In vitro phototoxicity of glycoconjugated porphyrins and chlorins in colorectal adenocarcinoma (HT29) and retinoblastoma (Y79) cell lines.

BACKGROUND: Retinoblastoma is the most common malignant intraocular tumor in children. The current treatment gives a good vital prognostic but there are several drawbacks to the arsenal of "classical antitumoral" therapies. Photodynamic therapy (PDT) could be an exciting non-toxic and non-mutagenic alternative protocol. METHOD: In this paper, we report about the screening of the in vitro photocytotoxicity of hydrophenylporphyrins and chlorins and their glycoconjugated derivatives in a human retinoblastoma cell line (Y79) and for comparison in a colorectal adenocarcinoma cell line (HT29). RESULTS: Despite lower photodynamic activity than that observed for hydroxylated photosensitizers, in particular Foscan(®) glycoconjugated derivatives display phototoxicity (IC50 2.4-0.05µM ±10%) against Y79 cells with examples of significant intrinsic cytotoxicity. Amongst them the triglucosyl porphyrin 10 is highly photocytotoxic (IC50 0.9µM ±10%) but is fully devoid of cytotoxicity (IC50>15µM). The photoactivity is highly modulated by the presence of a diethyleneglycol spacer between the chromophore and the glycoside (compounds 14-17, IC50 0.5, 0.6, 0.05 and 0.35µM ±10%) and by the anomeric configuration of the sugar (compound 15 and 17, IC50 0.6 and 0.05µM ±10% respectively). One of the main problems for the use of Foscan(®) is its poor solubility which might be improved by glycoconjugation. Moreover Foscan has been shown to induce necrosis after PDT leading to a possible ulceration of surrounding tissues unsuitable for a conservative treatment. A preferential mitochondrial subcellular localization which has been previously reported for some glycoconjugated photosensitizers could enhance the contribution of apoptosis process. CONCLUSION: Tri-α-O-galactosyl porphyrin 16 is a better candidate than Foscan(®) for a clinical application of PDT for a conservative therapy of retinoblastoma.

A study of the stability of tri(glucosyloxyphenyl)chlorin, a sensitizer for photodynamic therapy, in human colon tumoural cells: a liquid chromatography and MALDI-TOF mass spectrometry analysis.

Asymmetrical glycoconjugated tetrapyrrolic macrocycles are under study as efficient sensitizers for photodynamic therapy (PDT). In this context, tri(meta-O-beta-glucopyranosyloxyphenyl)chlorin [TPC(m-O-Glu)(3)] 2a/3a was found to be four times more photoactive in vitro than Foscan. In a further study of this interesting glycoconjugate, its metabolism by cellular glycosidases in HT29 cells has to be explored. Cellular extracts of HT29 cells incubated with TPC(m-O-Glu)(3) (24h, 6microM) were analyzed by MALDI-TOF mass spectrometry and high performance liquid chromatography (HPLC). In MALDI-TOF mass spectra, the presence of compounds distinct from TPC(m-O-Glu)(3) (m/z 1151) were observed at m/z 989, 827 and 665 corresponding to the loss of one, two or three glucose units (162u) and were be ascribed to TPC(m-OH)(m-O-Glu)(2) 2/3b,b',b", TPC(m-OH)(2)(m-O-Glu) 2/3c,c',c" and TPC(m-OH)(3) isomers 2d/3d, respectively. The porphyrins resulting from chlorin oxidation TPP(m-O-Glu)(3) 4a, TPP(m-OH)(m-O-Glu)(2) 4b,b", TPP(m-OH)(2)(m-O-Glu) 4c,c" and TPP(m-OH)(3) 4d were also observed. The HPLC profile (lambda(anal)=420 nm) showed eight peaks consistent with mass spectra. The kinetics of deglucosylation was studied from HPLC profiles between 1 and 48h incubation. The concentration of triglucoconjugated and diglucoconjugated molecules was maximum around 3 and 8h incubation, respectively, whereas, totally deglucosylated species appeared only after incubation for more than 10h. The fully deglycosylated porphyrin TPP(m-OH)(3) is the final metabolite, being observed at a concentration 15 times higher than that of the remaining TPC(m-O-Glu)(3) 2a/3a. Compared to the photobiological activity of the parent molecule [TPC(m-O-Glu)(3)], a three times higher TPP(m-OH)(3) concentration was necessary to observe a similar in vitro photoactivity.

Synthesis, cellular internalization and photodynamic activity of glucoconjugated derivatives of tri and tetra(meta-hydroxyphenyl)chlorins.

Glucoconjugated tri and tetra(meta-hydroxyphenyl)chlorins have been synthesized in order to explore how glucoconjugation of the macrocycle affects the photoactivity of the molecule. Internalization processes, photosensitizing efficacy of TPC(m-O-GluOH)(3) and TPC(m-O-GluOH)(4), in HT29 human adenocarcinoma cells have been compared to those of tetra(meta-hydroxyphenyl) chlorin (m-THPC, Foscan). The tetra glucoconjugated chlorin, TPC(m-O-GluOH)(4), was found to be poorly internalized and weakly photoactive. In contrast, the asymmetric and more amphiphilic compound TPC(m-O-GluOH)(3), exhibited superior phototoxicity compared to m-THPC. Drug concentration, temperature and sodium azide effects indicated that TPC(m-O-GluOH)(3) internalization partly proceeds via an active receptor-mediated endocytosis mechanism. Cellular uptake appeared as a saturable process and remained 30% lower than for mTHPC. However, a maximum phototoxicity in HT29 cells (survival fraction of 2+/-0.6%) were observed for concentration as low as 2 microM. A 4-fold higher concentration of m-THPC was necessary to observe the same level of photoactivity. This higher phototoxicity has been correlated to a greater mitochondrial affinity. On the basis of these results, work is in progress to further evaluate the potential of glycosylated chlorins in photodynamic therapy (PDT).

Five-coordinate iron-porphyrin as a model for the active site of hemoproteins. Characterization and coordination properties.

Preparation of iron(III)-deuteroporphyrin 6(7)-methyl ester, 7(6)-(histidine methyl ester) by coupling histidine methyl ester to deuterohemin has been performed using the mixed carboxylic/carbonic-acid-anhydride method. This compound, which is very soluble in various organic solvents, can be considered as a prosthetic group model for the active site of five-coordinate hemoproteins. In the oxidized state a basic, a neutral or an acid form can be isolated. The basic and acid forms are monomeric at all concentrations. The neutral form is found dimeric in concentrated solutions while it is monomeric at low concentration. The coordination state of iron in these various species is investigated. The neutral form reacts with ligands, such as nitrogenous organic bases, leading to six-coordinate well-known hemichromes which exhibit low-spin electron spin resonance (ESR) spectra. The reaction of anionic ligands, such as F-, CN-, NO-2 and N-3, with the neutral form model leads to unsymmetrical six-coordinate complexes whose optical and ESR spectra are similar to those of synthetic deuteromyoglobin. In benzene, toluene or dichloromethane solutions iron (II)-deuteroporphyrin 6(7)-methyl ester, 7(6)-histidine methyl ester), obtained from ferric forms by heterogeneous reduction with aqueous dithionite, exhibits an optical spectrum characteristic of a five-coordinate high-spin ferrous complex. At low temperature important spectral modifications are observed indicating a dimeric association. At room temperature it binds one nitrogenous base molecule leading to the well-known hemochrome. It reacts also with carbon monoxide with a very high affinity constant (4.5 X 10(8) M-1), comparable to that of the isolated human hemoglobin alpha and beta chains, but much higher than the values reported for other various models, suggesting that the side-chain length bearing the fifth ligand may have an important influence upon the reactivity of the sixth position of the iron ion. At low temperature in toluene or dichloromethane, this compound reversibly binds oxygen without oxidation of the iron ion while oxidation occurs at room temperature. The significance of these results is discussed in relation with the local environment, the electronic nature of the base and the immobilization of the heme group in hemoproteins.