Alexandre Marti

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.

OPTIMISATION OF THE FORMATION AND DISTRIBUTION OF PROTOPORPHYRIN IX IN THE UROTHELIUM: AN IN VITRO APPROACH

PURPOSE To optimize conditions for photodynamic detection (PDD) and photodynamic therapy (PDT) of bladder carcinoma, urothelial accumulation of protoporphyrin IX (PpIX) and conditions leading to cell photodestruction were studied. MATERIALS AND METHODS Porcine and human bladder mucosae were superfused with derivatives of 5-aminolevulinic acid (ALA). PpIX accumulation and distribution across the mucosa was studied by microspectrofluorometry. Cell viability and structural integrity were assessed by using vital dyes and microscopy. RESULTS ALA esters, especially hexyl-ALA, accelerated and regularized urothelial PpIX accumulation and allowed for necrosis upon illumination. CONCLUSIONS hexyl-ALA used at micromolar concentrations is the most efficient PpIX precursor for PDD and PDT.

Routine experimental system for defining conditions used in photodynamic therapy and fluorescence photodetection of (non-) neoplastic epithelia.

A common method to induce enhanced short-term endogenous porphyrin synthesis and accumulation in cell is the topical, systemic application of 5-aminolevulinic acid or one of its derivatives. This circumvents the intravenous administration of photosensitizers normally used for photodynamic therapy (PDT) of fluorescence photodetection. However, in the majority of potential medical indications, optimal conditions with respect to the porphyrin precursor or its pharmaceutical formulation have not yet been found. Due to ethical restrictions and animal right directives, the number of available test objects is limited. Hence, definition and use of nonanimal test methods are needed. Tissue and organ cultures are a promising approach in replacing cost intensive animal models in early stages of drug development. In this paper, we present a tissue culture, which can among others be used routinely to answer specific questions emerging in the field of photodynamic therapy and fluorescence photodetection. This technique uses mucosae excised from sheep paranasal sinuses or pig bladder, which is cultured under controlled conditions. It allows quasiquantitative testing of different protoporphyrin IX precursors with respect to dose-response curves and pharmacokinetics, as well as the evaluation of different incubation conditions and/or different drug formulations. Furthermore, this approach, when combined with the use of electron microscopy and fluorescence-based methods, can be used to quantitatively determine the therapeutic outcome following protoporphyrin IX-mediated PDT.

An Open Pharmacokinetic Study of Hexylaminolevulinate-Induced Photodiagnosis after Intravesical Administration

AbstractBackground and objectives: Hexylaminolevulinate-mediated photodiagnosis of superficial bladder cancer recently gained marketing authorisation in Europe and is therefore being used increasingly, especially to visualise flat tumours such as carcinoma in situ. Although no significant adverse effects related to the drug have been reported to date, precise information with respect to the safety of this procedure in clinical practice has not yet been published. In the present study, we investigated the possible systemic absorption of different hexylaminolevulinate concentrations and characterised the adverse effects after bladder instillation. Methods: Twelve patients with known bladder cancer received a 2-hour administration of 50mL of phosphate-buffered saline containing 4 mmol/L, 8 mmol/L or 16 mmol/L of hexylaminolevulinate. Safety was primarily evaluated by monitoring adverse effects. Standard haematology and clinical biochemistry were assessed by the local hospital laboratory. Measurements of hexylaminolevulinate, 5-aminolevulinate and protoporphyrin IX in plasma and solution collected after instillation were performed, as were fluorescence measurements in the urothelium and the skin by using an optical fibre-based spectrofluorometer. Results: No hexylaminolevulinate was found in the plasma. Furthermore, 5-aminolevulinate and protoporphyrin IX showed no significant increases in plasma after intravesical instillation of hexylaminolevulinate 4 mmol/L, 8 mmol/L or 16 mmol/L for 2 hours. This implies a very low systemic absorption at the administered doses with a hexylaminolevulinate uptake from the bladder of about 5%. Neither skin sensitivity nor adverse reactions that could be attributed to the drug were reported. Renal and liver function were not affected by the hexylaminolevulinate doses used in this study. Conclusion: We demonstrated for the first time that hexylaminolevulinate-mediated photodiagnosis is a safe procedure for a patient undergoing this examination, the drug being only minimally systemically absorbed after intravesical instillation. Therefore, no or only minimal adverse effects compared with those seen with conventional photosensitising agents can be expected.

On the influence of the instillation time on the results of HAL (Hexvix®) fluorescence detection of superficial bladder cancer

Hexyl aminolevulinate (HAL) fluorescence cystoscopy is being investigated as a new diagnostic tool for the detection of flat urothelial malignancies in bladder cancers. However, the influence of the bladder instillation time on the performance of this detection modality has not been addressed up to now. We report our initial experience comparing different instillation schedules of HAL cystoscopy in the diagnosis of superficial bladder cancer. A total of 718 fluorescent positive (433) and fluorescence negative (285) biopsies have been taken in the bladder of 143 patients using the Storz D-light fluorescence imaging system (Karl Storz, Tuttlingen, Germany) which allows both white and blue light (380-450 nm) bladder wall inspection. Following hospitalisation, 50 ml of HAL (8mM) phosphate buffer solution was instilled into the bladder of patients during one hour (1 hour protocol involving 57 patients), or during two hours followed by a two hours resting time after removal of the solution (2+2 hours protocol involving 86 patients). Both instillation subgroups were homogeneous in terms of proportion of high risk disease, previous BCG treatment and/or recurrent disease. This study indicates that the instillation duration does not influence the results of HAL (Hexvix) fluorescence cystoscopy in our conditions. Compared to the standard use of ALA, HAL (Hexvix) fluorescence cystoscopy allows a significant reduction of the instillation time (to less than one hour) without prejudicing the efficacy of the method, what represents a real advantage in daily clinical practice.

La cystoscopie en fluorescence dans le cancer de la vessie, quel avenir ?

Bladder transitional cell carcinoma, limited to the lamina propria, or superficial in 80% of the cases, is characterized by a high recurrence rate up to 70% in the three years following the first treatment. However, the constant evolution of the endoscopic techniques, useful to its diagnosis and treatment, contributed to reduce about by half this recurrence rate at three months. The actual development concerns new fluorescence detection methods, adapted to the standard endoscopic equipment. This new technique or fluorescence cystoscopy aims at improving the visual contrast between tumor and normal tissue in order to obtain a precise mapping of the diseased bladder. Thus, it allows to detect a carcinoma in situ (CIS) with a sensitivity around 80 to 90%. Its impact on the progression of the disease remains however to be demonstrated.