines, a thyroid carcinoma (TK‐6) and its lung metastasis (MPTK‐6). Whereas the cell lines are equally sensitive to the drug in the dark, if irradiated during incubation with daunomycin (86 J/cm2 at 488 nm), they become more and differently sensitive. Namely, the drug doses producing 50% mortality decrease by factors of about 22, 28 and 16 for FRTL‐5, TK‐6 and MPTK‐6 cell lines, respectively. This result correlates with differences in drug uptake and resistance observed in the normal and neoplastic cell lines.