In vivo pharmacokinetics and photodynamic effect of Foscan® (mTHPC) in tumor tissue of mice bearing human colon adenocarcinoma

H. 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,
URA CNRS D0821, 54511 Vandoeuvre-lès-Nancy Cedex, France

b Centre de Recherche en Automatique de Nancy CRAN URA CNRS DO821

c Russian State Medical University



Key words : Photodynamic therapy, Foscan¨ (mTHPC), Spectrofluorometry, cytotoxicity, nude mice, human colon adenocarcinoma.



Address for reprints and correspondence :

Lina Bezdetnaya
Laboratoire de Recherche en Oncologie
Centre Alexis Vautrin
Avenue de Bourgogne, 54511 Vandoeuvre-lès-Nancy Cedex, France
Tel. 03 83 59 85 06
Fax. 03 83 44 60 71
E. mail : bolotine@nancy.fnclcc.fr



Abstract
Introduction
Material and Methods
Results
Discussion
References


Abstract

Foscan® or meta-tetra (hydroxyphenyl) chlorin (mTHPC) pharmacokinetics was investigated using non invasive light induced mTHPC fluorescence emission analysis.
Tumor/healthy tissue mTHPC fluorescence ratios were analyzed to determine the optimal time for photodynamic therapy (PDT) after injection in nude mice xenografted with HT29 human colon adenocarcinoma cells. After recording autofluorescence spectra in tumor and normal surrounding tissues, the mice were injected intraperitoneally with mTHPC (0.3 mg/kg b.w).
In vivo fluorescence emission spectra normalized to the autofluorescence spectrum were recorded at different time (4 h - 10 days). The maximum fluorescence ratio (tumor/healthy tissue) of mTHPC (652 nm) was reached 72 h after injection.
Pharmacodynamic activity of mTHPC was then evaluated in HT29 tumor bearing mice injected intraperitoneally (0.3 mg/kg b.w) and irradiated 72 hours after injection at 650 nm, 10 J/cm2 using a dye laser. Two fluences rates were tested : 32 mW/cm2; and 160 mW/cm2. Responses were evaluated from tumor volume measurement. Tumor volume was measured every 2 day and normalized to the initial volume. A continuous growth was observed in the control groups (neither drug nor light, or drug only or light only). Tumors exposed to 160 mW/cm2 showed a growth delay (d2-d4). Regression was observed in tumors treated at 32 mW/cm2 between d2-and d6. Seventy-two hours time interval between injection and light exposure seems appropriate for mTHPC-PDT. In addition, this study confirms that under our experimental conditions the fluence rate is of major importance in mTHPC PDT, lower fluence rate yielding higher therapeutic effect.

1. Introduction

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. Material and Methods

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).
Three mice were injected with mTHPC (0.3 mg/kg i. p.). At each time interval the mTHPC fluorescence was measured in three points on the surface of both tumor and healthy tissues. The fiber was maintained to be in direct contact with the tissue (tumor through skin or muscle through skin). Fluorescence measurements were performed in unanesthetized animal in order to avoid possible changes in metabolism, that may affect fluorescence characteristics.

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)
where D1, D2 and D3 are three orthogonal diameters of the tumors that were measured every two days by a caliper.

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. Results

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.



Figures :

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). 




Fig.1


Fig.2


Table 1

Time (h) 0.3 mg/kg
4 1.5
8 1.5
24 1.4
48 1.6
72 2.0
96 1.1





4. Discussion

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 results of our previous work (Bossu 97 ) demonstrated, that the highest tumor/normal skin ratio was reached 72 hours after mTHPC administration (0.3 mg/kg, i. p.) and was equal to 5.1. As a tumor, the hairless mouse (SKh-1) bearing skin tumors, induced chemically, have been used. In our present study the ratio (tumor/healthy tissue) was found to be 2.5 fold lower. It should be noted, that the pharmacokinetics of the photosensitizer can be different between grafted tumor models (the present study) and the one induced chemically (Bossu, 97). The ratio (tumor/healthy tissue) found by different groups varied from the value of 5 to 18 (Bossu 97, Morlet 95, Peng 95). These discrepancy in results can be attributed to the different detection methods, as well as to the tumors of different origins, different vascularization systems etc. Also, one cannot exclude the influence of a temporal variation of the optical parameters of the tissue, for example due to temporal change of hemoglobin concentration in the tissue (Glanzman 95). Such a brief overview of the available literature data shows that each PDT-treatment protocol should be adapted to the given type of tumor model. That is why in our study the PDT-treatment was performed 72 hours after mTHPC injection, the interval of time which corresponds to the maximum ratio (tumor/healthy tissue) of the dye, and also the absolute maximum of dye in the tumor.
The tumor volume growth monitored under choosen conditions demonstated that PDT effects depend on the applied fluence rate. Low fluence rate (32 mW/cm2) appeared to be more efficient in comparison with high fluence rate (160 mW/cm2) : the doubling times were 13 days versus 9 days, respectively. The tumoral regrowth can be explained by our recently published results : low fluence-rate (32 mW/cm2) induced tumor necrosis of 3.0±0.3 mm depth, whereas no necrosis was observed at high fluence-rate (160 mW/cm2) (Rezzoug 97). Meanwhile, the depth of necrosis, obtained with 32 mW/cm2, was not sufficient to destroy completely the tumor, since the initial tumor diameter size was 8-10 mm; the tumor regrowth was observed one week (6 days) after PDT treatement. Peng (95), showed that the regrowth take a place 12 days after PDT. Compared to our results the long time, observed in the study of Peng (95) can be explained by the difference in the quantity of mTHPC injected (1 mg/kg) and the dose of irradiation (20 J/cm2).
As demonstrated in our previous study (Rezzoug 96, 97), the photodestruction of intratumoral mTHPC during PDT treatment does not depend on the fluence rates used (32 or 160 mW/cm2), therefore, the difference in the degree of necrosis, as well as in tumor regrowth induced by different fluence rates cannot be correlated with mTHPC photobleaching.
The difference in the response to mTHPC-PDT due to a non specific effect such as heat, should be excluded since the highest temperature (33¡C) was below the temperature at which hyperthermia (39¡C) act synergistically with PDT (Waldow, 95). Therefore, neither photobleaching nor temperature can explain our data in mTHPC-PDT response at different fluence rates. The higher mTHPC-PDT efficiency at low fluence rate is usually attributed to reduced oxygen consumption (Foster 91).

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 cancer such as Ïsophagus epidermoid carcinoma.





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