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Fluorescence Spectroscopy of Exogenous, Exogenously-Induced and Endogenous Fluorophores for the Photodetection and Photodynamic Therapy of Cancer

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Fluorescence Spectroscopy of Exogenous, Exogenously-Induced and Endogenous Fluorophores for the Photodetection and Photodynamic Therapy of Cancer FLUORESCENCE SPECTROSCOPY OF EXOGENOUS, EXOGENOUSLY-INDUCED AND ENDOGENOUS FLUOROPHORES FOR THE PHOTODETECTION AND PHOTODYNAMIC THERAPY OF CANCER. Thèse présentée au Département de Génie Rural Ecole Polytechnique Fédérale de Lausanne par Matthieu Zellweger Jury: Dr Georges Wagnières, rapporteur Prof. Hubert van den Bergh, corapporteur Dr Christian Depeursinge, corapporteur Prof. René Salathé, corapporteur Prof. Philippe Monnier, corapporteur Prof. Stanley Brown, corapporteur Lausanne, Février 2000 2 Foreword This work is the final report about my research activity as a Ph.D. student in the LPAS laboratory of Prof. Hubert van den Bergh, at the Swiss Federal Institute of Technology - Lausanne (EPFL). Some parts of this report have been published or submitted during the course of my Ph.D. thesis. I therefore included the text of the publications instead of an original chapter whenever relevant. In such a case, the reference is clearly stated. A brief introduction always precedes the resulting 'chapters'. Due to this inclusion, the reader might find some redundant information within this report. On the other hand, it should be emphasized that each chapter can be read as a stand-alone text and need not be linked to any other part of the whole work. However, I tried to avoid the unnecessary repetition of information and this is especially true for the references. Whenever the reader feels that a reference should have been included to support an information in the introduction, they should check the introduction of the following published chapter for a more detailed reference list. The same criticism applies to the introduction and, again, the reader should check the published section of a chapter if they feel that it lacks some information. As will be seen in the authors' list of the published chapters of this work, a lot has been done in close collaboration with other people. Whenever relevant (i.e. whenever I don't appear as the first author of a paper that has been included in my report), I stated in the introduction what my exact contribution was. It should be understood that those papers have been included in my report with full knowledge and agreement from my co-authors and Ph.D. supervisors. Finally, I would like to apologize to the native English speaking reader for my less than perfect command of their language. Lausanne, December 1999 3 To my parents, for their care and support. To Elena, my companion during these years as a Ph.D. student. Thesis A giant leap for a man A small step for mankind Lausanne, November 1999 4 Abbreviations List of abbreviations used throughout the text in alphabetical order: 2I-EtNBS 2-iodo-5-ethylamino-9-diethylaminobenzo[a]phenothiazinium chloride AF Autofluorescence ALA 5-Aminolaevulinic acid, ∂-Aminolaevulinic Acid AMD Age-related macular degeneracy BCC Basal cell carcinoma BPDMA Benzoporphyrin derivative monoacid ring A BSA Bovine serum albumin CCD Charge-coupled device CFDA Carboxyfluorescein diacetate CIS Carcinoma in situ CNV Choroidal neovascularization CT Computerized tomography ENT Ear, nose, throat EtNBA 5-ethylamino-9-diethylaminobenzo[a]phenoxazinium chloride EtNBS 5-ethylamino-9-diethylaminobenzo[a]phenothiazinium chloride FAD Flavin adenine dinucleotide (oxidized form) FDA Food and Drug Administration FMN Flavin mononucleotide FP Fluorophore FWHM Full width at half-maximum GI Gastro-intestinal (tract) h-ALA 5-Aminolaevulinic acid hexylester HPD Hematoporphyrin Derivative HPLC High performance liquid chromatography LIF Light-Induced fluorescence Lu-Tex Lutetium Texaphyrin MP Molecular Probes mTHPC 5,10,15,20-tetra(m-hydroxyphenyl)chlorin, Foscan® MRI Magnetic resonance imaging NADH Nicotinamide adenine dinucleotide (reduced form) NADPH Nicotinamide adenine dinucleotide phosphate (reduced form) PBS Phosphate buffered saline PDT Photodynamic therapy PET Positron emission tomography 5 Phe Phenylalanine PK Pharmacokinetics PPIX Protoporphyrin IX PPV Positive Predictive Value PS Photosensitizer RPE Retinal pigmented epithelium RuDPP Ruthenium-(4,7-diphenyl-1,10-phenanthroline) SCC Squamous cell carcinoma SNR Signal to noise ratio SPECT Single photon emission computed tomography Trp Tryptophane Tyr Tyrosine UADT Upper aerodigestive tract 6 Abstract The purpose of this thesis is to study different applications of fluorescence spectroscopy and imaging to detect early cancerous lesions in two organs: the bronchi and the urinary bladder. Early diagnosis of epithelial carcinomas is of major importance because the risk of the tumor infiltrating the underlying tissues and eventually spreading throughout the host increases as the lesion develops. Endoscopy is a central tool in achieving this goal as it facilitates access to hollow organs. The additional use of fluorescence spectroscopy provides otherwise unavailable information about the histopathological status of the tissue. Fluorescence spectroscopy requires a fluorophore. The fluorophores can be of three types, the exogenous (synthesized out of the patient's body), the exogenously induced (synthesized in the patient's body from exogenous precursors) and the endogenous fluorophores (synthesized in the patient's body). This work has consequently been divided in three parts according to the types of fluorophore. The exogenous fluorophore mTHPC was used in clinical routine in Lausanne as a photosensitizer for the photodynamic therapy of early carcinomas in the esophagus and the bronchi. Strong interpatient fluctuations in therapeutic response have been observed in patients treated with similar doses of mTHPC and light. This is thought to be due to metabolic differences between patients leading to various concentrations of mTHPC in the target organ. To some extent, this can be compensated by adjusting the light dose for each patient, according to the tissular mTHPC concentration. Several methods can provide this information, among them fluorescence spectroscopy. The usual method in Lausanne is to measure the mTHPC fluorescence level with an optical fiber on the target organ. This can be a difficult measurement, because the operator has limited control upon the fiber. We propose a simplified way of measuring this concentration in the oral cavity instead of on the target organ. This location is relevant because it is easily accessible and because there is a significant correlation between the mTHPC fluorescence signal in the bronchi or in the esophagus, and in the oral cavity. Five patients undergoing PDT were included in this study. The mTHPC fluorescence signal was measured in their oral cavity in the two hours preceding PDT. It was found that this signal is fairly reproducible and little sensitive to the exact location in the oral mucosa. It is not very sensitive to the pressure applied to the fiber either. This measurement location is easier to access than the bronchi or the esophagus, yields more reproducible results and yet does not result in any loss of useful information. The exogenous fluorophores Lu-Tex, EtNBS and 2I-EtNBS (the last two being Nile Blue derivatives) have been tested as potential tumor detection agents in a hamster animal model bearing an early cancerous lesion similar to those encountered in the human upper aero-digestive tract. The Lu-Tex and the Nile Blue derivatives were expected to localize in greater quantity in the lesion than in the surrounding healthy tissue. The fluorescence spectroscopy allowed us to measure a pharmacokinetics curve in both types of tissue and to evaluate the selectivity of these molecules for early lesions of the hamster. Twenty-six animals received a 12 mg/kg intracardiac injection of a Lu-Tex solution. The pharmacokinetics curve showed that Lu-Tex displays some selectivity (1.5:1) in this model. Moreover, it seems not to be detectable in the skin of the animals after 24 hours and this observation was confirmed by skin irradiation tests. This is a clear advantage of Lu-Tex over fluorophores such as mTHPC or HPD. A similar study was conducted with EtNBS and 2I-EtNBS on 30 hamsters. Despite promising preliminary results (3:1 selectivity 100-200 min after the injection of 2.5 mg/kg of 2I-EtNBS), this selectivity could not be reproduced. Sytox® Green is an impermeant metachromatic dye. This category of fluorophore represents a different type of approach to cancer detection as it does not rely on the accumulation of different quantities of fluorophore in the healthy or cancerous cells at a given point in time. Instead, it is sensitive to local environmental factors (in this case, the permeability of the membrane) that differ between the two types of cell. The fluorescence quantum yield of Sytox® Green increases 1000- fold when bound to DNA, an event likely to occur in cancer cells with damaged membranes. 7 Since Sytox® Green does not cross intact membranes, it will not bind to DNA in healthy cells. This fluorophore was injected intracardiacally into 9 hamsters (0.3 mg/kg). No pharmacokinetics curve could be measured, but images have been taken. They display contrasts (up to 10:1) between healthy and early cancerous cells. Moreover, it seems that there is a correlation between the histopathological status of the tissue and the position of the emission maximum of Sytox® Green. The exogenously induced fluorophore Protoporphyrin IX (PPIX) is synthesized in mammals from 5-aminolevulinic acid (ALA) among others. This highly fluorescent fluorophore is the immediate metabolic precursor of heme in its biosynthetic pathway. When in contact with an aqueous solution of ALA, cancer cells tend to temporarily
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