Georges Wagnières

IN VIVO FLUORESCENCE SPECTROSCOPY AND IMAGING FOR ONCOLOGICAL APPLICATIONS

Keywords: Photomedicine group Reference LPAS-ARTICLE-1998-003View record in Web of Science Record created on 2007-07-20, modified on 2016-08-08

Noninvasive determination of the optical properties of two-layered turbid media

Light propagation in two-layered turbid media having an infinitely thick second layer is investigated in the steady-state, frequency, and time domains. A solution of the diffusion approximation to the transport equation is derived by employing the extrapolated boundary condition. We compare the reflectance calculated from this solution with that computed with Monte Carlo simulations and show good agreement. To investigate if it is possible to determine the optical coefficients of the two layers and the thickness of the first layer, the solution of the diffusion equation is fitted to reflectance data obtained from both the diffusion equation and the Monte Carlo simulations. Although it is found that it is, in principle, possible to derive the optical coefficients of the two layers and the thickness of the first layer, we concentrate on the determination of the optical coefficients, knowing the thickness of the first layer. In the frequency domain, for example, it is shown that it is sufficient to make relative measurements of the phase and the steady-state reflectance at three distances from the illumination point to obtain useful estimates of the optical coefficients. Measurements of the absolute steady-state spatially resolved reflectance performed on two-layered solid phantoms confirm the theoretical results.

Photodetection of early human bladder cancer based on the fluorescence of 5-aminolaevulinic acid hexylester-induced protoporphyrin IX: a pilot study

Exogenous administration of 5-aminolaevulinic acid (ALA) is becoming widely used to enhance the endogenous synthesis of protoporphyrin IX (PpIX) in photodynamic therapy (PDT) and fluorescence photodetection (PD). Recently, results have shown that the chemical modification of ALA into its more lipophilic esters circumvents limitations of ALA-induced PpIX like shallow penetration depth into deep tissue layers and inhomogeneous biodistribution and enhances the total PpIX formation. The present clinical pilot study assesses the feasibility and the advantages of a topical ALA ester-based fluorescence photodetection in the human bladder. In this preliminary study 5-aminolaevulinic acid hexylester (h-ALA) solutions, containing concentrations ranging from 4 to 16 mM, were applied intravesically to 25 patients. Effects of time and drug dose on the resulting PpIX fluorescence level were determined in vivo with an optical fibre-based spectrofluorometer. Neither local nor systemic side-effects were observed for the applied conditions. All conditions used yielded a preferential PpIX accumulation in the neoplastic tissue. Our clinical investigations indicate that with h-ALA a twofold increase of PpIX fluorescence intensity can be observed using 20-fold lower concentrations as compared to ALA.

5-Aminolevulinic acid and its derivatives: physical chemical properties and protoporphyrin IX formation in cultured cells

Protoporphyrin IX (PpIX) is used as a fluorescence marker and photosensitizing agent in photodynamic therapy (PDT). A temporary increase of PpIX in tissues can be obtained by administration of 5-aminolevulinic acid (ALA). Lipophilicity is one of the key parameters defining the bioavailability of a topically applied drug. In the present work, octanol-water partition coefficients of ALA and several of its esters have been determined to obtain a parameter related to their lipophilicity. The influence of parameters such as lipophilicity, concentration, time, and pH value on PpIX formation induced by ALA and its esters is then investigated in human cell lines originating from the lung and bladder. ALA esters are found to be more lipophilic than the free acid. The optimal concentration (c(opt), precursor concentration at which maximal PpIX accumulation is observed) is then measured for each precursor. Long-chained ALA esters are found to decrease the c(opt) value by up to two orders of magnitude as compared to ALA. The reduction of PpIX formation observed at higher concentrations than c(opt) is correlated to reduced cell viability as determined by measuring the mitochondrial activity. Under optimal conditions, the PpIX formation rate induced by the longer-chained esters is higher than that of ALA or the shorter-chained esters. A biphasic pH dependence on PpIX generation is observed for ALA and its derivatives. Maximal PpIX formation is measured under physiological conditions (pH 7.0-7.6), indicating that further enhancement of intracellular PpIX content may be achieved by adjusting the pharmaceutical formulation of ALA or its derivatives to these pH levels.

Organometallic Ruthenium(II) Arene Compounds with Antiangiogenic Activity

The antimetastatic ruthenium(II) compounds [Ru(η(6)-p-cymene)Cl(2)(PTA)] (PTA = 1,3,5-triaza-7-phosphaadamantane) (RAPTA-C) and [Ru(η(6)-toluene)Cl(2)(PTA)] (RAPTA-T), as well as their analogues [Ru(η(6)-p-cymene)Cl(2)(DAPTA)] (DAPTA = (3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane)) (DAPTA-C) and [Ru(η(6)-toluene)Cl(2)(DAPTA)] (DAPTA-T), respectively, were tested in in vitro bioassays for endothelial cell function. All compounds showed low toxicity profiles and similar dose-dependent antiproliferative effects in endothelial cells at ≥100 μg/mL (∼200 μM). EC migration, measured 6 h after drug exposure, was also efficiently inhibited (ED(50) of ∼300 μg/mL, ∼500 μM, for all compounds). Since no cytostatic effect was noted, the inhibition of proliferation was considered mainly to consist of antiangiogenic activity. RAPTA-T and DAPTA-C were also tested in vivo in the chicken chorioallantoic membrane (CAM) assay and found to inhibit CAM development. Importantly, effective prevention of revascularization of the CAM after vaso-occlusive photodynamic therapy was observed. The reported ruthenium complexes show promising antimetastatic activity involving inhibition of angiogenesis and therefore are attractive agents for development of anticancer therapies based on combination of chemo- and angiostatic treatments.

Photodetection and photodynamic therapy of "early" squamous cell carcinomas of the pharynx, oesophagus and tracheo-bronchial tree

The efficacy of photodynamic therapy (PDT) alone was evaluated on 41 ‘early’ squamous cell carcinomas of the pharynx (10), oesophagus (15) and tracheo-bronchial tree (16). All lesions but two were synchronous second primaries in ENT-patients suffering from a more extensive cancer, governing the overall oncological prognosis.Photofrin I (3 mg/kg) or Photofrin II (2 mg/kg) were injected 72 h prior to the red light irradiation, supplied by an argon pumped dye laser. A diffusing cylinder was used to obtain a homogeneous light distribution at the tumour site (60 J to 150 J/cm2). In the oesophagus and bronchi, the results are good for cancers staged in situ or microinvasive at endoscopy (two recurrencies for 23 lesions treated). For more advanced cancers (submucosal in the oesophagus or invading the bronchial cartilage), the results are less satisfactory (three recurrencies for eight lesions treated). In the pharynx where light dosimetry is more difficult, the rate of recurrencies is higher (3/10 lesions treated). In the bronchi (one case) and oesophagus (one case), the longest disease-free survival is now 5 years.The irradiation of a non-cancerous zone of normal buccal mucosa on 25 patients having received HPD showed necrosis in all cases with light doses as low as 50mW/cm2 for 20 min (60 J cm−2), even with Photofrin II.We encountered six complications (three cicatricial stenosis, two fistulae, one severe sunburn), most of them resulting from the lack of selectivity of HPD. According to these experiments, PDT is efficient at destroying early squamous cell carcinomas in the pharynx, oesophagus and bronchi, but the tumour selectivity of HPD is poor in the digestive tract lined with squamous cell epithelium. The only hope for the future lies in the synthesis of a more selective and more stable photosensitizer. This discussion reviews possible directions of research for the development of new dyes (cationic dyes, dyes attached to monoclonal antibodies, etc), for PDT and hyperthermia, for photodetection of early cancers using a fluoro-endoscope, and finally, for tumour depth profiling in hollow organs using lasers of different wavelengths.

Clinical evaluation of a method for detecting superficial transitional cell carcinoma of the bladder by light-induced fluorescence of protoporphyrin IX following topical application of 5-aminolevulinic acid: Preliminary results

In bladder cancer, conventional white light endoscopic examination of the bladder does not provide adequate information about the presence of “flat” urothelial lesions such as carcinoma in situ. In the present investigation, we examine a new technique for the photodetection of such lesions by the imaging of protoporphyrin IX (PpIX) fluorescence following topical application of 5‐aminolevulinic acid (ALA).

Antibody–fluorescein conjugates for photoimmunodiagnosis of human colon carcinoma in nude mice

To improve the detectability of tumors by light‐induced fluorescence, the use of monoclonal antibodies (MoAb) as carriers of fluorescent molecules was studied. As a model for this approach, the biodistribution of an anticarcinoembryonic antigen (CEA) MoAb coupled to fluorescein was studied in mice bearing a human colon carcinoma xenograft. In vitro, such conjugates with fluorescein‐MoAb molar ratios ranging from four to 19, doubly labeled with 125I, showed more than 82% binding to immobilized CEA. In vivo, conjugates with a fluorescein–MoAb molar ratio of ten or less resulted in a tumor uptake of more than 30% of the injected dose of radioactivity per gram tumor at 24 hours. Tumor to liver, kidney, and muscle ratios of 20, 30 and 72, respectively, were obtained 48 hours after injection of the 125I‐MoAb–(fluorescein)10 conjugate. The highest fluorescence intensity was always obtained for the tumor with the anti‐CEA MoAb conjugate; whereas in control mice injected with fluoresceinated control immunoglobulin G1, no detectable increase in tumor fluorescence was observed. To compare these results with a classically used dye, mice bearing the same xenografts received 60 μg of Photofrin II. The intensity of the fluorescence signal of the tumor with this amount of Photofrin II was eight times lower than that obtained after an injection of 442 ng of fluorescein coupled with 20 μg of MoAb, which gave an absolute amount of fluorescein localized in the tumor of up to 125 ng/g of tumor. These results illustrate the possibility of improving the specificity of in vivo tumor localization of dyes for laser‐induced fluorescence photodetection and phototherapy by coupling them to MoAb directed against tumor markers.

Clinical pharmacokinetic studies of photofrin by fluorescence spectroscopy in the oral cavity, the esophagus, and the bronchi

Background. To optimize photodynamic therapy (PDT) and photodetection of cancer, two important variables that must be considered are the uptake of the dye and the dye contrast between normal and neoplastic tissue after injection.

Spectroscopic studies of photobleaching and photoproduct formation of meta(tetrahydroxyphenyl)chlorin (m-THPC) used in photodynamic therapy. The production of singlet oxygen by m-THPC

Meta(tetrahydroxyphenyl)chlorin (m-THPC) is a new photosensitizer currently undergoing clin. trials at Lausannes CHUV hospital for photodynamic therapy (PDT) of early cancer in the upper aerodigestive tract. The illumination of m-THPC with light at 650 nm in aq. soln. contg. 10% fetal calf serum (FCS) causes two simultaneously occurring processes: its photodegrdn. and the formation of a more stable photoproduct absorbing at 320 nm. The photodegrdn. quantum yield (FPb) of m-THPC is found to be of the order of 1.5*10-5 in 10% FCS. A strong dependence on oxygen concn. of the photodegrdn. and the formation of photoproducts has been obsd. Indeed, the m-THPC presents rather low FPb under N2-satd. conditions: 6.9*10-6. In aerobic conditions, the photodegrdn. as well as the formation of photoproducts, have been competitively inhibited by known singlet oxygen (1O2) quenchers. The addn. of superoxide dismutase (SOD), catalase or desferal, known quenching agents of type I mechanisms, has little or no effect on the rate of photobleaching and photoproduct formation of m-THPC. m-THPC generates 1O2 with a quantum yield of 0.3 in ethanol soln. as detd. by photo-oxidn. expts. using 1,3-diphenylisobenzofuran (DPBF) as substrate. The rate and quantum yield of DPBF photo-oxidn. are found to increase with increasing substrate concn. and decrease in phosphate buffer soln. (FD=0.01), due to the partially hydrophilic character of m-THPC. In addn., the reaction of 1O2 with TEMP (2,2,6,6-tetramethyl-4-piperidone) in combination with ESR (EPR) detection has been used to det. the formation of 1O2 by m-THPC in ethanol soln.