Assessment of a sheep animal model to ... - Wiley Online Library

Lasers in Surgery and Medicine 41:643–652 (2009)

Assessment of a Sheep Animal Model to Optimize Photodynamic Therapy in the Oesophagus Thomas M. Glanzmann, PhD,1 Matthieu P.E. Zellweger, PhD,1 Franc¸ois Borle, PhD,1 Ramiro Conde, PhD,1 Alexandre Radu, MD,2 Jean-Pierre Ballini, PhD,1 Yves Jaquet, MD,2 Raphae¨lle Pilloud, MD,2 Hubert van den Bergh, PhD,1 Philippe Monnier, MD,2 Snezana Andrejevic-Blant, MD,3 and Georges A. Wagnie`res, PhD1* 1 Institute of Chemical Sciences and Engineering, Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland 2 Department of Otolaryngology, Head and Neck Surgery, CHUV Hospital, Faculty of Biology and Medicine, University of Lausanne, CH-1011 Lausanne, Switzerland 3 Institute of Pathology, CHUV Hospital, Faculty of Biology and Medicine, University of Lausanne, CH-1011 Lausanne, Switzerland

Background and Objectives: Precursor lesions of oesophagus adenocarcinoma constitute a clinical dilemma. Photodynamic therapy (PDT) is an effective treatment for this indication, but it is difficult to optimise without an appropriate animal model. For this reason, we assessed the sheep model for PDT in the oesophagus with the photosensitiser meta-(tetra-hydroxyphenyl) chlorin (mTHPC). Materials and Methods: Twelve sheep underwent intravenous mTHPC injection, blood sampling and fluorescence measurements. mTHPC’s pharmacokinetics was measured in vivo and in plasma by fluorescence spectroscopy. Biopsies of sheep oesophagus were compared to corresponding human tissue, and the mTHPC’s biodistribution was studied under fluorescence microscopy. Finally, the sheep oesophageal mucosa was irradiated, 4 days after mTHPC’s injection. Results: Histologically, the sheep and human oesophagus were closely comparable, with the exception of additional fatty tissue in the sheep oesophagus. mTHPC’s pharmacokinetics in sheep and human plasmas were similar, with a maximum of concentration in the sheep 10 hours after i.v. injection. mTHPC’s pharmacokinetics in vivo reached its maximum after 30–50 hours, then decreased to background levels, as in humans under similar conditions. Two days after injection, mTHPC was mainly distributed in the lamina propria, followed by a penetration into the epithelium. The sheep and human tissue sensitivity to mTHPC PDT was similar. Conclusion: In conclusion, this model showed many similarities with humans as to mTHPC’s plasma and tissue pharmacokinetics, and for tissue PDT response, making it suitable to optimise oesophagus PDT. Lasers Surg. Med. 41:643–652, 2009. ß 2009 Wiley-Liss, Inc. Key words: cancer; fluorescence; mTHPC; PDT; photodynamic therapy; photosensitiser; sheep model; spectroscopy ß 2009 Wiley-Liss, Inc.

INTRODUCTION Photodynamic therapy (PDT) is a treatment modality for certain cancers and other non-oncological diseases [1–5]. It is minimally invasive, provoking little trauma and few side effects, especially in comparison to more radical oncological treatments. Thus, it is particularly suitable for patients who are in critical general condition, or for patients with early, often non-symptomatic, lesions, in whom the side effects of more aggressive treatments would be unnecessarily heavy [6]. For these reasons, amongst others, PDT is an interesting alternative for the treatment of early cancers in the oro-pharynx, the gastrointestinal tract and bronchi [2,7]. The benefit of PDT for advanced tumours of the oesophagus is less clear-cut, especially since any beneficial effect is counterbalanced by the induced skin photosensitisation [8,9]. However, there is a need for a minimally invasive, curative treatment of early stage squamous cell carcinoma of the oesophagus. This is increasingly important because of the frequent field cancerisation of this malignancy [4], and because of the very poor survival prognosis once the cancer infiltrates the submucosa of the oesophagus [10]. Fortunately PDT is an effective and safe treatment of these early malignancies [11–14], in particular for lesions on Barrett’s oesophagus. An increase of the prevalence of adenocarcinomas in the oesophagus has been observed during the last years in the Abbreviations: DLI, drug-light interval; mTHPC, meta-(tetrahydroxyphenyl) chlorin; PDT, photodynamic therapy; PS, photosensitiser; FWHM, full width at half maximum; PK, pharmacokinetics. Contract grant sponsor: Swiss National Science Foundation; Contract grant number: 205320-116556. *Correspondence to: Georges A. Wagnie`res, PhD, Institute of Chemical Sciences and Engineering, Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland. E-mail: [email protected] Accepted 1 September 2009 Published online 15 October 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/lsm.20844

644

GLANZMANN ET AL.

western world [15,16], potentially due to the prevalence of Barrett’s oesophagus, that can evolve from columnar cell metaplasia into dysplasia [17,18]. Considering the poor survival rate of advanced adenocarcinoma of the oesophagus, there is a clinical dilemma as to how to handle these dysplasias to avoid the development of oesophageal cancer. Oesophagectomy with its substantial mortality and morbidity rate is very unsatisfying as a preventive measure and regular surveillance is expensive and does not offer a definitive solution to the patient. Therefore, endoscopic ablative techniques are under investigation as minimally invasive alternatives [19–23]. Although several such therapies seem to be able to eradicate dysplasia and to replace at least partially Barrett’s metaplasia, Photofrin1 or 5-ALA PDT seem to have an advantage in terms of control and efficiency of the treatment, especially in the case of extended circumferential lesions [20,24–27]. On the other hand, some other studies show that the treatment of Barrett’s oesophagus with 5-ALA PDT is still insufficient for a complete eradication of Barrett’s epithelium [28,29]. This is problematic since persistent Barrett’s oesophagus is considered to be at risk of progression to adenocarcinoma [30]. Because the phototherapeutic action in PDT is an interplay between the photosensitiser (PS), its vehicle and its specificity for the targeted tissue, and the light parameters, success depends on a large number of factors such as the amount of injected drug, the intensity, dose and wavelength of the PS excitation light, as well as the druglight interval (DLI). The optimisation of these parameters in the clinic is very slow for ethical reasons, since effective treatment without side effects is required. Thus, there is a need for an animal model, which mimics as closely as possible the mucosa of the human oesophagus. We have already investigated the hamster cheek pouch model with a chemically induced squamous cell carcinoma [31]. Although this model shows a number of similarities with the corresponding human mucosa [32], it shows some limitations [31]. For instance, the difference in size, of the oesophagus in particular, between hamsters and humans, makes it difficult to investigate some aspects of PDT. One such aspect is the study of the depth of light penetration in the tissue. This is a central parameter because it determines, in part, the depth of tissue destruction. This is especially important in the case of the oesophagus to find the optimal ‘balance’ between a desired complete eradication of the lesion, and the unwanted risk of stenosis and/or perforation of the oesophageal wall, which may lead to tracheo-bronchial or mediastinal fistulae [12,14,33]. Most PSs currently under investigation or approved for clinical use allow their photoactivation by irradiation at different wavelengths with different penetration depths. Therefore, the use of less penetrating wavelengths than the red light were proposed to improve the safety of PDT in the oesophagus while preserving its efficacy [33,34]. Moreover, if the light could be overdosed in a safe way, to achieve a minimal tissue response without risking complications, PDT could be substantially simpli-

fied and would avoid the current inter-individual fluctuations of the response due to the variability of the drug concentration in the target tissue [35]. The oesophageal mucosa of the sheep oesophagus is histologically comparable to that of the human oesophagus both in terms of histology and thickness [36–39]. We used this model to determine the optimal illumination wavelength to improve the safety of PDT [37,40]. In the study presented here, we assessed the validity of this animal model in terms of biodistribution and pharmacokinetics of meta(tetra-hydroxyphenyl) chlorin (mTHPC), the active compound of Foscan1, its approved formulation commercialised for the treatment of carcinoma in the oral cavity, by comparing the results with the known pharmacokinetics (PK) and biodistribution of this PS in humans. MATERIALS AND METHODS Photosensitiser The PS mTHPC was kindly provided by QuantaNova, Ltd (Guildford, UK), a company that sold its assets, including its PDT technology, to Biolitec AG (Jena, Germany). It was obtained as a powder and prepared for injection by dissolving 20 mg of dry powder in 5 ml of a mixture of H2O, polyethylene glycol 400 and ethanol (5:3:2 vol/vol/vol). A drug dose of 0.15 mg per kg bodyweight was administered to the sheep by injection in the jugular vein. This dose corresponds to that injected to the patients for mTHPC PDT in the clinic. Animal Model Twelve sheep (Swiss White Alpine) were selected with a weight ranging between 40 and 90 kg. They were kept in a farm (Farm Eric Pavillard, Orny, Switzerland) and were brought to the laboratory only for PS administration, regular blood sampling and endoscopic measurements. The mTHPC fluorescence spectra measured in vivo showed interference from endogenous tissue fluorophores, called autofluorescence, and from the food given to the animals. The usual nutrition of a sheep living in a farm includes a mixture of corn leaves, alfalfa, cereals and hay. The corn leaves and, to a lesser degree, the alfalfa, show a very high and sharp emission peak at 660 nm upon irradiation at 420 nm. Therefore, a diet consisting of hay and cereals was given to the animals a few days before and until the end of the fluorescence measurements to minimise the exposure of the mucosa to these contaminants. In addition, a systematic cleaning of the mucosal surface prior to the measurements was performed. The sheep received a xylesine injection as premedication to relax the animal before blood sampling or fluorescence measurements in the oral cavity. The fluorescence measurements were then carried out by placing the tip of the optical fibre-based spectrofluorometer described below in gentle contact with the oral mucosa. During the measurements in the oesophagus, the animal was under anaesthesia, and the optical fibre was passed through the biopsy channel of the oesophagoscope and brought in contact with the mucosa. All experiments were realised in accordance

ASSESSMENT OF A SHEEP ANIMAL MODEL

with protocols approved by the local Ethics Committee for animal experiments of the CHUV hospital. In Vivo Fluorescence Pharmacokinetics Measurements The pharmacokinetics of mTHPC in the oral mucosa and in the oesophagus of the sheep were measured in vivo by light-induced fluorescence spectroscopy. These measurements were performed with an optical fibre-based spectrofluorometer that has previously been described in detail [41,42]. Briefly, the excitation light of a high-pressure xenon lamp was spectrally filtered by a spectrograph and coupled into an optical fibre. This fibre was held in gentle contact with the tissue under investigation. The emitted fluorescence of the tissue was collected by the same fibre, separated from the excitation path by a dichroic filter and spectrally dispersed by a second spectrograph. The fluorescence spectrum was recorded by a Peltier-cooled CCD camera and processed by a microcomputer. A fluorescence standard (Rhodamine B, 1 mM in ethanol) was used to take into account possible variations of the excitation intensity and of the light collection efficiency of the optical system. The absorption peak of mTHPC at 415 nm was chosen as excitation wavelength in order to be sensitive to the uppermost layers of the mucosa [43,44]. The spectrum of the tissue autofluorescence, obtained before the mTHPC administration, was subtracted from the recorded spectrum and the height of the peak at 652 nm of the mTHPC fluorescence was used as a relative ‘measure’ of the PS concentration. This value was corrected for the signal of the fluorescence standard. The fluorescence units obtained in this way are termed relative units (r.u.) in the following figures. For each measurement performed in the oral cavity, 16 spectra were recorded by slightly shifting the fibre on the mucosa in order to reduce the influence of local inhomogeneities. Between each group of measurement, the fibre tip was cleaned with ethanol. The given relative PS fluorescence was defined as the mean value of these measurements, and is reported together with the corresponding standard deviations in the following figures. The in vivo tissue pharmacokinetics of mTHPC was investigated in six sheep during 4 days. The fluorescence spectra of the endogenous fluorophores of the oesophageal mucosa and in the oral cavity were comparable in shape and in intensity and very stable. The maximum of this autofluorescence was situated at around 520 nm and the intensity decreased for longer wavelengths. Because the shape of these autofluorescence spectra was always very stable, it could be subtracted from the measured spectra as described in detail by Braichotte et al. [45]. Plasma Pharmacokinetics of mTHPC The plasma pharmacokinetics of mTHPC was obtained from three animals. Blood samples were collected between 10 minutes and 3 days after i.v. injection of the PS. The blood samples (5–7 ml) taken on the jugular vein of the sheep were collected in heparinated tubes (10 ml; Monovette Sarstedt, Nu¨ mbrecht, Germany) and directly centrifuged (10 minutes at 4,000g). The supernatants were

645

removed and stored at low temperature (48C). The mTHPC concentration in the blood plasma was determined by recording the absorbance spectra using a double beam spectrophotometer (Cary-Varian 500 Scan (Varian Australia Pty Ltd, Mulgrave, Victoria, Australia)) with a sample of plasma, obtained before the injection of mTHPC, as a reference. The calibration of the absorbance was carried out by the addition of five determined amounts of mTHPC in a plasma sample followed by successive measurements of its absorption. Histology Biopsies of both, the oral and oesophageal mucosa were performed in vivo in five animals to analyse the morphology and depth of the different tissue layers, thus providing useful information to interpret the PDT effects produced in the sheep as compared with its human counterpart. The oesophagus of three animals was excised, fixed in 5% buffered formalin (pH 7.0) and processed in order to analyses the histological structure of the different layers of the oesophageal wall, and to compare it with its human counterpart. Fluorescence Microscopy Measurements Biopsies of the oral and oesophageal mucosa were obtained in vivo at different times after i.v. injection of the mTHPC. The samples, were processed and analysed following a procedure that has been described in detail elsewhere [46]. Briefly, tissue sections were prepared in the dark to avoid photobleaching. The frozen tissue blocks were mounted in OCT medium (Tissue Tek II embedding compound BDH) and a series of sections was cut in a cryostat. From each frozen section, one or two images were recorded from two different parts of the slice, to minimise photobleaching. An Olympus BH-2 epifluorescence microscope equipped with a filtered 100 W mercury lamp as excitation light source was used to generate the fluorescence images. These images were detected with a cooled slow-scan 16-bit CCD camera (EEV P86231, Wright Instr., Endfield, Middx, UK). For excitation, an interference 420DF30 band pass filter (Omega Optical, Brattelboro, VT; transmission wavelength: 420 nm; full width half maximum (FWHM): 30 nm) and a dichroic mirror (cut-off wavelength: 470 nm) were used. A RG630 long pass filter (Schott, Mainz, Germany; cut-on wavelength: 630 nm) was used to transmit only the fluorescence of mTHPC, and a 560DF40 interference band pass filter (Omega Optical; transmission wavelength: 560 nm; FWHM: 40 nm) was used to record the tissue autofluorescence. This combination allowed the subtraction of the background autofluorescence. Using a solid sample fluorescence reference, the different measurements were normalised to allow comparison between different samples. Finally, the histological location of the PS was identified by subsequent haematoxylin and eosin staining of the slide and analysis of the corresponding tissue compartments. This approach allowed investigation of the relative PS amount in the different mucosal layers. Due to the size of the biopsies, this

646

GLANZMANN ET AL.

analysis was mainly limited to the different mucosal layers, such as the epithelium and the lamina propria. Tissue Damage After PDT in the Oesophagus of the Sheep In order to ascertain the potency of mTHPC in the sheep model, the mucosa of the oesophagus of sensitised animals was irradiated under conditions identical to the clinical treatment of humans. Three spots of the healthy oesophageal mucosa of four sheep were irradiated with light doses ranging from 10 to 150 J/cm2 at 514 nm, 4 days after injection of 0.15 mg/kg of mTHPC. This wavelength was chosen because it demonstrated a similar efficacy and a better safety for the clinical treatment of dysplasia and early carcinoma in the oesophagus [34]. The light was emitted by an argon ion laser (Spectra Physics, CA, Mountain View, model 2045) operated in the ‘single-line’ mode, and delivered at an irradiance of 100 mW/cm2 by means of a semi-rigid light distributor (Type OE15.40.180; 1808, length ¼ 40 mm, diameter ¼ 15 mm, Medlight SA, Ecublens, Switzerland). Since the diameter of the sheep oesophagus is larger than that of the human oesophagus, it was possible to attach the light distributor to a rigid oesophagoscope for visual control of the contact with the tissue. This allowed a better stretch of the oesophageal wall, thus improving the homogeneity of the illumination. The mucosal damages were assessed macroscopically

during endoscopy 1 week after PDT, as in humans, according to the previously described ‘five-grade’ damage scale described by Savary et al. [4]: 0: no reaction, 1: erythema, 2: erythema with fibrin whose extension is smaller than the irradiated spot, 3: fibrinous reaction equal to the irradiated spot size, 4: fibrinous surface larger than that of the irradiated spot size. RESULTS Histology of the Sheep Oesophagus The different layers of the sheep oesophageal wall are shown in Figure 1a (Masson’s Trichrome stained tissue section). The morphology of the sheep oesophagus is comparable to that of humans (Fig. 1b). The mean thickness of the mucosa and submucosa of the sheep depends on the animal’s weight, and ranges between 1.2 and 1.8 mm (for sheep between 40 and 90 kg), which is comparable to the mean value of 1.5 mm reported for humans, depending on the thickness of fatty tissue in the submucosa [36]. However, the ratio of the thickness of the mucosa and submucosa compared to that of the muscularis propria is 1 in humans and 1.8 in sheep. Thus, with an oesophageal wall of comparable size, the mucosa and submucosa are relatively thicker in sheep than in humans. This is probably due to the presence of fatty tissue, especially in the submucosa, that is usually

Fig. 1. Histology of the sheep (a), and human (b) oesophageal wall (Masson trichrome stained tissue sections). Details (c) of the sheep epithelium.


647

observed on sheep oesophagus. The keratinisation of the upper epithelial layer in the sheep, as observed in the Figure 1c, is not usually seen in humans. In addition, the oral mucosa of the sheep shows numerous spikes, typical of ruminants, which are not observed in the oral mucosa of humans. PK of mTHPC in Sheep Plasma The PK of mTHPC in the sheep plasma after i.v. injection is shown in Figure 2. It is expressed as the mean concentration value measured in three sheep, with the error bar representing the 65% confidence interval. The mTHPC concentration in sheep plasma starts at a level of 1 mg/ml 10 minutes after injection, decreases to a minimum at 0.3 mg/ml after around 20 minutes, and then increases steadily to reach a maximum of 1.1 mg/ml at around 4–6 hours after injection. From this point on, the plasma concentration decreases over the next 24 hours, with a halflife of about 12 hours. A slower decaying component remains measurable over 70 hours, with a half-life of about 36 hours. Compared to the mTHPC plasma PK in humans [35], it shows a similar profile but with an earlier maximum of plasmatic concentration (4–6 hours vs. 10 hours after i.v. injection in humans). Pharmacokinetics of mTHPC in the Oral Mucosa The PK of the mTHPC measured in the oral mucosa of four sheep is depicted in Figure 3 in the form of mean fluorescence values expressed in relative units (r.u.). The curve of the fluorescence intensity I(t) was fitted for visual

Fig. 3. Fluorescence pharmacokinetics of mTHPC measured with an optical fibre-based spectrofluorometer in the oral cavity of the sheep. The curve is fitted for visual support only.

support only. The mTHPC fluorescence in the oral mucosa reached its maximum between 30 and 50 hours after injection, and decreased to levels comparable to the background 150 hours after injection. The subsequent clearance of the mTHPC from the mucosa was slower, with a halflife of about 50 hours after maximum concentration. The fluorescence spectra of the endogenous fluorophores of the mucosa in the oesophagus and in the oral cavity were comparable in shape and intensity. The maximum of this autofluorescence was situated at around 520 nm and the intensity decreased for longer wavelengths. Because the shape of this autofluorescence spectrum was very stable, it could be subtracted from the measured spectra as previously described [45]. The PK observed in Figure 3 is similar to that observed in humans with similar conditions [31]. Correlation Between mTHPC Fluorescence in the Oral Cavity and the Oesophagus It is important to know if the mTHPC levels in the mucosa of the oral cavity and the mucosa of the oesophagus are correlated in order to assess the usefulness of the measurements in the oral cavity for individualised PDT dosimetry in the oesophagus [40]. The values of fluorescence intensity measured in the oral cavity and in the oesophagus during the time range of 24–100 hours after injection of mTHPC do not present a clear correlation, as seen in Figure 4. This is probably because large intra- and inter-individual variations of the fluorescence intensity in the oesophagus seem to be frequent. It is interesting to note that this drawback may be solvent-dependent or formulation-dependent, as the fluorescence intensity of a modified formulation of mTHPC has the potential to predict photodynamic activity in some animal models [47].

Fig. 2. Plasma pharmacokinetics of mTHPC after i.v. injection of 0.15 mg/kg. * In sheep; } in humans (adapted from Ref. [35]).

Fluorescence Microscopy The distribution of mTHPC in the sheep oesophageal mucosa was studied by fluorescence microscopy in different

648

GLANZMANN ET AL.

mination of the mTHPC fluorescence was possible in this compartment by integrating over a ‘large’ area (diameter: 30 mm). The data points presented in Figure 6 correspond to the fluorescence mean values measured on several (5–9 microsections, depending on availability) frozen tissue sections of a sheep mucosa together with the corresponding standard deviation. The presence of mTHPC as the main fluorophore was confirmed by spectroscopic measurements. In spite of the limitation caused by the autofluorescence background as well as the inter- and intra-sheep fluctuations, Figure 6 indicates that the maximum of the mTHPC fluorescence in the lamina propria is probably between 1 and 2 days after injection, or just before that point, which is comparable to the results obtained in humans [45], and in good agreement with the PK shown in Figure 3. Fig. 4. Comparison of mTHPC fluorescence intensity measured with an optical fibre-based spectrofluorometer in the oesophagus and in the oral cavity of the sheep, 2 days after injection of 0.15 mg/kg.

Tissue Damages Upon PDT Comparison Between the Sheep and Human Oesophagus

tissue compartments 2 days after injection, as presented in Figure 5. An inhomogeneous mTHPC fluorescence was observed in the fibrovascular core of the lamina propria, probably located in small blood vessels (Fig. 5a), and a low fluorescence was detected in the underlying epithelium (Fig. 5b,c). As presented in Figure 5c, the mTHPC fluorescence was observed in the cytoplasm, whereas no fluorescence was observed in the nuclei. Although mTHPC was quite inhomogeneously distributed in the lamina propria, a semi-quantitative deter-

Four sheep were subjected to three irradiations in the oesophageal mucosa with various light doses at 514 nm, 4 days after injection of 0.15 mg/kg of mTHPC. The macroscopic tissue reaction upon oesophageal PDT is shown in Figure 7 for different light doses in sheep (open squares) and compared to that of the mucosa of humans who were treated for early intra-epithelial cancer with PDT under the same conditions (full circles; data derived from the Ref. [31]), using a macroscopic damage scale described elsewhere [4], and summarised in Tissue Damage After PDT in the Oesophagus of the Sheep Section. The results of 29 patients are presented as mean values with the corresponding standard deviations. Although the light dose range could not be extended, for obvious ethical reasons,

Fig. 5. Photomicrographs obtained by fluorescence microscopy showing the localisation of mTHPC in different parts of the sheep oesophagus, 2 days after injection. The tissue compartments observed include: the lamina propria (A), a region located between the epithelium and the underlying lamina

propria, including fibrovascular cores (B), as well as the intermediate and suprabasal epithelial layer (C): A 20 objective was used to detect these 160 mm250 mm images. a: Lamina propria. b: Epithelium with fibrovascular core of lamina propria. c: Epithelium.


649

DISCUSSION Comparison Between the Sheep and the Human Normal Mucosa in Terms of Histology and Autofluorescence

Fig. 6. Microscopic fluorescence intensity of the lamina propria measured in histological sections (average of four microsections) of the oral cavity of the sheep as a function of the delay after injection.

since this would over- or under-treat the patients, this figure shows clearly that the tissue sensitivity of sheep and humans to mTHPC PDT under the applied conditions is similar. The tissue reaction was quite comparable in function of the light dose, with a reaction level between 2 and 3 for light doses ranging between 60 and 130 J/cm2.

Fig. 7. Tissue response in the oesophagus 10 days after PDT with mTHPC at 0.15 mg/kg as a function of the light dose at 514 nm: Comparison in the sheep (&) and in human (*). The data for the human are derived from Ref. [31]. The tissue reaction scale is described in Tissue Damage After PDT in the Oesophagus of the Sheep Section.

This histological analysis of the sheep mucosa of the oesophagus and the oral cavity showed that both of them are comparable to their human counterparts in terms of structure and thickness of the different layers, although the sheep model exhibited more variability of the submucosal layer as a function of the animal’s weight. The main differences lay in the keratinisation of sheep epithelia in both organs, in the presence of spikes on the surface of the oral mucosa, and in the presence of more fatty tissue in the submucosa of the sheep than in its human counterpart. The autofluorescence spectra of the oesophagus and oral cavity mucosa of the sheep, excited at 420 nm, were comparable in shape to those of humans, with a fluorescence intensity approximately five times higher for the latter [41]. This is in agreement with the microscopic findings that the fluorescing compartments, namely the lamina propria and the submucosa, exhibited a lower autofluorescence in the sheep mucosa, partly due to the presence of non-fluorescing adipose tissues. The tissue autofluorescence of sheep fed with standard food was variable, however. As mentioned above, some components of the usual nutrition for sheep living in a farm present a very high and sharp emission peak at 660 nm upon irradiation at 420 nm and should therefore be absolutely avoided. As hay is a mixture of dry plants, different types of chlorophyll emit fluorescence between 660 and 750 nm. Finally, cereals present a significant fluorescence signal between 455 and 555 nm. As sheep are ruminants, hay cannot be bypassed, thus a diet consisting of hay and cereals was administered from a few days before, and until the end of the fluorescence measurements. Depending on the time of food intake and regurgitation of the sheep, the oesophagus and the oral cavity were more or less contaminated with food fluorophores during the measurements. The systematic cleaning of the mucosal surface prior to measurements reduced this effect, but could not totally suppress these interferences. Indeed, the mastication reduced the food to fine particles that disseminated in the whole digestive tract. Therefore, the spectra had to be checked individually for interference with food fluorophores and to be rejected if the latter were present. This variable autofluorescence/food fluorescence background interfered with the measurements of PS fluorescence intensity, thus limiting the accurate determination of the relative mTHPC concentration in sheep from a few hours up to 5 days after injection of 0.15 mg/kg. Comparison of mTHPC Plasma PK Between Sheep and Human The PK of mTHPC in the sheep plasma showed the same unusual behaviour as in humans with the formulation used in this study and by Glanzmann et al. [35]. In both species, the mTHPC concentration in the plasma first decreased, then increased, and showed a delayed maximum several

650

GLANZMANN ET AL.

hours after injection. In the case of the sheep, the maximum seemed to be reached earlier than in humans, followed by a faster clearance process. After 1 day, the mTHPC concentration in the sheep plasma was below 25% of the maximum, whereas in humans, this level is reached only after about 4 days. Lipoproteins are reported to carry mTHPC in the blood of humans [44,48,49]. This different clearance time might thus be due to a decreased binding of mTHPC to the lipoproteins in the sheep plasma, and suggests that mTHPC circulated for a shorter time in the blood vessels of the sheep [50,51]. The physiological levels measured in the sheep blood were clearly under the usual limits for humans, suggesting that the content in lipoproteins was rather low. However, the PK of mTHPC in sheep plasma mimicked that of humans better than was the case in a rodent animal model [45]. The fact that most PSs are fluorescent provides the opportunity to measure a PK in vivo in the target tissue. The correlation between mTHPC fluorescence and its concentration was verified in an earlier study [41]. Although an entire PK could not be measured in the oesophagus because each measurement would require general anaesthesia, it was expected that the PK measured in the oral cavity would be quite similar to the one in the oesophagus considering the similarity of both mucosae. If the above presented PK of mTHPC observed in the sheep is compared to that reported in humans [35], it can be stated that the sheep PK is slightly faster than the human PK, with a maximum located between 4 and 6 hours (between 10 and 12 hours in humans). With a half-life after the maximum of around 2–3 days, the clearance of mTHPC is also faster in the sheep mucosa than in the human mucosa, the latter presenting a half-life of 9 days [45]. This is in agreement with the plasma PK indicating that mTHPC circulates during a longer time in the human blood, and diffuses in the mucosa during an extended time. The intensity of the fluorescence maximum is approximately twice larger in the mucosa of humans as compared to the sheep. This indicates either somewhat higher concentrations of the PS in the human target tissue, or a tissue architectural effect. mTHPC Localisation in the Mucosa and Correlation Between Measurements in the Oral Cavity and the Oesophagus Fluorescence microscopy revealed that mTHPC was mainly located in the lamina propria of the sheep, 2 days after injection. mTHPC fluorescence in the epithelium was always considerably lower than that in the lamina propria, up to 4 days after injection (data not shown). This is in contrast to the observation made in humans, where a high uptake of mTHPC in the normal epithelium was observed 4 days after injection, with a much lower fluorescence in the lamina propria [46]. This difference might partly explain the much higher mTHPC fluorescence (up to a factor > 2 at 4 days after injection) measured in humans [32,35,37]. The fluorescence intensity measured in the lamina propria of the sheep confirmed the hypothesis that mTHPC fluorescence signal measured in vivo stems mainly from

various structure of the lamina propria in this model. One possible mechanism explaining the difference observed between the sheep model and its human counterpart regarding the mTHPC fluorescence level in the epithelium could be due to the fact that the submucosa is deeper in the sheep due to fat. Since the mTHPC is somewhat lipophilic, it is possible that its transfer to the epithelial cells might be hindered by its retention in the interstitium/adipocytes, which would explain why mucosal fluorescence levels remain much lower in the sheep than in humans. The correlation between mTHPC levels in the mucosa of the oral cavity and the oesophagus is important for the concept of individualised light dosimetry [35], and for the extrapolation to the oesophagus of pharmacokinetic data from the easily accessible oral cavity. The fluctuations of mTHPC fluorescence measured on the same site were larger than those found in humans with the same apparatus and geometry [41]. This is in agreement with the finding that mTHPC was rather heterogeneously distributed in the lamina propria of the sheep, whereas a more homogeneous distribution was found in the normal epithelium of humans [46]. This is why the fluorescence intensity of mTHPC in the sheep’s oral mucosa and the oesophagus were only weakly correlated. It should be noted that this partial correlation could also be due to variations of the keratinised layer’s thickness. Indeed, this tissue layer lining the sheep, but not the human, oesophageal wall could influence the mTHPC fluorescence excitation light. Tissue Reaction to PDT The oesophagus of the sheep was irradiated under conditions used in the clinical setting (0.15 mg/kg; DLI ¼ 4 days). In spite of the lower mTHPC fluorescence measured in sheep vs. humans at that DLI, the tissue reaction was quite comparable, with both species showing a reaction between 2 and 3 for light doses ranging between 60 and 130 J/cm2. The identical sensitivity of the sheep mucosa to PDT for lower mTHPC in vivo fluorescence may be explained by a different distribution of mTHPC in the mucosa layers of the sheep, thus inducing a relatively stronger tissue reaction upon PDT than in humans. It should be noted that this sensitivity of the sheep mucosa could also be due to different tissue optical properties as compared to human tissue. At light doses larger than 100 J/cm2, the tissue response no longer increased. This observation can be explained by the photobleaching of the PS in the mucosa, as the fluorescence of mTHPC is reduced by photobleaching to 40% of the initial intensity at light dose of 100 J/cm2 (data not shown). Concluding Remarks mTHPC is a hydrophobic PS which binds principally to HDL and LDL in the blood [52]. The sheep model showed many similarities with humans as far as mTHPC PK in the plasma and tissue, and tissue response to PDT, are concerned. The PKs in the oral mucosa were qualitatively identical, and even the unusual plasma PK of mTHPC


found in humans was reproduced in this animal model. However, there were a certain number of limitations of the model, namely the faster PK in sheep, the lower mTHPC fluorescence in the tissue, and the different preferential localisation in the epithelium and lamina propria. In spite of these differences, the PDT-induced tissue reaction was very similar in the sheep model and in humans. These elements, together with the fact that the mucosa of the sheep is very similar to that of its human counterpart in terms of histology and size, render the sheep a very interesting model for testing tissue destruction with new irradiation parameters or new PSs. We therefore used this model in another study to investigate the safe therapeutic light dose range and the possibility to limit the complications of mTHPC PDT in the oesophagus by using violet or green light irradiation [37]. Another advantage is the possibility to perform several irradiations in the sheep oesophagus at any one DLI due to the length of the sheep’s oesophagus. It should however be noted that this, in turn, requires long endoscope and light distributors. The sheep model also allows to easily measure the blood drug PK in a single animal, which is not possible with, for example small rodents. However, when investigating new PSs with the sheep model, it should be kept in mind that differences in blood lipoprotein concentrations may influence the PK. In summary, we can conclude that the sheep enables more realistic tests than small rodents to study PDT on the mucous membrane of the oesophagus.

ACKNOWLEDGMENTS The authors are grateful to QuantaNova, Ltd and Biolitec AG, for kindly providing the mTHPC and advices, to Marco Burki (CHUV), to J.-D. Horisberger (IPT-UNIL), and to the Swiss National Science Foundation grant #205320-116556.

10. 11.

12.

13.

14. 15. 16.

17. 18. 19.

20. 21. 22. 23.

REFERENCES 1. Dougherty TJ. An update on photodynamic therapy applications. J Clin Laser Med Surg 2002;20(1):3–7. 2. Hopper C, Kubler A, Lewis H, Tan IB, Putnam G. mTHPCmediated photodynamic therapy for early oral squamous cell carcinoma. Int J Cancer 2004;111(1):138–146. 3. Monnier P. Potential of photodynamic therapy in the treatment of pharyngeal tumours: The clinical point of view. J Photochem Photobiol B 1991;9(1):122–125. 4. Savary JF, Monnier P, Fontolliet C, Mizeret J, Wagnieres G, Braichotte D, van den Bergh H. Photodynamic therapy for early squamous cell carcinomas of the esophagus, bronchi, and mouth with m-tetra (hydroxyphenyl) chlorin. Arch Otolaryngol Head Neck Surg 1997;123(2):162–168. 5. Triesscheijn M, Baas P, Schellens JH, Stewart FA. Photodynamic therapy in oncology. Oncologist 2006;11(9):1034– 1044. 6. Barr H, Kendall C, Bazant-Hegemark F, Stone N. Endoscopic photodynamic therapy for oesophageal disease. Photodiagnosis Photodynamic Ther 2006;3:102–105. 7. Sheski FD, Mathur PN. Endoscopic treatment of early-stage lung cancer. Cancer Control 2000;7(1):35–44. 8. Lightdale CJ. Role of photodynamic therapy in the management of advanced esophageal cancer. Gastrointest Endosc Clin N Am 2000;10(3):397–408. 9. van Boxem AJ, Westerga J, Venmans BJ, Postmus PE, Sutedja G. Photodynamic therapy, Nd-YAG laser and electro-

24.

25.

26.

27.

28.

651

cautery for treating early-stage intraluminal cancer: Which to choose? Lung Cancer 2001;31(1):31–36. Wobst A, Audisio RA, Colleoni M, Geraghty JG. Oesophageal cancer treatment: Studies, strategies and facts. Ann Oncol 1998;9(9):951–962. Etienne J, Dorme N, Bourg-Heckly G, Raimbert P, Flejou JF. Photodynamic therapy with green light and m-tetrahydroxyphenyl chlorin for intramucosal adenocarcinoma and highgrade dysplasia in Barrett’s esophagus. Gastrointest Endosc 2004;59(7):880–889. Radu A, Wagnieres G, van den Bergh H, Monnier P. Photodynamic therapy of early squamous cell cancers of the esophagus. Gastrointest Endosc Clin N Am 2000;10(3):439– 460. Saidi RF, Marcon NE. Nonthermal ablation of malignant esophageal strictures. Photodynamic therapy, endoscopic intratumoral injections, and novel modalities. Gastrointest Endosc Clin N Am 1998;8(2):465–491. Sibille A, Lambert R, Souquet JC, Sabben G, Descos F. Longterm survival after photodynamic therapy for esophageal cancer. Gastroenterology 1995;108(2):337–344. Claydon P, Ackroyd R. Barrett’s oesophagus and photodynamic therapy (PDT). Photodiagnosis Photodynamic Ther 2004;1:203–209. Pera M. Trends in incidence and prevalence of specialized intestinal metaplasia, barrett’s esophagus, and adenocarcinoma of the gastroesophageal junction. World J Surg 2003;27(9):999–1008; discussion 1006–1008. Flejou JF. Barrett’s oesophagus: From metaplasia to dysplasia and cancer. Gut 2005;54(Suppl 1):i6–i12. Spechler SJ. Clinical practice. Barrett’s Esophagus. N Engl J Med 2002;346(11):836–842. Conio M, Cameron AJ, Chak A, Blanchi S, Filiberti R. Endoscopic treatment of high-grade dysplasia and early cancer in Barrett’s oesophagus. Lancet Oncol 2005;6(5): 311–321. Foroulis CN, Thorpe JA. Photodynamic therapy (PDT) in Barrett’s esophagus with dysplasia or early cancer. Eur J Cardiothorac Surg 2006;29(1):30–34. Gossner L. The role of endoscopic resection and ablation therapy for early lesions. Best Pract Res Clin Gastroenterol 2006;20(5):867–876. Mitton D, Ackroyd R. Photodynamic therapy in oesophageal carcinoma: An overview. Photochem Photobiol Sci 2004; 3(9):839–850. Radu A, Grosiean P, Jaquet Y, Pilloud R, Wagnieres G, van den Bergh H, Monnier P. Photodynamic therapy and endoscopic mucosal resection as minimally invasive approaches for the treatment of early esophageal tumors: Pre-clinical and clinical experience in Lausanne. Photodiagnosis Photodynamic Ther 2005;2:35–44. Gossner L, May A, Sroka R, Stolte M, Hahn EG, Ell C. Photodynamic destruction of high grade dysplasia and early carcinoma of the esophagus after the oral administration of 5-aminolevulinic acid. Cancer 1999;86(10):1921–1928. May A, Gossner L, Pech O, Muller H, Vieth M, Stolte M, Ell C. Intraepithelial high-grade neoplasia and early adenocarcinoma in short-segment Barrett’s esophagus (SSBE): Curative treatment using local endoscopic treatment techniques. Endoscopy 2002;34(8):604–610. Overholt BF, Lightdale CJ, Wang KK, Canto MI, Burdick S, Haggitt RC, Bronner MP, Taylor SL, Grace MG, Depot M. Photodynamic therapy with porfimer sodium for ablation of high-grade dysplasia in Barrett’s esophagus: International, partially blinded, randomized phase III trial. Gastrointest Endosc 2005;62(4):488–498. Overholt BF, Panjehpour M, Halberg DL. Photodynamic therapy for Barrett’s esophagus with dysplasia and/or early stage carcinoma: Long-term results. Gastrointest Endosc 2003;58(2):183–188. Hage M, Siersema PD, van Dekken H, Steyerberg EW, Haringsma J, van de Vrie W, Grool TE, van Veen RL, Sterenborg HJ, Kuipers EJ. 5-Aminolevulinic acid photodynamic therapy versus argon plasma coagulation for ablation of Barrett’s oesophagus: A randomised trial. Gut 2004;53(6):785–790.

652

GLANZMANN ET AL.

29. Kelty CJ, Ackroyd R, Brown NJ, Stephenson TJ, Stoddard CJ, Reed MW. Endoscopic ablation of Barrett’s oesophagus: A randomized-controlled trial of photodynamic therapy vs. argon plasma coagulation. Aliment Pharmacol Ther 2004; 20(11–12):1289–1296. 30. Hage M, Siersema PD, Vissers KJ, Steyerberg EW, Haringsma J, Kuipers EJ, van Dekken H. Molecular evaluation of ablative therapy of Barrett’s oesophagus. J Pathol 2005; 205(1):57–64. 31. Glanzmann T, Forrer M, Blant SA, Woodtli A, Grosjean P, Braichotte D, van den Bergh H, Monnier P, Wagnieres G. Pharmacokinetics and pharmacodynamics of tetra (m-hydroxyphenyl)chlorin in the hamster cheek pouch tumor model: Comparison with clinical measurements. J Photochem Photobiol B 2000;57(1):22–32. 32. Blant SA, Glanzmann TM, Ballini JP, Wagnieres G, van den Bergh H, Monnier P. Uptake and localisation of mTHPC (Foscan) and its 14C-labelled form in normal and tumour tissues of the hamster squamous cell carcinoma model: A comparative study. Br J Cancer 2002;87(12):1470–1478. 33. Lovat LB, Jamieson NF, Novelli MR, Mosse CA, Selvasekar C, Mackenzie GD, Thorpe SM, Bown SG. Photodynamic therapy with m-tetrahydroxyphenyl chlorin for high-grade dysplasia and early cancer in Barrett’s columnar lined esophagus. Gastrointest Endosc 2005;62(4):617–623. 34. Grosjean P, Wagnieres G, Fontolliet C, van den Bergh H, Monnier P. Clinical photodynamic therapy for superficial cancer in the oesophagus and the bronchi: 514 nm compared with 630 nm light irradiation after sensitization with Photofrin II. Br J Cancer 1998;77(11):1989–1995. 35. Glanzmann T, Hadjur C, Zellweger M, Grosiean P, Forrer M, Ballini JP, Monnier P, van den Bergh H, Lim CK, Wagnieres G. Pharmacokinetics of tetra(m-hydroxyphenyl)chlorin in human plasma and individualized light dosimetry in photodynamic therapy. Photochem Photobiol 1998;67(5):596–602. 36. Bays R, Wagnieres G, Robert D, Braichotte D, Savary JF, Monnier P, van den Bergh H. Light dosimetry for photodynamic therapy in the esophagus. Lasers Surg Med 1997; 20(3):290–303. 37. Radu A, Conde R, Fontolliet C, Wagnieres G, Van den Bergh H, Monnier P. Mucosal ablation with photodynamic therapy in the esophagus: Optimization of light dosimetry in the sheep model. Gastrointest Endosc 2003;57(7):897–905. 38. Taniguchi DK, Martin RW, Trowers EA, Dennis MB, Jr., Odegaard S, Silverstein FE. Changes in esophageal wall layers during motility: Measurements with a new miniature ultrasound suction device. Gastrointest Endosc 1993;39(2): 146–152. 39. Taniguchi DK, Martin RW, Trowers EA, Silverstein FE. Simultaneous M-mode echoesophagram and manometry in the sheep esophagus. Gastrointest Endosc 1995;41(6): 582–586. 40. Radu A, Grosjean P, Fontolliet C, Monnier P. Endoscopic mucosal resection in the esophagus with a new rigid device: An animal study. Endoscopy 2004;36(4):298–305. 41. Zellweger M, Grosjean P, Monnier P, van den Bergh H, Wagnieres G. Stability of the fluorescence measurement of

42.

43.

44. 45.

46.

47.

48.

49.

50.

51.

52.

Foscan in the normal human oral cavity as an indicator of its content in early cancers of the esophagus and the bronchi. Photochem Photobiol 1999;69(5):605–610. Zellweger M, Radu A, Monnier P, van den Bergh H, Wagnieres G. Fluorescence pharmacokinetics of Lutetium Texaphyrin (PCI-0123, Lu-Tex) in the skin and in healthy and tumoral hamster cheek-pouch mucosa. J Photochem Photobiol B 2000;55(1):56–62. Georgakoudi I, Jacobson BC, Van Dam J, Backman V, Wallace MB, Muller MG, Zhang Q, Badizadegan K, Sun D, Thomas GA, Perelman LT, Feld MS. Fluorescence, reflectance, and light-scattering spectroscopy for evaluating dysplasia in patients with Barrett’s esophagus. Gastroenterology 2001;120(7):1620–1629. Wagnieres GA, Star WM, Wilson BC. In vivo fluorescence spectroscopy and imaging for oncological applications. Photochem Photobiol 1998;68(5):603–632. Braichotte D, Savary JF, Glanzmann T, Westermann P, Folli S, Wagnieres G, Monnier P, Van den Bergh H. Clinical pharmacokinetic studies of tetra(meta-hydroxyphenyl)chlorin in squamous cell carcinoma by fluorescence spectroscopy at 2 wavelengths. Int J Cancer 1995;63(2):198– 204. Andrejevic Blant S, Grosjean P, Ballini JP, Wagnieres G, van den Bergh H, Fontolliet C, Monnier P. Localization of tetra(m-hydroxyphenyl)chlorin (Foscan) in human healthy tissues and squamous cell carcinomas of the upper aerodigestive tract, the esophagus and the bronchi: A fluorescence microscopy study. J Photochem Photobiol B 2001;61(1–2): 1–9. Tran N, Krueger T, Pan Y, Yan H, Cheng C, Altermatt HJ, Ballini JP, Borle F, Ris HB, Andrejevic-Blant S. Correlation of photodynamic activity and fluorescence signaling for free and pegylated mTHPC in mesothelioma xenografts. Lasers Surg Med 2007;39(3):237–244. Hopkinson HJ, Vernon DI, Brown SB. Identification and partial characterization of an unusual distribution of the photosensitizer meta-tetrahydroxyphenyl chlorin (temoporfin) in human plasma. Photochem Photobiol 1999;69(4):482– 488. Sasnouski S, Zorin V, Khludeyev I, D’Hallewin MA, Guillemin F, Bezdetnaya L. Investigation of Foscan interactions with plasma proteins. Biochim Biophys Acta 2005;1725(3): 394–402. Guyard-Dangremont V, Desrumaux C, Gambert P, Lallemant C, Lagrost L. Phospholipid and cholesteryl ester transfer activities in plasma from 14 vertebrate species. Relation to atherogenesis susceptibility. Comp Biochem Physiol B Biochem Mol Biol 1998;120(3):517– 525. Terpstra AH, Sanchez-Muniz FJ, West CE, Woodward CJ. The density profile and cholesterol concentration of serum lipoproteins in domestic and laboratory animals. Comp Biochem Physiol B 1982;71(4):669–673. Michael-Titus AT, Whelpton R, Yaqub Z. Binding of temoporfin to the lipoprotein fractions of human serum. Br J Clin Pharmacol 1995;40(6):594–597.