In vivo pharmacokinetics and photodynamic effect of Foscan® (mTHPC) in tumor tissue of mice bearing human colon adenocarcinomaH. Rezzoug a, O. A'Amar a,b, L. Bezdetnaya * a,c and F.Guillemin a,b a Centre Alexis Vautrin, Unité de Recherche en Thérapie
Photodynamique, b Centre de Recherche en Automatique de Nancy CRAN URA CNRS DO821 c Russian State Medical University Lina Bezdetnaya AbstractFoscan® or meta-tetra (hydroxyphenyl) chlorin (mTHPC)
pharmacokinetics was investigated using non invasive light
induced mTHPC fluorescence emission analysis. Photodynamic therapy (PDT) involves the administration of a photosensitizer which induces a cytototoxic effect after exposure to light at the appropriate wavelength. The photosensitizer is retained in the tumor as well as in the surrounding tissue. Estimation of the maximum dye accumulation in the tumor is one of the way to optimize PDT-treatment. Non-invasive fluorescence spectroscopy is a particularly attractive technique for pharmacokinetical evaluation. This technique is based on the photosensitizer fluorescence induced by a monochromatic light. Determination of the maximum fluorescence ratio (tumor/ healthy tissue) should allow to optimize time interval between drug administration and tumor irradiation. Fluorescence measurements give a semi-quantitative estimate of tissue photosensitizer content. However, a correlation was shown between fluorescence detection of mTHPC and internal tumor mTHPC content measured by extraction and reverse-phase high-performance liquid chromatography (HPLC) (Grahn 97). Another major parameter affecting PDT response is fluence rate. During the last years, different groups studied the effect of the fluence rate on the in vitro and in vivo efficiency of different photosensitizers. The recents studies on the mTHPC efficiency in vivo demonstrated that the fluence rate plays a significant role in mTHPC-PDT (Andrejevic 96 a, Van Geel 96). In this study we report the mTHPC distribution (tumor/healthy tissue) based on light induced fluorescence (LIF). This technique allows to find the maximum intratumoral dye concentration and, taking this as endpoint, to choose optimal time interval for photodynamic treatment. To define the irradiation conditions sufficient to induce tumor response, we have performed irradiation test at different fluence rates. 2.1. Photosensitizers Foscan¨ (mTHPC) was kindly provided by Scotia Pharmaceuticals (Guilford, UK) and was dissolved according to laboratory recommendation in polyethylene glycol (PEG) 400 (30%), ethanol (20%) and water (50 %). 2.2. Tumor models HT-29 colon adenocarcinoma cell line was maintained in Dulbecco's MEM (Gibco BRL, Cergy Pontoise, France) supplemented with 10 % heat inactived fetal calf serum (FCS) (Dutscher, France), 1% penicillin / streptomycin (Gibco BRL) at 37¡C in a 5% CO2 atmosphere to reach exponential growth, then trypsinized to obtain cell suspensions. Three-week athymic female mice (Swiss, nu/nu, IFFA CREDO, France) were inoculated subcutaneously on the right side with 8.106 HT 29 cells. Three to four weeks later, as the tumor reached a diameter of 8-10 mm, mTHPC (0.3 mg/kg body weight) was injected intraperitoneally. Control groups of animals received either mTHPC alone, or light alone. 2. 3. Light induced fluorescence measurements In vivo quantification of mTHPC fluorescence was performed
using a CP 200 optical multichannel analyser (Jobin-Yvon) managed
by Spectramax software. Three optical fiber probe was used for
excitation (550 µm of core diameter), fluorescence collection
and backscattered power measuring (200 µm of core diameters).
The distance between the axis of optical fibers was 375 µm. The
410 nm excitation light was selected by passing the light of 300W
xenon lamp through a bandpass filter (FWHM = 9 nm) (Guillemin
96). 2. 4. Photoirradiation Light (650 nm) was emitted from a kiton red dye laser (Spectra-Physics 375 B, Les Ulis, France) pumped by an argon laser (Spectra-Physics 2030). The wavelength used for irradiation was controlled with a monochromator (Jobin Yvon, France). The laser beam was transmitted through a 600 µm silica-silicon optic fiber (SEDI, Evry, France). The output power was fixed to 100 mW and 500 mW (to give 32 mW/cm2 or 160 mW/cm2, respectively) and controlled before each irradiation using a power-meter. Homogenous light spot was adjusted to 20 mm in diameter and exposure time varied in order to obtain a light fluence of 10 J/cm2. 2. 5. Photodynamic activity To evaluate the mTHPC activity, mTHPC was injected in groups of 6 to 9 mice. Three days after mTHPC injection, each animal was anaesthetized by i.m. injection of ketalar 12.5 mg/ml (2 x 0.25 ml) (Parke Davis, Courbevoie, France), then photoirradiated at 650 nm with 10 J/cm2 either at 32, or at 160 mW/cm2 with the dye laser. The photodynamic activity was evaluated by direct measurement of tumor volume every two days. The tumor volume was calculated using the following formula : V = ¹/6 (D1xD2xD3) 2. 6. Temperature measurements Six animals were anesthetized with ketalar, as described above. For each fluence rate, the temperature (¡C) was measured on 3 animals at the surface of the treated areas before and during laser delivery, using a thermocouple (chromel-alumel, DIGISENSE, Bioblock, Illkirch, France). 3. 1. mTHPC fluorescence in tumor The in vivo mTHPC fluorescence appears 4 hours after i.p. injection and increases linearly with the time. The plateau reached at 48 hours and lasts up to the 240 hours (Fig. 1). Afterwards, the decrease in fluorescence signal was observed (data not shown). In the healthy tissue the plateau was also achieved at 48 hours (Fig. 1), but the fluorescence intensity of mTHPC in tumor (tumor + skin) was higher than that in the healthy tissue (muscle+skin). The fluorescence ratios of tumor/healthy tissue at various time intervals after injection of mTHPC are presented in Table 1. The maximum fluorescence ratio (2.0) was obtained 72 hours after mTHPC injection. 3. 2. Influence of light fluence rate on tumor regrowth Growth curves of tumors exposed to laser light 72 h after i.p. injection of 0.3 mg/kg of mTHPC are shown in Fig. 2. A growth delay was observed in the group of mice treated at 160 mW/cm2 (10 J/cm2) during the first four days after irradiation in comparison with control group (neither light, nor drug). So, the doubling time of about 4 days was observed in the control group, and about 9 days for the group treated at 160 mW/cm2. The tumor exposed to 32 mW/cm2 (10 J/cm2) showed a tumor decrease during first 7 days following PDT with a doubling time of about 13 days. 3. 3. Temperature measurements The temperature measured on the surface of the treated areas
before light delivery was 30¡C. During light delivery at fluence
rate of 32 mW/cm2, no temperature changes were
observed (ÆT ¡C = 0). On the contrary, for 160 mW/cm2
the temperature increased at the tumor surface (ÆT ¡C = 3) , as
well as in the deeper layers (ÆT ¡C = 2). All temperatures were
below the threshold for thermal tissue damage. Figure 1 : mTHPC fluorescence intensity at 652 nm as a function of time after injection of 0.3 mg/kg as observed in the healthy tissue (open symboles),with comparison of in tumor (close symboles). Excitation at 410 nm. The points are reported on 3 animals (3 measurements by animal). Figure 2 : Regrowth curves of HT29 tumors after PDT with 0.3 mg/kg of mTHPC. Tumor were exposed at laser light 72 h after injection (650 nm, 32 or 160 mW/cm2, 10J/cm2). Table. 1 : Fluorescence ratio of tumor:healthy tissue in mice
bearing HT29 colon adenocarcinoma at various times after an i.p.
injection of mTHPC ( 0.3mg/kg).
Non-invasive fluorescence spectroscopy is an attractive
technique providing indication of photosensitizer distribution in
the tissue. Our results showed, that mTHPC was retained in tumor
for a long time period (d2-d10). The study of Andrejevic (96 b),
using a 2-fold lower mTHPC dose (0.15 mg/kg) showed that the
maximum fluorescence signal of mTHPC in tumor was reached between
d2-d7 and the fluorescence signal detected in tumor was higher in
comparison with healthy tissue. As a tumor model, the authors
used squamous cell carcinomas induced on the hamster cheek pouch
mucosa and fluorescence detection was performed by means of
fluorescence microscopy in tissue. The results of work of Peng
(95) showed that the maximum mTHPC concentration was achieved
between 24 and 48 hours after i. p. injection of 1 mg/kg of
photosensitizer, and very low level of mTHPC was seen in the
normal muscle tissue. In this case mammary carcinomas were used
and mTHPC was quantify after chemical extraction of
photosensitizer by means of fluorescence detection. The phototoxicity of mTHPC is realized in the neoplasic cells and in the vascular wall, the sites of preferential mTHPC accumulation. Indeed, the study of Peng (95) showed that 24 and 96 h after PDT treatment with mTHPC both vascular walls and tumor cells were destroyed. However, the vasculature damage was considered to be the initial event bringing to the photochemical destruction of tumor (Henderson 85). The haemorrhage observed at the surface of tumor treated at 32 mW/cm2, but not at 160 mW/cm2 (data not shown) can be taken as an indication of tumor vascularity destruction for the low fluence rate. The low mTHPC-PDT effect observed at 160 mW/cm2 could be attributed to the slowdown of blood flow, occurring during the first secondes after irradiation. Decrease in blood flow leads to an oxygen depletion and protection the cells from PDT-damage. In conclusion, a non-invasive spectrofluorometry seems to be
appropriate to detect the biodistribution of the dye in the
tissue and to determine the optimal time interval for mTHPC-PDT
treatment. The proposed technique can be used in clinical
endoscopy to follow the mTHPC pharmacokinetics to optimize
light-drug-interval. Using low fluence-rate PDT-Foscan¨ may
improve therapeutical index, especially for a thin superficial
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