Matthieu Zellweger

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.

In vivo autofluorescence spectroscopy of human bronchial tissue to optimize the detection and imaging of early cancers

We are developing an imaging system to detect pre-/early cancers in the tracheo-bronchial tree. Autofluorescence might be useful but many features remain suboptimal. We have studied the autofluorescence of human healthy, metaplastic and dysplastic/CIS bronchial tissue, covering excitation wavelengths from 350 to 480 nm. These measurements are performed with a spectrofluorometer whose distal end is designed to simulate the spectroscopic response of an imaging system using routine bronchoscopes. Our data provide information about the excitation and emission spectral ranges to be used in a dual range detection imaging system to maximize the tumor vs healthy and the tumor vs. inflammatory/metaplastic contrast in detecting pre-/early malignant lesions. We find that the excitation wavelengths yielding the highest contrasts are between 400 and 480 nm with a peak at 405 nm. We also find that the shape of the spectra of healthy tissue is similar to that of its inflammatory/metaplastic counterpart. Finally we find that, when the spectra are normalized, the region of divergence between the tumor and the nontumor spectra is consistently between 600 and 800 nm and that the transition wavelength between the two spectral regions is around 590 nm for all the spectra regardless of the excitation wavelength, thus suggesting that there might be one absorber or one fluorophore. The use of backscattered red light enhances the autofluorescence contrast.

Pharmacokinetics of tetra(m-hydroxyphenyl)chlorin in human plasma and individualized light dosimetry in photodynamic therapy

The pharmacokinetics of the photosensitizer 5,10,15,20‐tetra(m‐hydroxyphenyl) chlorin(mTHPC) was investigated in the plasma of 20 patients by absorption and fluorescence spectroscopy. The temporal behavior was characterized by a rapid decrease in concentration during the first minutes after intravenous injection of 0.15 mg/kg mTHPC. A minimum concentration in the plasma was reached after about 45 min. The drug concentration then increased again, attaining a maximum after about 10 h, after which it decreased again with a halflife of about 30 h. Irradiation tests in the oral cavity at different time intervals after the injection revealed that the tissue re‐action was only partially correlated with the mTHPC plasma level. The tissue response was stronger at later drug‐light intervals (1–4 days) than during the first hours after injection even though the mTHPC plasma concentration was higher at the shorter times. Relative mTHPC concentrations were also measured in the mucosae of the oral cavity, the esophagus and the bronchi of 27 patients by light‐induced fluorescence spectroscopy using an optical fiber‐based spectrometer. These measurements were performed prior to photodynamic therapy (PDT), 4 days after injection of the photosensitizer. Highly significant linear correlations were found between the relative mTHPC concentrations in the mucosae of these three organs. Likewise, the plasma levels of mTHPC measured just before PDT were significantly correlated with the mTHPC concentrations in the three types of mucosae mentioned above. These results indicate that mTHPC plasma levels measured just before PDT can be used for PDT light dosimetry.

In vivo autofluorescence imaging of early cancers in the human tracheobronchial tree with a spectrally optimized system

The changes in the autofluorescence characteristics of the bronchial tissue is of crucial interest as a cancer diagnostic tool. Evidence exists that this native fluorescence or autofluorescence of bronchial tissues changes when they turn dysplastic and to carcinoma in situ. There is good agreement that the lesions display a decrease of autofluorescence in the green region of the spectrum under illumination with violet-light, and a relative increase in the red region of the spectrum is often reported. Imaging devices rely on this principle to detect early cancerous lesions in the bronchi. Based on a spectroscopic study, an industrial imaging prototype is developed to detect early cancerous lesions in collaboration with the firm Richard Wolf Endoskope GmbH, Germany. A preliminary clinical trial involving 20 patients with this spectrally optimized system shows that the autofluorescence can help to detect most lesions that would otherwise have remained invisible to an experienced endoscopist under white light illumination. A systematic off line analysis of the autofluorescence images pointed out that real-time decisional functions can be defined to reduce the number of false positive results. Using this method, a positive predictive value (PPV) of 75% is reached using autofluorescence only. Moreover, a PPV of 100% is obtained, when combining the white light (WL) mode and the autofluorescence (AF) mode, at the applied conditions. Furthermore, the sensitivity is estimated to be twice higher in the AF mode than in WL mode.

Absolute autofluorescence spectra of human healthy, metaplastic, and early cancerous bronchial tissue in vivo

Autofluorescence is emerging as a useful tool for the detection of early cancers in the bronchi. It has already produced interesting results, which have been implemented in commercial imaging devices. Their design relies on the spectroscopy of the tissues of interest. However, a large majority of these autofluorescence spectroscopy studies have been presented in arbitrary units. This is a drawback for, in particular, the designing of imaging devices based on autofluorescence. Using correction factors and a spectral sensitivity correction curve, we determined the absolute spectral distribution of the tissue autofluorescence in vivo. These measurements were performed on healthy, metaplastic, and dysplastic bronchial tissues at several excitation wavelengths ranging from 350 to 495 nm. Moreover, we measured at a fixed distance between the tissue and the probe to avoid geometric distortions of the spectra that are due to the optical characteristics of tissue. We found that the order of magnitude of the autofluorescence brightness was stable as the excitation wavelengths varied (on the order of 5 pW/muW x nm at the maximum of the fluorescence emission spectra).

Stability of the fluorescence measurement of Foscan in the normal human oral cavity as an indicator of its content in early cancers of the esophagus and the bronchi

Abstract— Photodynamic therapy (PDT) with Foscan® (mTHPC) is used to cure early cancers of the esophagus or the tracheobronchial tree. However, fixed PDT parameters (drug dose, light dose, etc.) do not permit an accurate prediction of the tissue damage. Large interpatient fluctuations in tissue drug level, at the time of light application, suggest that the light dose must be adjusted to the drug dose shortly before the PDT. This drug dose can be measured endoscopically by light‐induced fluorescence spectroscopy, but this measurement is inconvenient and somewhat difficult. A better test site, yielding comparable information, is needed. The oral cavity seems ideal. However, it first had to be established to what extent the estimation of the drug dose was dependent upon the location of the measurement and the pressure applied to the probe. These measurements prove to be not only correlated to similar measurements in the esophagus or the bronchi but also more consistent and less sensitive to the location and the applied pressure. The buccal mucosa is therefore recommended as a test site for measuring the Foscan(r) fluorescence signal at the time of PDT in the esophagus or the bronchi. This measurement is accurate enough for use in light‐dose adjustment.

Comparison of ALA- and ALA hexyl-ester-induced PpIX depth distribution in human skin carcinoma.

Photodynamic therapy (PDT) based on the use of photoactivable porphyrins, such as protoporphyrin IX (PpIX), induced by the topical application of amino-levulinic acid (ALA) or its derivatives, ALA methyl-ester (m-ALA), is a treatment for superficial basal cell carcinoma (BCC), with complete response rates of over 80%. However, in the case of deep, nodular-ulcerative lesions, the complete response rates are lower, possibly related to a lower bioavailability of PpIX. Previous in vitro skin permeation studies demonstrated an increased penetration of amino-levulinic acid hexyl-ester (h-ALA) over ALA. In this study, we tested the validity of this approach in vivo on human BCCs. An emulsion containing 20% ALA (w/w) and preparations of h-ALA at different concentrations were applied topically to the normal skin of Caucasian volunteers to compare the PpIX fluorescence intensities with an optical fiber-based spectrofluorometer. In addition, the PpIX depth distribution and fluorescence intensity in 26 BCCs were investigated by fluorescence microscopy following topical application of 20% ALA and 1% h-ALA. We found that, for application times up to 24h, h-ALA is identical to ALA as a PpIX precursor with respect to PpIX fluorescence intensity, depth of penetration, and distribution in basal cell carcinoma, but has the added advantage that much smaller h-ALA concentrations can be used (up to a factor 13). We observed a non-homogenous distribution in BCCs with both precursors, independent of the histological type and depth of invasion in the dermis.

Correlation between Protoporphyrin IX Fluorescence Intensity, Photobleaching, Pain and Clinical Outcome of Actinic Keratosis Treated by Photodynamic Therapy

Background: Photodynamic therapy (PDT) with Metvix® is a good therapeutic option to treat actinic keratosis, but it presents drawbacks (pain, lesion recurrences, heterogeneous outcome), emphasizing the possible need to individualize treatment. Objective: We assessed whether PDT clinical outcome and pain during treatment were correlated with protoporphyrin IX fluorescence intensity and photobleaching. Methods: 25 patients were treated by Metvix PDT. The outcome was evaluated after 1.3 (±0.4), 7.6 (±1.8), 13.2 (±1.2) and 33.6 (±3.0) months. After administration of Metvix, red light (632 ± 10 nm) was delivered with a light-emitting diode panel device. The outcome was assessed on a cosmetoclinical scale. Results: All patients who showed a fluorescence level before PDT treatment above a certain threshold had a complete recovery at 33.6 (±3.0) months. Conclusion: Our approach could be used to individualize PDT treatment based on the pretreatment fluorescence level, and to predict its long-term outcome.

Fluorescence pharmacokinetics of Lutetium Texaphyrin (PCI-0123, Lu-Tex) in the skin and in healthy and tumoral hamster cheek-pouch mucosa

We have investigated the pharmacokinetics (PK) of Lutetium Texaphyrin (Lu-Tex), a second-generation photosensitizer, in the Syrian hamster cheek pouch early cancer model. Ten male hamsters, five with chemically induced early squamous cell cancer of the left cheek pouch, received an intracardiac injection of a 10 mg/ml Lu-Tex solution, resulting in a dose of 12 mg Lu-Tex per kg of bodyweight. The PK of the dye have been measured during the 24 h following the injection with an optical-fiber-based spectrofluorometer on the ventral skin, the healthy and the tumoral cheek-pouch mucosa. The Lu-Tex fluorescence is excited at 460 nm and detected around 740 nm. All the measurements yield very similar pharmacokinetic curves. The fluorescence intensity reaches a maximum between two and three hours after the injection and, at its maximum, it is consistently higher (up to 1.5 times) on the tumor than on the healthy mucosa. It remains smaller on the skin than on cheek-pouch mucosa. After 24 h, the Lu-Tex fluorescence is no longer detectable either on the skin, on the lesion or on the healthy mucosa. Moreover, Lu-Tex clearly displays a significant fluorescence selectivity between early carcinoma and healthy mucosa in this model. Furthermore, the inter-animal fluctuations of the fluorescence signal are small (+/-16% on the tumor-bearing mucosa). Eight-minute-long skin-irradiation tests have been performed 24 h after the injection of the Lu-Tex on the ventral skin of 16 additional animals with a solar simulator. No reaction is observed, either macroscopically or microscopically, which further demonstrates, as suggested by the fluorescence measurements, that this photosensitizer is significantly cleared from the skin after 24 h.