Jocelyne Blais

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

BACKGROUNDnRetinoblastoma 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.nnnMETHODnIn 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).nnnRESULTSnDespite 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.nnnCONCLUSIONnTri-α-O-galactosyl porphyrin 16 is a better candidate than Foscan(®) for a clinical application of PDT for a conservative therapy of retinoblastoma.

Intracellular pH of Candida albicans blastospores as measured by laser microspectrofluorimetry and 31P-NMR

The intracellular pH (pHi) of Candida albicans blastospores harvested from 8 h or 48 h cultures was determined under identical experimental conditions by two different techniques: 31P-NMR and laser microspectrofluorimetry. Time dependence of pHi was monitored by 31P-NMR on the whole cell population. Microspectrofluorimetry, after loading of the cells with SNARF-1, enabled the determination of pHi in isolated cells and its distribution among the cell population. By this method, the vacuolar pH could not be distinguished from the cytoplasmic pH in C. albicans blastospores, but alkalization of pHi was observed at the beginning of germ tubes. The absolute values of pHi determined by 31P-NMR were slightly different from those obtained by laser microspectrofluorimetry. However, the pH distributions in the cell population were converging. For blastospores in exponential phase a gaussian distribution of pHi was observed with both methods, the cells maintained a steady pHi value when the external pH was varied from 5.5 to 8.5. For cells in stationary phase two pools were identified: the combination of the two techniques demonstrated the presence of two different subpopulations. One of these population (with lower pH) was able to commute to the other one with time as shown by 31P-NMR kinetics. This information is reported here for the first time in C. albicans.

In vivo efficacy of photodynamic therapy in three new xenograft models of human retinoblastoma.

PURPOSEnRetinoblastoma is the most common primary intraocular tumor in children. In industrialized countries, 95% of patients are cured by chemotherapy and conservative treatments. However, these treatments can increase the risk of secondary tumors in patients with a constitutional alteration of the RB1 gene. Photodynamic therapy represents a nonmutagenic therapeutic approach, and may reduce the incidence of secondary tumors. To study the in vivo efficacy of photodynamic therapy, human retinoblastoma xenografts were established in nude mice.nnnMETHODSnThree xenografted cell lines, RB102-FER, RB109-LAK and RB111-MIL, were characterized and used for therapeutic evaluation. Mice were randomly divided into control and treatment groups with 5-8 mice in each group. Treatment groups received irradiation alone, photosensitizer alone or both in 2 of the 3 models and in the third model, photosensitizer plus irradiation was compared to untreated controls. mTHPC was injected intraperitoneally at a dose of 0.6mg/kg and verteporfin intravenously at a dose of 1mg/kg. Illuminations were performed 24h after mTHPC and 1h after verteporfin injections.nnnRESULTSnA transient but significant response to mTHPC was observed for RB102-FER (p=0.03) and a significant response to mTHPC for RB111-MIL (p<10(-4)) with partial regression maintained for more than 60 days. No significant difference between the different groups was observed for RB109-LAK, except in the verteporfin plus laser group (p=0.01).nnnCONCLUSIONSnThe studies confirmed the suitability of the three xenograft models for the evaluation of photodynamic therapy in retinoblastoma. Our findings suggest that PDT may represent an alternative conservative treatment for these tumors.

5-Aminolevulinic acid ester-induced protoporphyrin IX in a murine melanoma cell line

The use of 5-aminolevulinic acid (5-ALA) ester derivatives as precursors of endogenous protoporphyrin IX (PpIX) has been proposed as a good strategy for improved drug diffusion across biological membranes. In the present work, the 5-ALA ester derivatives hexyl-ALA (h-ALA), octyl-ALA (o-ALA), and decyl-ALA (d-ALA) were synthesized, and their efficacy to induce endogenous PpIX was explored in a murine melanoma cell line (B-16) as compared with that of 5-ALA. The maximum level of PpIX induced in cells treated with 5-ALA, h-ALA, o-ALA, and d-ALA was reached at optimal concentrations of 0.3, 0.075, 0.1, and 0.075xa0mM, respectively. The derivatives h-ALA and o-ALA appear as the most efficient PpIX precursors in this cell line, since a higher or similar PpIX production could be achieved with a fourfold and threefold lower dose of these precursors compared with 5-ALA. The phototoxicity effect of h-ALA and o-ALA ester derivatives showed the same phototoxicity behavior detected for 5-ALA but at much lower drug doses. Our study suggests that h-ALA and o-ALA esters improve intracellular PpIX formation in B-16 cells at reduced concentrations. This should enable clinical applications at lower precursor doses with reduced effective costs.

Photofrin-induced fluorescence in progressive and regressive murine colonic cancer cells: correlation with cell photosensitivity

Microspectrofluorometry and fluorescence imaging were used to investigate the intracellular fluorescence of two murine colonic cancer cell lines--a progressive cell line (PROb) and a regressive cell line (REGb)--incubated with Photofrin. These two cell lines, which were initially cloned from the same chemically induced colonic murine cancer, differ in their metastatic properties and have been considered as models to mimic the tumoral cell heterogeneity. The fluorescence from cytoplasmic area of cells incubated with Photofrin appeared as a complex emission, with two maxima at 632 and 695 nm assigned to monomer species, and a poorly resolved band around 665 nm assigned to aggregates. The spectral distribution was shown to depend on the incubation time, with an aggregate contribution increasing for extended periods. The amount of Photofrin uptake, as determined from the total fluorescence intensity, was found for PROb to be twice that for REGb. However, the phototoxicities were quite similar for both cell lines, suggesting that drug concentration may not be the only determining factor in photobiological efficiency.

Glycodendrimeric phenylporphyrins as new candidates for retinoblastoma PDT: Blood carriers and photodynamic activity in cells

Photodynamic therapy (PDT) has recently been proposed as a possible indication in the conservative treatment of hereditary retinoblastoma. In order to create photosensitizers with enhanced targeting ability toward retinoblastoma cells, meso-tetraphenylporphyrins bearing one glycodendrimeric moiety have been synthesized. The binding properties to plasma proteins and photodynamic activity of two monodendrimeric porphyrins bearing three mannose units via monoethylene glycol (1) or diethylene glycol (2) linkers have been compared to that of the non-dendrimeric tri-substituted derivative [TPP(p-Deg-O-α-ManOH)(3)]. The dendrimeric structure was found to highly increase the binding affinity to plasma proteins and to modify to some extent plasma distribution. HDL and to a lesser extent LDL have been shown to be the main carriers of dendrimeric and non-dendrimeric compounds. The phototoxicity observed for the two glycodendrimers (1) and (2) (LD(50)=0.5 μM) in Y79 cells is of the same order of magnitude that for TPP(p-Deg-O-α-ManOH)(3) (LD(50)=0.7 μM), with a similar cellular uptake level for (1) and a lower for (2). A serum content increase from 2% to 20% (v/v) in the incubation medium was found to inhibit both cellular uptake and photoactivity of dendrimeric derivatives, whereas those of TPP(p-Deg-O-α-ManOH)(3) remained little affected. Specificities of glycodendrimeric porphyrins, combining a lower cellular uptake together with a higher affinity toward plasma proteins, make these derivatives possible candidates for a vascular targeting PDT.

Enhancement of 5,10,15,20-Tetra(m-Hydroxyphenyl)chlorin Fluorescence Emission by Inclusion in Natural and Modified Cyclodextrins

Fluorescence properties of 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (m-THPC), a sensitizer used in photodynamic therapy (PDT), were studied in the presence of three native cyclodextrins (CDs) -α, -β, and γ-CD—and two modified cyclodextrins—heptakis (2,6-di-O-methyl) β-CD (Me-β-CD) and heptakis (2-O-hydroxy-propyl) β-CD (HP-β-CD). The CDs studied have been shown to undergo the formation of an inclusion complex with m-THPC, leading to a large enhancement (up to 300 times) of m-THPC fluorescence intensity depending on the CD used. Stoichiometry and association constants have been determined on the basis of the variation of fluorescence intensity. A 1:2 stoichiometry has been found for m-THPC complexes with γ-CD, Me-β-CD, and HP-β-CD. Association constants as high as K = 9.5 × 108 M−2 in the case of m-THPC/2Me-β-CD and K = 2.7 × 109 M−2 in the case of m-THPC/ 2HP-β-CD complex were determined, whereas a lower value (K = 2.4 × 104 M−2) was found in the case of m-THPC/2γ-CD. Because of the highest fluorescence enhancement (a factor of about 300) observed with Me-β-CD, this compound would be the most suitable to improve the detection of m-THPC in further pharmacokinetics and biodistribution studies.

A direct sensitized fluorimetric determination of 5,10,15,20-tetra(m-hydroxyphenyl)chlorin [m-THPC (Foscan)] in human plasma using a cyclodextrin inclusion complex.

The 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (m-THPC) (Foscan) is a photosensitizer used in the photodynamic therapy (PDT) of cancers which is currently under clinical trial. The formation of a m-THPC inclusion complex with dimethyl-beta-cyclodextrin (Me-beta-CD) in solution was demonstrated on the basis of circular dichroism experiments. A 1:2 complex stoichiometry was found and an inclusion constant beta 2 = 2.8(+/- 0.4) x 10(10) M-2 was determined. The formation of such a complex was shown to enhance the m-THPC fluorescence intensity. It could be exploited to improve the sensitivity of the direct m-THPC detection in human plasma. Optimization of the operating conditions shows that the best results were obtained by the addition of 100 microL of a concentrated Me-beta-CD solution (3.2 x 10(-2) M) to 1 mL plasma samples. Compared to the standard conditions, a 90% increase in sensitivity was obtained. The proposed analytical method which showed a linear response function [0-300 ng mL-1 (440 pM)] and a low limit of detection [1.5 ng mL-1 (2 pM) (S/N = 3)] appears, especially due to the absence of metabolism, a simple and specific method suitable for pharmacokinetics studies in patients.

Laser-Induced Auto Fluorescence of Normal and Tumor Bladder Cells and Tissues

Urothelial carcinoma in situ (CIS) is clearly related to tumor recurrence and subsequent cancer progression1. CIS detection remains a challenge for urologists since this lesion, which is only a few cell layers in thickness, may be asymptomatic and/or not visible under conventional white light cystoscopy.