Photodiagnosis and Photodynamic Therapy (2004) 1, 181—190

Tissue detection of diphenylchlorin sensitizer (SIM01) by fluorescence and high-performance liquid chromatography S. Thibauta, L. Bourr´ ea, A. Bendarraza, S. Juillardb, G. Simonneauxb, Y. Lajata, T. Patrice MD, PhDa,∗ a

LASER D´ epartement, Laboratoire de Photobiologie des Cancers, Neurochirurgie, CHU Nantes, 44093 Nantes, France b Laboratoire de Chimie Organom´ etallique et Biologique, UMR CNRS 6509, Universit´ e de Rennes 1, 35042 Rennes Cedex, France

KEYWORDS Photodynamic therapy; SIM 01; Pharmacokinetics; Cancer; Elimination kinetics; HPLC; Fluorescence

Summary Cancer is today a major problem of public health. Unfortunately, the current treatments remain still too often impotent or too heavy compared to the gross national product of many countries. The use of PDT in the treatment of the malignant tumours currently raises great hopes. This physicochemical method is based on the combined action of a nontoxic drug given systematically to the patient and of the visible light delivered locally to the tumour using optical fibres. The radiation will activate the significant substance preferentially fixed on cancerous cells and will cause the death of the tumoral cells while releasing from the toxic ridicalizing species which then will deteriorate vital cellular targets. Tissue distribution and elimination kinetics of the SIM01 were analysed in biological samples from mice tissues by spectrofluorometry and HPLC. Measurements were performed 4, 6, 12, 24 and 48 h after an intraperitoneal injection for SIM01 doses of 2, 5 and 15 mg kg−1 . Elimination seemed to concern essentially gallbladder, liver and stools, where maximum fluorescence reached, respectively, 20,000, 2800 and 15,000 cps for 5 mg kg−1 , 6 h after injection. Among the tissues examined with HPLC, the highest SIM01 levels were found in stools, urine, liver, gallbladder and spleen. Liver, gallbladder, and stool homogenates from drug-treated animals contained an additional peak (16, 7 min) detectable only after injection of at least 15 mg kg−1 . Our HPLC determinations and in vivo fluorescence detection of SIM01 gave comparable kinetic profiles. These techniques should be considered as complementary rather than exclusive for kinetic profiles determination. © 2004 Elsevier B.V. All rights reserved.

Introduction * Corresponding author. Tel.: +33 2 40 16 56 75/53 37;

fax: +33 2 40 16 59 35. E-mail address: [email protected] (T. Patrice).

Tissue distribution of the new photodynamic therapeutic agent, 2,3-dihydro-5,15-di(3,5-dihydroxyphenyl)porphyrin (SIM01, patent PCT/FR01/02470)

1572-1000/$ — see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/S1572-1000(04)00043-2

182 was analysed in tissues biopsies by optical fibre spectrofluorometry (OFS) and high-performance liquid chromatography (HPLC). Photodynamic therapy (PDT), an innovative approach to cancer treatment, is currently performed in non-operable patients with small tumours for whom conventional treatments (radiotherapy and chemotherapy) are ineffective or not indicated [1]. This treatment is based on photoactivation at the specific wavelength of a sensitizer relatively retained by cancer cells. In general, PDT mechanisms are thought to be initiated when a photoexcited membrane-localized photosensitizer transfers energy of the excited triplet state to molecular oxygen to generate singlet oxygen [1]. Subsequent redox reactions can also produce superoxide anions, hydrogen peroxide and hydroxyl radicals. Photochemical interaction of the photosensitizing agent, light and oxygen leads to formation of reactive oxygen species (ROS) that can destroy tumour cells [2,3]. The short half-life of ROS, especially of singlet oxygen and hydroxyl radicals, means that they react with cellular components within a few nanometers of their formation sites in membranes. Once the tissue distribution of the photosensitizer is determined, it is possible to optimize PDT treatments having already received gouvernment approvals and eventually to define tissue targets susceptible to a new therapeutic approach. Previous studies in our laboratory evaluated the photosensitizing properties of SIM01 in vitro and in vivo for PDT applications [4]. Human tumour from a human colorectal adenocarcinoma (HT29) or a human prostate adenocarcinoma (PC3) was used for in vivo experiments, and kinetics were assessed by OFS measurements. Kinetic studies of muscle, skin and tumour were performed during these earlier experiments. The purpose of the present study was to assess the tissue distribution of SIM01 and its metabolites as well as its elimination kinetics. OFS and HPLC with UV—vis detection were used for determination of SIM01 extracted from biological samples. The accuracy of fluorometric measurements was evaluated as compared to HPLC as it is regarded as the gold standard, but is much more complicated to perform than OFS.

Materials and methods Chemicals Methanol, and water, for HPLC applications were supplied by Merck Eurolab (Fontenay-sous-bois,

S. Thibaut et al. France, Nos.133512500 and 23595294). Tetrabutylammonium dihydrogen solution was from Fluka Chemicals (Saint Quentin Fallavier, France, No. 86842), sodium dihydrogenophosphate (Rectapur® , No. 28013264) from Prolabo (Paris, France), and dimethyl sulfoxide from Sigma (Saint Quentin Fallavier, France) (D5879). The new photosensitizer, 2,3-dihydro-5,15di(3,5-dihydroxyphenyl)porphyrin (SIM01), was obtained from UMR 6509 CNRS (Rennes, France). One milligram of SIM01 was dissolved in 1 mL of PEG 400 (30%), ethanol (20%) and water (50%). Further concentrations were obtained by successive dilutions in isotonic saline.

Animals OF-1 male mice (weighting 25—30 g) used for this experiment were obtained from Iffa-Credo (L’Arbresle, France). Animals were fed with nonfluorescence-inducing food for 7 days before the experiment. Four mice were used for each experimental condition. Mice were killed by cervical dislocation, under general anaesthetic.

Injection procedure After being weighed, mice received an intraperitoneal injection of SIM01. Spectrofluorometric measurements were performed 4, 6, 12, 24, and 48 h after injection for doses of 2 and 5 mg kg−1 . Once injected, the mice and tissue samples were protected from direct light until fluorometry was performed.

Absorption and fluorescence spectrometry Absorption spectrometry was performed with an 8500PC double-beam UV—vis scanning spectrometer (Fischer, France), and absorption spectra were obtained between 300 and 800 nm. Fluorescence spectrometry was performed using a Spectrofluo JY3D (Jobin-Yvon, France), and fluorescence spectra were recorded between 550 and 850 nm after excitation at 488 nm. One milligram of SIM01 was dissolved in 1 mL of PEG 400, ethanol and water (30:20:50, v/v/v). A 10 ␮g mL−1 solution for absorption measurements, and a 100 ␮g mL−1 solution for fluorescence measurements were prepared by further dilution with isotonic saline.

Tissue detection of diphenylchlorin sensitizer (SIM01)

Optical fibre spectrofluorometer (OFS) An OFS method was used to evaluate SIM01 incorporation into different tissues [5,6]. Light (514 nm) from an argon laser was used to excite fluorescence, which was then attenuated by reflection on a glass plate and fed into a 600 ␮m core plastic-clad silica fiber with perpendicular polished end-faces, using a small 45◦ metal mirror and an achromatic lens. The distal fiber end was brought into direct contact with target tissue. This system provides simultaneous delivery of excitation light and a gathering of fluorescence light. Light leaving the fiber is imaged into a glass fiber bundle (Oriel 77402) through the above-mentionned achromatic lens, a second achromatic lens located behind the metal mirror, and a high-pass filter (Schott OG 530), which virtually eliminates back-scattered laser light. The fibre bundle converts the circular beam into a rectangular one. Convenient spectral resolution was obtained when the system was coupled to a monochromator (Jobin Yvon CP 200). Spectra were recorded using a cooled 1024-element diode array interfaced with a 386 personal computer (Jobin Yvon Quickview software). Electromechanical shutters were placed in the excitation and detection paths to avoid excessive irradiation and allow determination of the dark current of the diodes. Recording of a single spectrum required 20 ms, but the integration period covered 3 s. Power density was 0.5 mW cm−2 (514 nm) at the fibre tip.

Pharmacokinetic studies For solid tissues, organs were divided into four parts. Two parts were assigned randomly for fluorescence measurements, and the other two were used for SIM01 extraction and HPLC measurements. SIM01 (2, 5 or 15 mg kg−1 ) was injected intraperitoneally (a total of 0.2 mL for each mouse). Muscle, skin, liver, heart, urine, stool, kidney, lung, gallbladder, brain and spleen were removed and stored at −20 ◦ C until analysis, for a maximum of 20 days. Freezing had been shown to have no influence on fluorescence spectra. Fluorometric measurements were performed 4, 6, 12, 24, and 48 h after injection for a SIM01 dose of 2 and 5 mg kg−1 . Stored samples were processed in parallel for HPLC. The spectra obtained after 15 mg kg−1 doses (a concentration used only for HPLC purposes) showed no differences (except in intensity) compared to those obtained after 2 and 5 mg kg−1 , which were regarded as the doses of interest for PDT and solely presented.

183 Measurements were performed through the optical fibre placed directly in contact with the tissue. A minimum of four spectra per tissue and mouse were recorded, and at least four mice were used for each experimental condition. Thus each kinetic point was obtained from a minimum of 16 spectra. Muscle, skin, liver, heart, urine, stool, kidney, lung, gallbladder, brain and spleen were studied successively. The fluorescence emission spectra of untreated mouse tissue were recorded in three separate experiments in order to estimate intermouse variations. The fluorescence peak was observed at 640 nm. The results of spectrofluorometry are expressed in counts per second (cps), an arbitrary unit. SIM01 fluorescence intensity in each organ of treated mice was evaluated at the emission peak (640 nm). Peak values were obtained by subtracting the baseline measured in spectra recorded in the control group for each organ from fluorescence values obtained in treated mice.

Extraction of SIM01 before HPLC analysis [7—9] Samples from mice given 2, 5 or 15 mg kg−1 had been processed by HPLC. 15 mg kg−1 dose had been used to make sure that metabolites did not appear even at high SIM01 doses. The following procedure was used for all tissues. To avoid artefactual results due to variations in sample size from different organs, each tissue was weighed and then extracted in medium with the appropriate volume. One mL of homogenizing medium (methanol:DMSO:water 32:8:1, v/v/v) was added for 100 mg of tissue. When the organ weighed less than 100 mg then the volume of homogenizing medium was proportionnally reduced. Thus, all results consist of Fluorescence or Absorbance expressed as arbitrary unit per 100 mg of tissue. The treatment with methanol/DMSO allowed simultaneous protein precipitation and drug extraction [10]. Samples were homogenized mechanically for 10 s (Ultra Turax, 24,000 rpm, Janke & Kundel, Staufen, Germany) in a glass tube. The homogenate was then centrifuged twice at 2600 × g for 10 min before the supernatant was removed and placed in another glass tube stored at 4 ◦ C until use. Twenty microlitres of supernatant were then injected into the chromatograph. Correct separation of haemin from SIM01 is crucial, as all tissues contain blood that releases haemin during homogenization. To avoid heavy haemin contamination, the surface blood of tissues

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needs to be removed by washing in distilled water prior to homogenization. Removal of blood at the tissue surface is also important, because SIM01 in blood may contribute to the tissue concentration if not removed.

High performance liquid chromatography Apparatus The Perkin-Elmer Series 200 LC pump was used together with a Perkin-Elmer Series 200 UV—vis detector (Perkin-Elmer Instruments, Courtaboeuf, France) set at 400 nm. Separation was carried out on a 100 mm × 4.6 mm SPHERI-5 RP-18 column (Perkin-Elmer Instruments, Courtaboeuf, France). Chromatographic conditions The multilinear gradient used was slightly modified from that described elsewhere [11—12]. The elution programme was started with a mixture of aqueous phosphate buffer (40 mM NaH2 PO4 , pH 5.6, in water) 55% and organic phase (12.5 mM tetrabutylammonium dihydrogen phosphate in Methanol, pH 6.6) 45% for 10 min. The organic phase was increased linearly over 5 min to 75% and then, over another 5 min, to the final concentration of 98% (an aqueous phase of 2%), which was maintained for 15 min. Finally, the column was reconditioned for 10 min with the starting mixture. The photosensitizer was quantified by an external standard containing SIM01 solutions at different concentrations. The flow rate was set at 1 mL min−1 .

Results Absorption and fluorescence spectrometry The absorption spectrum of SIM01 without foetal calf serum or human serum, obtained immediately after preparation of the solution, showed four main peaks at 417, 511, 595 and 647 nm, (Fig. 1). The fluorescence spectrum of SIM01 showed a peak at 649 nm with a shoulder between 700 and 750 nm.

SIM01 kinetics determined by optical fibre spectrofluorometer In in vivo studies, spectra were reproducible for both background and sensitizer fluorescence in a given organ. The fluorescence spectra of SIM01 homogenates from tissues showed a maximum intensity at 643 nm, with a shoulder between 700 and

Figure 1 Absorption (10 ␮g mL−1 ) and fluorescence (100 ␮g mL−1 ) spectra of SIM01 in isotonic saline solution.

750 nm (Fig. 2a—c). No fluorescence peak (except in stools) was observed at 640 nm in the absence of sensitizer administration in mouse tissues. The general profile of the fluorescence curves was similar for each tissue. We observed a maximum between 4 and 6 h, followed by a rapid decrease until 12 h, this decrease continuing until 48 h with a slight increase around 24 h. The fluorescence in the elimination organs (Fig. 3) was dose-dependent and varied according to the interval after injection. With a dose of 2 mg kg−1 , maximum fluorescence was observed 4 h after injection in liver, kidney and urinary bladder, and 6 h after injection in gallbladder and stool. For all elimination organs, maximum fluorescence was followed by a rapid decrease until 12 h, followed by a slower decrease to a value close to that of control mice after 48 h. With a 5 mg kg−1 dose, maximum fluorescence was observed 6 h after injection in liver, gallbladder, stools, kidney, and urinary bladder, followed by a rapid decrease until 12 h. Elimination seemed to concern essentially gallbladder, liver and stool. In these organs, maximum fluorescence reached respectivelly 20,000, 2800 and 15,000 counts per second (cps) for 5 mg kg−1 at 6 h after injection. In comparison, maximum fluorescence was 2300 and 10,400 cps in kidney and urinary bladder, respectively. In spleen, maximum fluorescence for the 2 mg kg−1 dose was observed 4 h after injection, reaching an intensity close to 1600 cps, followed by a rapid decrease to a lower level after 12 h, which was maintained until 48 h. With the 5 mg kg−1 dose, fluorescence was also maximum at 4 h (750 cps) and then decreased slowly. In heart and lung, the kinetic profile was similar, but fluorescence in lung was much higher. For

Tissue detection of diphenylchlorin sensitizer (SIM01)

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example, after 6 h of incubation with a dose of 5 mg kg−1 , maximum fluorescence reached 300 cps for heart and 600 cps for lung. At 2 mg kg−1 , maximum fluorescence for these organs was observed from 4 to 6 h (approximately 250 cps for heart and 700 cps for lung). The kinetic profile was similar for skin and muscle, showing maximum fluorescence of 300 cps at 5 mg kg−1 at 6 h for both. The fluorescence level for these tissues was similar to that of control mice after 48 h. In brain, no significant signal was detected for any incubation period tested.

SIM01 kinetics determined by HPLC The ion-pair reversed-phase HPLC technique allowed identification and quantification of SIM01 in mouse organs. The SIM01 peak was positively identified by co-injection of authentic solution of SIM01 mixed with an extract. Fig. 4a—c show the separation of SIM01 in heart, liver and gallbladder extracts. No interfering peaks were observed at the retention time of SIM01 when these extracts from control mice were analysed. For liver and gall bladder only, a second peak was observed ahead of the SIM01 peak. This was not detected in any of the other tissues. The sample preparation used produced clean chromatograms not obscured by interfering substances, with the haemin peak appearing after 25 min. The haemin peak was detected in spleen, liver, heart and lung. Cervical dislocation used for mice sacrifice under general anaesthetic, was responsible for the presence of blood in lung bronchioli and alveoli. SIM01 doses of 2, 5 and 15 mg kg−1 were used for mouse injections. As 2 and 5 mg kg−1 gave only tiny peaks, 15 mg kg−1 was also used to avoid mistakes in interpretation. Among the tissues examined, the highest SIM01 levels were found in stool, urine, liver, gallbladder and spleen (Fig. 5). The highest initial levels were obtained after 6 h except for heart (4 h), skin (24 h) and urine (4 h). In the brain, regardless of dose, the levels remained at the detection limit. After this initial maximum, a regular decrease was found in stools, urine, heart, skin and kidney. A second increase was clearly observed at 24 h for lung and muscle, but less apparent for liver and spleen. In gallbladder and spleen, may be due to artefacts related to secretion eliminates varying with feeding, the decrease was slower, even progressing up to 48 h. Liver, gallbladder, and stools homogenates from drug-treated animals contained an additional

Figure 2 Fluorescence emission spectra of a heart (a), a liver (b) and a gallbladder (c) extract 4 h after injection from a mouse receiving a 5 mg kg−1 sample of SIM01.

Figure 3 Fluorescence levels recorded per 100 mg of tissue in different mouse tissues 4, 6, 12, 24 and 48 h after injection of 2 mg kg−1 () and 5 mg kg-1 () of SIM01.

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Figure 3 (Continued ).

peak detectable only after injection of 15 mg kg−1 . The concentration of this unidentified compound was proportional to the SIM01 peak, being maximal

at 3 h for liver and 6 h for gallbladder and continuing to increase in stool until at least 48 h. It disappeared after 6 h in liver, remained more or less stable in gallbladder for 24 h (kinetic ‘‘accidents’’

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Figure 4 Chromatogram 4 h after injection of a heart (a), a liver (b) and a gallbladder (c) extract at 400 nm from a mouse receiving a 15 mg kg−1 sample of SIM01.

for the unidentified compound were supposedly related to gallbladder emptyings after meals), and then disappeared (Table 1). The concentration of the unidentified compound was found to be dosedependent in stool and liver, but not apparently so in gallbladder (again probably due to random bile excretion). A peak corresponding eventually to the unidentified compound was not found in fluorescence spectra from corresponding tissues.

Discussion

sitizer approved by the U.S. Food and Drug Administration. PDT research is ongoing for the design and synthesis of new sensitizers and the evaluation of their tissue pharmacokinetics in vivo, which is critical from several points of view: • First, PDT effects are induced by the light irradiation of tissues having retained a photosensitizer and these effects depend on sensitizer concentration at the time of irradiation. • Second, PDT sensitizers are non-toxic themselves. As for any medical drug, it is important to ensure that they are not metabolized into a new toxic and possibly phototoxic compound.

Although PDT was introduced in the 1970s, it was not until 1996 that Photofrin® became the first senTable 1 HPLC.

‘‘Metabolite’’/SIM01 as determined by

Time (h)

Liver

Gallbladder

Stool

3 6 12 24 48

0.11 0.08 0.00 0.00 0.00

0.16 0.24 0.04 0.22 0.00

0.01 0.06 0.01 0.02 0.78

SIM01 and a putative metabolite were extracted from liver, gallbladder and stool of drug-treated mice, and the ‘‘metabolite’’/SIM01 ratio was calculated for HPLC peaks.

Figure 5 Absorbance intensity per 100 mg of tissue at 400 nm for 18 ± 0.3% minutes retention time recorded in different mouse tissues 4, 6, 12, 24, and 48 h after injection of 2 mg kg−1 () and 5 mg kg−1 () of SIM01.

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Figure 5 (Continued ).

• Finally, the retention of photosensitizers by cancer tissues is considered to be relatively selective, which means that determination of the maximal concentration allowable in normal tissues could help avoid adverse effects during treatments.

As SIM01 is a fluorescent compound, in vivo fluorescence experiments were performed to estimate its biodistribution. If the fluorescence of a compound can be induced by exposure to photons of appropriate wavelength, it may be possible to identify a compound of interest by its characteristic

Tissue detection of diphenylchlorin sensitizer (SIM01) fluorescence emission or excitation spectrum and then monitor its synthesis and/or degradation in vivo by measuring changes in fluorescence intensity. If a sensitizer is a pure chemical, and if the active species is also the fluorescent one, then an in vivo spectrofluorometry laser technique would appear to have significant advantages over other methods. This technique is highly sensitive and can record complete fluorescence emission spectra so rapidly that it is now feasible to obtain a series of such spectra in anaesthetized or non-anaesthetized animals [6]. The spectra recorded are reproducible for both background and sensitizer in a given organ. It is important to determine tissue concentration for SIM01, which is a very powerful and potentially dangerous sensitizer for normal tissues, if irradiated accidentally. However, if the photosensitizing agent is not SIM01 but a metabolite, then interpretation of results based on SIM01 assays alone would be misleading. In addition, such as metabolite could have a specific toxicity in dark that could impair SIM01 safetyness? As SIM01 is easily identified by HPLC or fluorescence, metabolites can be distinguished from the unchanged drug. SIM01 has a chemical structure close to that of m-THPC for which tissue distribution and kinetics have been determined by OFS in our laboratory [5]. With a SIM01 dose of 2 mg kg−1 , maximum fluorescence intensities after injection were observed at 4 h in urinary bladder and 6 h in gallbladder and stool. However, with m-THPC administered at a dose close to that of SIM01 (1.6 mg kg−1 ), maximum fluorescence intensities were observed in these elimination organs 24 h after injection. With a dose of 2 mg kg−1 of SIM01, fluorescence intensity was maximal in kidney, liver, lung and spleen after 4 h. However, with an injection of 1.6 mg kg−1 of m-THPC, maximum fluorescence occurred at 72 h for kidney, lung and spleen versus 24 h for liver. In other organs, fluorescence was weak, but a maximum was obtained 6 h after injection of SIM01 and 72 h after injection of m-THPC. Fluorescence in brain was not increased after injection of either SIM01 or m-THPC as compared to untreated animals, regardless of the dose administered or the time interval. In other experiments currently in press, a period of 12 h between injection and irradiation was found to be the best time interval for SIM01, as compared to 24 or 48 h for m-THPC [4]. The faster kinetics of SIM01 seems to be related to more rapid tissue uptake and could be due to the presence of diphenyl

189 around the chlorin center, whereas m-THPC is a tetraphenyl molecule. For HPLC assays, the excitation wavelength chosen was 400 nm, i.e. where porphyrins have an intense absorption band (Soret band). Simultaneous sensitive detection of a wide range of porphyrins in biological samples could thus be achieved by setting the UV—vis detector at a compromise wavelength of 400—405 nm [13]. To avoid faulty interpretation of chromatograms, an injection of blood, collected and centrifuged at 1000 × g for 10 min at 4 ◦ C, was tested first. The haemin peak corresponded to a specific peak appearing in HPLC only in certain tissues (liver, heart, lung, kidney, spleen) 25 min after injection. Our extraction procedure allowed correct separation of haemin from SIM01, which is of critical importance as all tissues contain blood [7—9]. A mixture of methanol, DMSO and water was used to extract SIM01 from tissues. SIM01 is highly soluble in methanol, but this solvent did not allow the extraction of SIM01 from tissues as the photosensitizer is probably co-precipitated with the proteins at physiological pH. SIM01 was easily obtained after addition of DMSO, indicating that the hydrophobic tetrapyrroles can be extracted from proteins [10]. The extraction procedure with methanol, DMSO and water (38:8:2, v/v/v) did not modify SIM01 retention time as compared to native SIM01 in solution. To ensure that the peak observed at 18 ± 0.3% min was actually due to the presence of SIM01, a solution of SIM01 was added to tissue extracts for which the ‘‘suspected’’ SIM01 peak appeared or did not appear. A complete co-elution of the two peaks was observed when SIM01 was present in the extract, whereas the peak appeared 18 min after injection when no SIM01 was found in the extract (data not shown). Regardless of the organ tested, HPLC concentrations of SIM01 were very low, which excluded any possibility of truly accurate quantification, particularly for SIM01 doses found to be useful in previous in vivo PDT assays [4]. HPLC detected a new compound present in some tissues (liver, stool and gallbladder), which may have been an SIM01 metabolite. An unidentified peak (retention time: 16.7 min) was observed in liver, stool and gallbladder homogenates from drugtreated animals. The maximal concentration of this compound, whose characterisation and isolation are now in progress, varied according to the dose in liver and stool. This unidentified compound is likely to be an SIM01 metabolite. For high doses of SIM01, it could

190 appear at a limited concentration in liver (around 10% of native SIM01) and then be stored and excreted by the gallbladder before being eliminated slowly in stool where it would be concentrated due to water reabsorption. If this compound is of porphyrin structure, it may be weakly fluorescent or not fluorescent at all at the 640 nm wavelength, or its concentration may be too low to be detected in fluorescent spectra. It would seem that SIM01 2 and 5 mg kg−1 is eliminated by both the liver-bile pathway and the kidney-urine pathway. The relative importance of each pathway is difficult to assess, but elimination is probably similar for both. Similar findings to ours with SIM01 have been obtained with m-THPC [5], using fluorescence and HPLC methods [7—9]. m-THPC fluorescence was found in both gallbladder and urinary bladder, which implies that there are two elimination pathways [5] m-THPC was separated by HPLC in a mouse liver extract 4 h after administration. Three peaks were observed as possible metabolites, but appeared only in liver homogenates [7]. Experiments by Whelpton et al. [9] confirmed a putative metabolite in liver homogenates detectable in liver and small intestine, which suggests that, once formed, it was excreted via bile and may have undergone enterohepatic cycling (although stool analysis is not provided). This hypothesis cannot obviously fit to our observations since the unknown peak had also been found in stool. Our HPLC determinations and in vivo fluorescence detection of SIM01 gave comparable results. In particular, kinetic profiles were similar, except for that of the spleen, whereas maximal concentrations were reached between 4 and 6 h depending on the method used. Sensitivities were comparable in degree, being about 1000 times less for HPLC, regardless of organ.

S. Thibaut et al. Even though a good correlation was found between HPLC and fluorometric measurements for SIM01, these techniques should be considered as complementary rather than exclusive. The peak corresponding to the unidentified compound was found only with HPLC, whereas sensitivity was greater for fluorometry. A good preclinical pharmacokinetic analysis requires the use of both methods.

Acknowledgements The authors are grateful to Dr. J. Gray for reviewing the English text. This study was supported by grants from the ‘‘Comit´ e de Loire Atlantique de la Ligue Nationale contre le Cancer’’.

References [1] Dougherty TJ, Gomer CJ, Henderson BW, et al. J Nat Cancer Inst 1998;90:889. [2] Weishaupt KR, Gomer CJ, Dougherty TJ. Cancer Res 1976;36:2326. [3] Mitchell J, McPherson S, Degraff W, Gamson J, Zabell A, Russo A. Cancer Res 1985;45:2008. [4] Bourr´ e L, Simonneaux G, Ferrand Y, Thibaut S, Lajat Y, Patrice T. J Photochem Photobiol B: Biol 2003;69:179—92. [5] Morlet L, Vonarx-Coinsmann V, Lenz P, et al. J Photochem Photobiol B: Biol 1995;28:25. [6] Vonarx-Coinsmann V, Cordel S, Lenz P, et al. J Phys (Paris) 1994;4:199. [7] Wang Q, Altermatt HJ, Ris HB, et al. Biomed Chromatogr 1993;7:155. [8] Wang Q, Ris HB, Altermatt HJ, et al. Biomed Chromatogr 1993;7:45. [9] Whelpton R, Michael-Titus AT, Basra SS, Grahn M. Photochem Photobiol 1995;61:397. [10] Li F, Lim CK, Peters TJ. Biochem J 1987;243:863. [11] Szocs K, Gabor F, Csik G, Fidy J. J Photochem Photobiol 1999;50:8. [12] Luppa P, Jacob K, Ehret W. J Med Microbiol 1993;39:262. [13] Lim CK, Li F, Peters TJ. J Chromatogr 1988;429:123.

Tissue detection of diphenylchlorin sensitizer (SIM01) by fluorescence and high-performance liquid chromatography.

Cancer is today a major problem of public health. Unfortunately, the current treatments remain still too often impotent or too heavy compared to the g...
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