Thomas Glanzmann

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).

In vivo determination of the optical properties of muscle with time-resolved reflectance using a layered model

We have investigated the possibility of determining the optical coefficients of muscle in the extremities with in vivo time-resolved reflectance measurements using a layered model. A solution of the diffusion equation for two layers was fitted to three-layered Monte Carlo calculations simulating the skin, the subcutaneous fat and the muscle. Relative time-resolved reflectance data at two distances were used to derive the optical coefficients of the layers. We found for skin and subcutaneous fat layer thicknesses (l2) of up to 10 mm that the estimated absorption coefficients of the second layer of the diffusion model have differences of less than 20% compared with those of the muscle layer of the Monte Carlo simulations if the thickness of the first layer of the diffusion model is also fitted. If l2 is known, the differences are less than 5%, whereas the use of a semi-infinite model delivers differences of up to 55%. Even if l2 is only approximately known the absorption coefficient of the muscle can be determined accurately. Experimentally, the time-resolved reflectance was measured on the forearms of volunteers at two distances from the incident beam by means of a streak camera. The thicknesses of the tissues involved were determined by ultrasound. The optical coefficients were derived from these measurements by applying the two-layered diffusion model, and results in accordance with the theoretical studies were observed.

Time-resolved spectrofluorometer for clinical tissue characterization during endoscopy

Time-resolved fluorescence spectroscopy has the potential to provide more information for the detection of early cancer than continuous wave spectroscopy. A new optical fiber-based spectrofluorometer for time-resolved fluorescence spectroscopy of biol. tissue during clin. endoscopy is presented. The app. is based on a nitrogen laser pumping a dye laser as excitation source and a streak camera coupled with a spectrograph as time-resolved spectrometer. The excitation and fluorescence light is carried by an optical fiber to the tissue under investigation and back to the detector, resp. This optical fiber can be inserted into the biopsy channel of a conventional endoscope. Hence, the app. can be used to perform in situ tissue characterization during endoscopy. The instrument enables the measurement of the decays of entire fluorescence spectra within 15 s with a dynamic range of the spectro-temporal images of up to three orders of magnitude. Luminescence lifetimes from the sub ns up to the ms range can be measured. Spectral and temporal resoln., sensitivity, and dynamic range of the instrumentation were detd. The accuracy of the app. was checked by the measurement of the fluorescence lifetimes of various fluorophores with known lifetimes. For the first time, two-dimensional time-resolved spectra with sub-ns temporal resoln. of tissue fluorescence of the human bladder, the bronchi, and the esophagus taken during endoscopy are presented as a demonstration of performance of the instrumentation. The excitation wavelengths were 337 nm in the case of the bladder and the esophagus and 480 nm in the case of the bronchi. Lifetime contrasts between normal and neoplastic tissue were found in all three organs. The spectral anal. of the fluorescence decays showed that the fluorescence between 370 and 490 nm, excited at 337 nm, consisted in several overlapping spectra. In the case of the esophagus, the contrast between normal and tumoral tissue was inverse in two different spectral bands proving the importance of the choice of the appropriate spectral range for time-resolved autofluorescence measurements for an optimal contrast. The in vivo fluorescence decay of the photosensitizers 5-aminolevulinic acid hexylester hydrochloride-induced protoporphyrin IX was measured in the human bladder and found to be mono-exponential with a lifetime of 15.9 (+-1.2) ns. An in vivo fluorescence lifetime of 8.5 (+-0.8) ns was found in the case of the photosensitizer 5, 10, 15, 20-tetra(m-hydroxyphenyl)chlorin (mTHPC) in the esophagus.

Long circulating half-life and high tumor selectivity of the photosensitizer meta-tetrahydroxyphenylchlorin conjugated to polyethylene glycol in nude mice grafted with a human colon carcinoma

In a model of nude mice bearing a human colon carcinoma xenograft, the biodistribution and tumor localization of meta‐tetrahydroxyphenylchlorin (m‐THPC) coupled to polyethylene glycol (PEG) were compared with those of the free form of this photosensitizer used in photodynamic therapy (PDT). At different times after i.v. injection of both forms of 125I‐labeled photosensitizer, m‐THPC‐PEG gave on average a 2‐fold higher tumor uptake than free m‐THPC. In addition, at early times after injection, m‐THPC‐PEG showed a 2‐fold longer blood circulating half‐life and a 4‐fold lower liver uptake than free m‐THPC. The tumor to normal tissue ratios of radioactivity concentrations were always higher for m‐THPC‐PEG than for free m‐THPC at any time point studied from 2 to 96 hr post‐injection. Significant coefficients of correlation between direct fluorescence measurements and radioactivity counting were obtained within each organ tested. Fluorescence microscopy studies showed that m‐THPC‐PEG was preferentially localized near the tumor vessels, whereas m‐THPC was more diffusely distributed inside the tumor tissue. To verify whether m‐THPC‐PEG conjugate remained phototoxic in vivo, PDT experiments were performed 72 hr after injection and showed that m‐THPC‐PEG was as potent as free m‐THPC in the induction of tumor regression provided that the irradiation dose for m‐THPC‐PEG conjugate was adapted to a well‐tolerated 2‐fold higher level. The overall results demonstrate first the possibility of improving the in vivo tumor localization of a hydrophobic dye used for PDT by coupling it to PEG and second that a photosensitizer conjugated to a macromolecule can remain phototoxic in vivo. Int. J. Cancer 76:842–850, 1998.© 1998 Wiley‐Liss, Inc.

Investigation of two-layered turbid media with time-resolved reflectance

Light propagation in two-layered turbid media that have an infinitely thick second layer is investigated with time-resolved reflectance. We used a solution of the diffusion equation for this geometry to show that it is possible to derive the absorption and the reduced scattering coefficients of both layers if the relative reflectance is measured in the time domain at two distances and if the thickness of the first layer is known. Solutions of the diffusion equation for semi-infinite and homogeneous turbid media are also applied to fit the reflectance from the two-layered turbid media in the time and the frequency domains. It is found that the absorption coefficient of the second layer can be more precisely derived for matched than for mismatched boundary conditions. In the frequency domain, its determination is further improved if phase and modulation data are used instead of phase and steady-state reflectance data. Measurements of the time-resolved reflectance were performed on solid two-layered tissue phantoms that confirmed the theoretical results.

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 measurement of fluorescence bleaching of meso-tetra hydroxy phenyl chlorin (mTHPC) in the esophagus and the oral cavity

In vivo spectrofluorometric analysis during photodynamic therapy (PDT) is a tool to obtain information about fluorophore bleaching kinetics in tissue. Using a cylindrical esophageal light distributor for PDT with an integrated sensing fiber, together with a fluorescence detection setup, we can obtain tissue fluorescence spectra endoscopically in a clinical environment. This study was performed on patients with early squamous cell carcinomas in the esophagus. Patients were injected intravenously with 0.15 mg/kg of mTHPC and underwent PDT (lambda equals 514 nm, fluence rate equals 100 mW/cm2) 96 hours after injection. Bleaching kinetics of mTHPC and tissue autofluorescence at different wavelengths were recorded in real time and showed decreases in the observed fluorescence intensity in the 652 nm band of about 60% for light doses around 100 J/cm2. Additional information on bleaching kinetics induced at another excitation wavelength, lambda equals 652 nm, was obtained by irradiations at much lower doses in the buccal cavity. The data are analyzed using a simplified mechanism in which singlet oxygen is the hypothetical reactive intermediate which can both bleach the mTHPC and the autofluorescent molecules. The differential equations are solved by applying the quasi stationary state approximation for the reactive intermediate. The experimental data at least do not appear to contradict this oversimplified mechanism.

Optimizing light dosimetry in photodynamic therapy of the bronchi by fluorescence spectroscopy

Under identical conditions (drug and light dose, timing), the results of photodynamic therapy (PDT) of carcinomas of the bronchi with tetra(meta-hydroxyphenyl)chlorin (mTHPC) show large variations between patients. Before patients underwent PDT treatment, the mTHPC level was measured in the lesion, the normal surrounding tissue and the oral cavity, with an apparatus based on fluorescence spectroscopy. The fluctuations in degree of tissue reaction and tumour destruction between patients could be explained by individual variations in the mTHPC level in the mucosa of the bronchi. The patients who showed the highest mTHPC fluorescence signal also had the strongest response to PDT. In addition, a correlation between the mTHPC level in the oral cavity and bronchial mucosa was found. It is concluded that PDT can be improved by measuring the mTHPC level in the bronchi or the oral cavity before treatment by fluorescence spectroscopy, and then by adjusting the light dose to be applied to the observed mTHPC level.

In vivo time-resolved spectroscopy of the human bronchial early cancer autofluorescence

Time-resolved measurements of tissue autofluorescence (AF) excited at 405 nm were carried out with an optical-fiber-based spectrometer in the bronchi of 11 patients. The objectives consisted of assessing the lifetime as a new tumor/normal (T/N) tissue contrast parameter and trying to explain the origin of the contrasts observed when using AF-based cancer detection imaging systems. No significant change in the AF lifetimes was found. AF bronchoscopy performed in parallel with an imaging device revealed both intensity and spectral contrasts. Our results suggest that the spectral contrast might be due to an enhanced blood concentration just below the epithelial layers of the lesion. The intensity contrast probably results from the thickening of the epithelium in the lesions. The absence of T/N lifetime contrast indicates that the quenching is not at the origin of the fluorescence intensity and spectral contrasts. These lifetimes (6.9 ns, 2.0 ns, and 0.2 ns) were consistent for all the examined sites. The fact that these lifetimes are the same for different emission domains ranging between 430 and 680 nm indicates that there is probably only one dominant fluorophore involved. The measured lifetimes suggest that this fluorophore is elastin.

Pharmacokinetics and pharmacodynamics of tetra(m-hydroxyphenyl)chlorin in the hamster cheek pouch tumor model: comparison with clinical measurements

The pharmacokinetics (PK) of the photosensitizer tetra(m-hydroxyphenyl)chlorin (mTHPC) was measured by optical fiber-based light-induced fluorescence spectroscopy (LIFS) in the normal and tumoral cheek pouch mucosa of 29 Golden Syrian hamsters with chemically induced squamous cell carcinoma. Similar measurements were carried out on the normal oral cavity mucosa of five patients up to 30 days after injection. The drug doses were between 0.15 and 0.3 mg per kg of body weight (mg/kg), and the mTHPC fluorescence in the tissue was excited at 420 nm. The PK in both human and hamster exhibited similar behavior although the PK in the hamster mucosa was slightly delayed in comparison with that of its human counterpart. The mTHPC fluorescence signal of the hamster mucosa was smaller than that of the human mucosa by a factor of about 3 for the same injected drug dose. A linear correlation was found between the fluorescence signal and the mTHPC dose in the range from 0.075 to 0.5 mg/kg at times between 8 and 96 h after injection. No significant selectivity in mTHPC fluorescence between the tumoral and normal mucosa of the hamsters was found at any of the applied conditions. The sensitivity of the normal and tumoral hamster cheek pouch mucosa to mTHPC photodynamic therapy as a function of the light dose was determined by light irradiation at 650 nm and 150 mW/cm2, 4 days after the injection of a drug dose of 0.15 mg/kg. These results were compared with irradiations of the normal oral and normal and tumoral bronchial mucosa of 37 patients under the same conditions. The reaction to PDT of both types of human mucosae was considerably stronger than that of the hamster cheek pouch mucosa. The sensitivity to PDT became comparable between hamster and human mucosa when the drug dose for the hamster was increased to 0.5 mg/kg. A significant therapeutic selectivity between the normal and neoplastic hamster cheek pouch was observed. Less selectivity was found following irradiations of normal mucosa and early carcinomas in the human bronchi. The pharmacodynamic behavior of mTHPC was determined by test irradiations of the normal mucosa of hamsters and patients between 6 h and 8 days after injection of 0.5 and 0.15 mg/kg in the hamsters and the patients, respectively. The normal hamster cheek pouch showed a maximum response to irradiation 6 h after injection and then decreased continuously to no observable reaction at 8 days after injection. The reaction of the normal human oral mucosa, however, showed an increasing sensitivity to the applied light between 6 h and 4 days after mTHPC injection and then decreased again at 8 days. The hamster model with the chemically induced early squamous cell cancer in the cheek pouch thus showed some similarity to the early squamous cell cancer of the human oral mucosa considering the PK. However, a quantitative difference in fluorescence signal for identical mTHPC doses as well as a significant difference in pharmacodynamic behavior were also observed. The suitability of this animal model for the optimization of PDT parameters in the clinic is therefore limited. Hence great care must be taken in screening new dyes for PDT of early squamous cell cancer of the upper aerodigestive tract based upon observables in the hamster cheek pouch model.