Matrix-assisted laser desorption/Ionization coupled to Fourier transform ion cyclotron resonance mass spectrometry: a method to characterize temoporpfin photoproducts

Abstract

Photodynamic therapy (PDT) is a new rapidly developing method for cancer treatment, which is based on the systemic administration of a tumor-localizing photosensitizer (PS), followed by irradiation of the tumor with visible spectrally adapted light.1-3 The photochemical interaction of the PS, light and molecular oxygen results in the formation of singlet oxygen ( 1 O 2 ) and other reactive oxygen species (ROS), which are believed to be toxic species responsible for induction of tumor necrosis.2,3 5,10,15,20-m-(Tetrahydroxyphenyl)chlorin (m-THPC), one of the second generation photosensitizers, 4 has recently been proposed as a new sensitizer for future use in the PDT of cancer.5,6 m-THPC is a single compound, derived from 5,10,15,20-meso-(tetrahydroxyphenyl)porphyrin (m-THPP), 4 with a maximum absorption in the red region of visible light (! max 652 nm). The latter property favors deeper light penetration into tissue and allows the method to achieve 6-10 mm depth of necrosis in tumor-bearing mice. As is the case for the majority of photosensitive drugs, m-THPC is degraded upon light illumination. This process, usually called 'photobleaching', involves a decrease in absorbance for certain spectral bands, variations of the fluorescence intensity or the formation of new absorption and emission bands.7 The m-THPC photodegradation has been observed in aqueous 8 and organic solutions, 9 in vitro, 10 in vivo in tumor-bearing mice 11 and in clinical practice. 12Studyies of the photobleaching reaction are particularly important for two reasons: (1) Identification of photobleaching products, which can be photodynamically active, thus opening the possibility for 'extended' photodynamic therapy; (2) Measurement of the photochemical reactions that reflect the potential therapeutic effect and thus help to match the light dose. This paper describes the first results concerning the application of matrix-assisted laser desorption/ionization coupled to Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICRMS) for the analysis of the parent m-THPC molecule and the products of photochemical m-THPC oxidation. We have studied the formation of photoproducts in organic solvents as a result of two consecutive irradiations separated by a dark storage period. The kinetics of photoproduct formation, assessed by MALDI, have been compared with that obtained by spectroscopic measurements. EXPERIMENTALReagents and standard solutions m-THPC (Fig. 1) was kindly provided by Scotia Pharmaceuticals Ltd. (Guildford, UK) as crystalline powder (specification: content of m-THPC: 98.4% w/w; water content: 0.20% w/w; residual solvents: ethanol 0.18% w/w; content of impurities: m-THPP 0.7% w/w). Unless otherwise indicated, the photosensitizer was dissolved in methanol or in ethanol (Prolabo SA, Fontenay sous bois, France), which had been distilled beforehand twice under an inert atmosphere. IlluminationIllumination was performed by exposing 3.5 mL of 7 mg/mL (10 À5 mol/L) of m-THPC in ethanol or methanol in a quartz cuvette (pathlength 1 c...

MALDI‐TOF mass spectrometric analysis for the characterization of the 5,10,15,20‐tetrakis‐ (m‐hydroxyphenyl)bacteriochlorin (m‐THPBC) photoproducts in biological environment

“…4). The ion at m / z 713.24 was attributed to the protonated dihydroxylated m ‐THPC ([ m ‐THPC + (O) 2 + H] + ) as has been described by several authors12, 29 and the ion at m / z 714.24 was attributed to dihydroxy m ‐THPBC ([ m ‐THPBC + (O) 2 ] +• ) due to a photo‐substitution mechanism. Two explanations can be proposed to clarify the nature of the ion at m / z 715.24: It could arise from the photo‐addition (photo‐hydratation) of the m ‐THPC, giving the protonated dihydro dihydroxy m ‐THPC noted on the figures [ m ‐THPC + (OH) 2 + H] + .…”

Intracellular Photobleaching of 5,10,15,20-Tetrakis(m-hydroxyphenyl) chlorin (Foscan®) Exhibits a Complex Dependence on Oxygen Level and Fluence Rate¶

“…It is generally assumed that photodegradation of dyes such as PDT sensitizers can proceed via Type-I or singlet oxygen-mediated Type-II reactions (or both) (1), with one or the other predominating according to the sensitizer. In the solution phase, the relative contribution of the two mechanisms is also strongly dependent on the experimental conditions, such as the solvent used (32,33) or on the presence of biological substrates such as proteins. Therefore, some photoproducts detected, for example, in ethanolic solution of mTHPC (32) may not necessarily be generated in proteincontaining solution or in cells.…”

“…Therefore, some photoproducts detected, for example, in ethanolic solution of mTHPC (32) may not necessarily be generated in proteincontaining solution or in cells. Another aspect demonstrating the need for care when comparing solution phase results with cellular studies is the continuation of mTHPC degradation and product formation in the dark after irradiation, as reported for ethanolic solution (33). We did not observe any change of fluorescence intensity in the dark period after irradiation in the macrophage system.…”

Temoporfin (Foscan®, 5,10,15,20‐Tetra(m‐hydroxyphenyl)chlorin)—A Second‐generation Photosensitizer†,‡

“…Angotti et al. found evidence for both Type I and Type II reactions being involved in the photobleaching of m THPC (74) and identified m THPP as the primary photoproduct (75). Similar results were obtained using the HPLC separation of m THPC photoproducts after irradiation with 514 nm laser light in combination with MALDI‐TOF MS and UV/Vis spectroscopy.…”

“…A combined use of HPLC and electrospray ionization tandem MS (ESI-MS) identified mTHPP and several hydroxylated derivatives of both mTHPC and mTHPP as initial photobleaching products (73). Angotti et al found evidence for both Type I and Type II reactions being involved in the photobleaching of mTHPC (74) and identified mTHPP as the primary photoproduct (75). Similar results were obtained using the HPLC separation of mTHPC photoproducts after irradiation with 514 nm laser light in combination with MALDI-TOF MS and UV ⁄ Vis spectroscopy.…”