Biomedical subjects
Publications and source records attributed to Y Lajat.
The synthesis and characterization of 2,3-dihydro-5,15-di(3,5-dihydroxyphenyl) porphyrin is reported. The phototoxicity on C6 cell lines and the pharmacokinetics are also reported as preliminary results showing a very high tumor to skin ratio and short retention time in tissues, and thus promising activity in photodynamic therapy.
This paper reports the synthesis of a new diphenylchlorin photosensitizer, 2,3-dihydro-5,15-di(3,5-dihydroxyphenyl)porphyrin (SIM01). The photodynamic properties, cell uptake and localization of SIM01 were compared with those of structurally related meso-tetra(hydroxyphenyl)chlorin (m-THPC). In vitro studies were conducted on rat glioma cells (C6) and human adenocarcinoma (HT-29), and in vivo studies on human colon adenocarcinoma cells (HT-29) and human prostate adenocarcinoma cells (PC3). Both dyes showed an absorption maximum at around 650 nm, with a molar extinction coefficient of 13017 M(-1) cm(-1) for SIM01 and 22718 M(-1) cm(-1) for m-THPC. Their capacity to generate singlet oxygen was identical, but differences in partition coefficients indicated that SIM01 was slightly more hydrophilic. In vitro, SIM01 was slightly more phototoxic than m-THPC for C6 cells (4.8 vs. 6.8 microg ml(-1)). However, phototoxicities were nearly identical for HT29 cells (0.45 microg ml(-1) for 5 h incubation followed by 300 mW, 20 J cm(-2)). Pharmacokinetics in vivo in mice, as determined by fibre spectrofluorimetry, showed that the SIM01 fluorescence signal in the tumor was maximal between 6 and 12 h after injection, as compared to 72 h for m-THPC. With a 2 mg kg(-1) dye dose and laser irradiation at 300 J cm(-2) (650 nm, 300 mW), the optimal PDT response occurred when the interval between injection and irradiation was 6 h for SIM01 and 24 h for m-THPC. For SIM01 with 5 mg kg(-1) injection, the optimal PDT response occurred with a 12 h delay and with the same irradiation parameters as described above, in this case the tumor response showing 40% growth. Considering the tumor volume doubling time, the value was 6.5 days in the control group and increased to 13.5 days with SIM01. Thus, SIM01 may be a powerful sensitizer characterized by strong in vitro and in vivo phototoxicity and faster tissue uptake and elimination than m-THPC.
As many types of cells exposed to photodynamic therapy (PDT) appear to undergo apoptosis, various apoptosis inhibitors have already been used in studies of PDT-induced apoptosis. Although these inhibitors decrease apoptosis, their real effect on the phototoxic efficacy of photosensitisers is unclear. The good phototoxicity of m-THPC was confirmed on murine melanoma B16-A45 cells. Toxicity and phototoxicity studies were then carried out using four apoptosis inhibitors: BAPTA-AM, Forskolin, DSF, and Z.VAD.fmk. Although all inhibitors tested blocked PDT-induced apoptosis, none produced a significant modification of the phototoxic effect of m-THPC on B16 cells. It has been suggested that apoptosis and necrosis share common initiation pathways and that the final outcome is determined by the presence of an active caspase. This implies that apoptosis inhibition reorients cells to necrosis, i.e. those cells sufficiently damaged by PDT appear to be killed, regardless of the mechanism involved.
The purpose of this study was to estimate the efficacy of an endogenous sensitizer (delta-aminolevulinic acid (or ALA) induced protoporphyrin IX (or PpIX)) and an exogenous sensitizer (meta(tetrahydroxyphenyl)chlorin or m-THPC) on two different cell lines, rat colon adenocarcinoma PROb cells and murine melanoma B16A45 (B16) cells, in apoptosis production. After sensitizer incubation, cells were irradiated with an argon dye laser. LD(50) with m-THPC was 2.8 microg/ml and 4.7 microg/ml under irradiation of 25 J/cm(2) respectively for PROb and B16 cells. With ALA, LD(50) was 150 microg/ml and 175 microg/ml under 25 J/cm(2) respectively for PROb and B16 cells. Apoptosis induction by m-THPC or ALA-PDT was detected by DNA gel electrophoresis and quantified using an ELISA assay 24 h after PDT. The maximal apoptosis enrichment factor (MAEF) was reached for 6 microg/ml m-THPC at 10 J/cm(2) for PROb and B16 cells and for 50 microg/ml ALA at 25 J/cm(2) for PROb or B16 cells. Both m-THPC and PpIX are efficient photosensitizers and apoptosis inducers. However, MAEF is obtained by sensitizer or laser doses inducing very different phototoxic effects: MAEF was obtained after m-THPC-PDT with LD(78) for PROb cells and LD(30) for B16 cells and after ALA-PDT with LD(22) for PROb cells and LD(18) for B16 cells. However the overall m-THPC/PDT apoptotic induction (under the curve surface analysis) was not different whatever the cell line for 10 and 25 J/cm(2). On the contrary, ALA-PpIX/PDT apoptotic induction was twice for 25 J/cm(2) as compared to 50 J/cm(2) (p < 0.01) for both the PROb and B16 cells. These results indicate that the apoptosis rate in PDT cell killing varies considerably according to cell type and sensitizer.
Photodynamic therapy (PDT) induces cell-membrane damage and alterations in cancer-cell adhesiveness, an important parameter in cancer metastasis. These alterations result from cell sensitivity to photosensitizers and the distribution of photosensitizers in cells. The efficacy of photosensitizers depends on their close proximity to targets and thus on their pharmacokinetics at the cellular level. We studied the cellular distribution of photosensitizers with a confocal microspectrofluorimeter by analysing the fluorescence emitted by benzoporphyrin derivative-monoacid ring A (BPD-MA) and Photofrin relative to their cell sensitivity. Two cancer cell lines of colonic origin, but with different metastatic properties, were used: PROb (progressive) and REGb (regressive). For BPD-MA (1.75 microg/ml), maximal fluorescence intensity (8,300 cts) was reached after 2 h for PROb and after 1 h (4,900 cts) for REGb. For Photofrin (10 microg/ml), maximal fluorescence intensity (467 cts) was reached after 5 h for PROb and after 3 h (404 cts) for REGb. Intracellular studies revealed stronger cytoplasmic than nuclear fluorescence for both BPD and Photofrin. Both of the sensitizers induced a dose-dependent phototoxicity; LD50 with BPD-MA was 93.3 ng/ml for PROb and 71.1 ng/ml for REGb, under an irradiation of 10 J/cm2. With Photofrin, LD50 was 1,270 ng/ml for PROb and 1,200 ng/ml for REGb under an irradiation of 25 J/cm2. The photosensitizer effect within PROb and REGb cancer cells was assessed by incorporation kinetics and toxicity-phototoxicity tests. The intracellular concentration of the photosensitive agent was one important factor in the effectiveness of PDT, but not the only one contributing to the photodynamic effect. In conclusion, this study showed that there was a clear difference between sensitizer uptake and phototoxicity, even in cancer cells of the same origin. This could induce cell-killing heterogeneity in clinics.
Photodynamic therapy (PDT) with Photofrin has already been authorized for certain applications in Japan, the USA and France, and powerful second-generation sensitizers such as meta-(tetrahydroxyphenyl) chlorin (m-THPC) are now being considered for approval. Although sensitizers are likely to localize within the cytoplasm or the plasma membrane, nuclear membrane can be damaged at an early stage of photodynamic reaction, resulting in DNA lesions. Thus, it is of critical importance to assess the safety of m-THPC-PDT, which would be used mainly against early well-differentiated cancers. In this context, m-THPC toxicity and phototoxicity were studied by a colorimetric MTT assay on C6 cells to determine the LD50 (2.5 microg/ml m-THPC for 10 J/cm2 irradiation and 1 microg/ml for 25 J/cm2 irradiation) and PDT doses inducing around 25% cell death. Single-cell electrophoresis (a Comet assay with Tail Moment calculation) was used to evaluate DNA damage and repair in murine glioblastoma C6 cells after LD25 or higher doses for assays of PDT. These results were correlated with m-THPC nuclear distribution by confocal microspectrofluorimetry. m-THPC failed to induce significant changes in the Tail Moment of C6 cells in the absence of light, whereas m-THPC-PDT induced DNA damage immediately after irradiation. The Tail Moment increase was not linear (curve slope being 43 for 0-1 microg/ml m-THPC and 117 for 1-3 microg/ml), but the mean value increased with the light dose (0, 10 or 25 J/cm2) and incubation time (every hour from 1 to 4 h) for an incubation with m-THPC 1 microg/ml. However, cultured murine glioblastoma cells were capable of significant DNA repair after 4 h, and no residual DNA damage was evident after 24-h post-treatment incubation at 37 degrees C. An increase in the light dose appeared to be less genotoxic than an increase in the m-THPC dose for similar toxicities. Our results indicate that m-THPC PDT appears to be a safe treatment since DNA repair seemed to not be impaired and DNA damage occurred only with lethal PDT doses. However, the Comet assay cannot give us the certainty that no mutation, photoadducts or oxidative damage have been developed so this point would be verified with another mutagenicity assay.
Delta-aminolevulinic acid (ALA)-PDT efficacy is particularly dependent on the quality of protoporphyrin IX (PpIX)-induced synthesis. The purpose of this study was to determine the ability of cells from two human cancer types to synthesise PpIX after ALA administration. Biopsies of glioma cells have been obtained from patients with glioblastomas that have or have not been given ALA IV (ex vivo incubation). Peripheral blood lymphocytes, obtained from leukemic patients, have also been ALA-incubated in vitro. In glioma cells, fluorescence heterogeneity was extensive either in ALA infused patients or in ex vivo ALA incubated cells. Mean intensities after 3 h were 110 cts (range 0-340) and 1000 cts (range 0-3600). Similar results were found in leukemic lymphocytes where cell fluorescence varied from 0 to 480 cts with a percentage of fluorescent cells varying with time and from one patient to another. Furthermore, PpIX was not detectable in two patients with CLL. These observations suggest that a marked heterogeneity of ALA uptake and/or PpIX synthesis exists in a given human cancer cell population particularly after systemic administration. Improvements for ALA transformation into PpIX are strongly recommended to ensure the efficacy of ALA/PpIX-PDT.
Synthesis of delta-aminolevulinic acid (ALA) derivatives is a promising way to improve the therapeutic properties of ALA, particularly cell uptake or homogeneity of protoporphyrin IX (PpIX) synthesis. The fluorescence emission kinetics and phototoxic properties of ALA-n-pentyl ester (E1) and R,S-ALA-2-(hydroxymethyl) tetrahydrofuranyl ester (E2) were compared with those of ALA and assessed on C6 glioma cells. ALA (100 micrograms/mL), E1 and E2 (10 micrograms/mL) induced similar PpIX-fluorescence kinetics (maximum between 5 and 7 h incubation), fluorescence being limited to the cytoplasm. The 50% lethal dose occurred after 6 h with 45, 4 and 8 micrograms/mL of ALA, E1 and E2, respectively. ALA, E1 and E2 induced no dark toxicity when drugs were removed after 5 min of incubation. However, light (25 J/cm2) applied 6 h after 5 min incubation with 168 micrograms/mL of each compound induced 85% survival with ALA, 27% with E1 and 41% with E2. Increasing the incubation time with ALA, E1 and E2 before washing increased the phototoxicity, but E1 and E2 remained more efficient than ALA, regardless of incubation time. ALA-esters were more efficient than ALA in inducing phototoxicity after short incubation times, probably through an increase of the amount of PpIX synthesized by C6 cells.
PpIX synthesis after incubation with delta-aminolevulinic acid (ALA) is highly variable from one cell to another within a single cell population and in human glioblastomas in vivo. To improve PpIX synthesis, we attempted to modify the PpIX synthesis pathway in a C6 glioma cell model. To perform this experiment we used confocal microspectrofluorometry to analyse the effects of a highly purified form of sulfentrazone (FP846) on the kinetics of PpIX synthesis after ALA administration to living C6 cells. Our results show that PpIX fluorescence was maximal (seven-fold higher than basal values) 3 to 4 hrs. after the beginning of incubation with ALA. FP846 depressed this increase in fluorescence nearly to basal levels not only in C6 cells but also in HT29 and HepG2 cells. Fluorescence spectra shape were not affected by FP846, except for intensity. ALA/PpIX-induced photocytoxicity was perfectly correlated with fluorescence intensity recorded in cell cytoplasm. ALA alone (100 microg/ml) did not induce a significant decrease in cell survival, but irradiation of 25 J/cm2 leading to an overall cell death of 60%. FP846 added together with ALA suppressed ALA/PpIX-induced phototoxicity. The fact that the FP846-induced decrease in PpIX synthesis was not the same in animal and plant cells suggests that the porphyrin metabolic pathway differs due to the relative amounts of substrate or the effect of inhibitor and that another chemical would be needed alone or in combination with FP846 to improve PpIX synthesis.
Photodynamic therapy (PDT) could be a useful adjuvant in glioblastoma treatment. The fact that epidermal growth factor (EGF) and its receptor are involved in glioblastoma growth control led us to investigate the relationships between EGF and PDT with respect to three different glioma cell lines (C6, T98 G, U87 MG) responsive to growth stimulation by EGF. Flow cytometric analysis revealed that each cell line expressed EGF receptors. PDT was then applied to the cells using haematoporphyrin derivative (HPD) as photosensitizer and argon laser irradiation. When cells were incubated for 2 h with HPD (0.1-10 micrograms/ml) and then laser-irradiated (lambda = 514 nm; energy density 25 J/cm2), all three cell lines showed photosensitivity. The median lethal dose was respectively 3, 4.5 and 2.7 micrograms/ml for C6, T98 G and U87 MG. EGF (2-50 ng/ml) had no effect on HPD- and laser-induced toxicity when added to cells before PDT, whereas toxicity decreased for all three cell lines when EGF was added after PDT. HPD (1-2 micrograms/ml, incubation times 30-180 min) also induced an increase in EGF receptor expression for the C6 line.
Photofrin is a potent sensitizer which localizes, among other sites in membranes of malignant cells. To evaluate the effect of photodymanic therapy (PDT) on specific antitumoral immunological response, we used a chromium release assay to compare the specific cytolytic activity (CLA) of primed mouse spleen T lymphocytes sensitized against syngeneic mastocytoma P511 cells. P511 cells or lymphocytes or both were treated or not with Photofrin and/or light (514 nm). Photofrin alone (1 microg/ml, 2 h) reduced CLA by 59% when P511 cells were treated although this decrease was not drug dose dependent. Photofrin (1 microg/ml, 2 h) followed by light (25 J/cm2) reduced CLA by 35% in a drug dose dependent manner. Longer incubation times led to reduced CLA inhibition (10% for 3 h incubation) after Photofrin followed by light. The light dose (25, 37, 50 J/cm2) did not influence CLA for a given Photofrin concentration. Photofrin alone (0.5 microg/ml, followed by light (25 J/cm2 for 2 h) reduced CLA respectively by 8 and 45% only when lymphocytes were treated. When lymphocytes and P511 cells were treated with Photofrin alone or followed by light (25 J/cm2), CLA was also reduced (by 19 and 41% respectively). This type of damage can be evaluated in terms of antigen expression on the target cells, on the lymphocyte T receptor, on H-2 (histocompatibility major complex), or on lymphocyte activity after PDT.
Endogenously generated protoporphyrin IX (PpIX) from exogenous delta-aminolevulinic acid (ALA) has the photodynamic capacity to inactive cancer cells of different origins. The aim of this study was to characterize the ability of normal lymphocytes to transform ALA into PpIX in order to appreciate through further studies changes in pathologic lymphocytes. We investigated in this study PpIX synthesis by normal human lymphocytes using a confocal laser microspectrofluorometer. Live lymphocytes were identified by monoclonal antibody fluorescent labeling. B and T lymphocytes synthesized PpIX (80-100 counts), with a maximum being reached after 4 h ALA incubation. When T subpopulations of lymphocytes were labeled, T4 and T8 changes in fluorescence kinetics were similar, reaching a maximum after 5 h ALA incubation. The influence of monoclonal antibody labeling on this delayed increase for maximum fluorescence is considered. Phytohemagglutinin (PHA, incubation for 72 h) lymphocyte stimulation induced a 100% increase in PpIX fluorescence for T lymphocytes, whereas pokeweed mitogen activation produced an increase of about 50% in the B- or T-lymphocyte signal. Finally, the scanning fluorescence image clearly indicated the inhomogeneity of cytoplasmic ALA-induced PpIX fluorescence, which was probably due to the distribution of mitochondria. The influence of this heterogeneity on PpIX photosensitivity effects is discussed.
A delta 5-aminolevulinic acid (ALA) bioadhesive gel has been developed and evaluated in an in-vivo mouse model for photodynamic treatment of gastric cancer or Barrett's oesophagus. Four gels were tested: noveon AA-1, keltrol T, lutrol and blanose. An initial in-vitro study of gel adhesion showed that noveon and keltrol had longer polyethylene transit times than lutrol and blanose. In-vivo assays indicated that protoporphyrin IX was synthesized by gastric mucosa when ALA-noveon and ALA-lutrol were used (preferable results for noveon). Keltrol was eliminated from the study after these investigations. Only ALA-noveon gel was retained for studies of the relationship between ALA dose and fluorescence. Fluorescence measurements in-vivo showed that ALA concentration and application time had an influence on protoporphyrin IX synthesis. Maximum intensity (2091 counts s-1) was found with 2 mg mL-1 ALA, and fluorescence intensities differed with application time, reaching 1805 counts s-1 after 240 min. ALA-noveon, showing good adhesion and enabling efficient diffusion of ALA at a pH < 6, was considered the best formulation for maintaining ALA stability.
We present an integrated imaging system which enables CT and MRI data to blend with angiographic or ventriculographic films obtained by teleradiography and digitized. From all the data fed to the calculator, orthogonal, frontal, lateral, simple or double-obliquity trajectories can be determined easily and rapidly.
Explore the source record for details and available documents.
Talairach's method remains the most universal of all stereotactic methods. It makes it possible to go back to an examination left interrupted, but above all, it provides multiple lateral and coronal approaches in matters of epilepsy, radio-isotopes or photobiology. The advances achieved in modern imaging methods, notably CT and MRI, and the performance of modern computers have enabled us to devise an integrated imaging system meant to accelerate and make feasible calculations of penetrating trajectories, according to the position of the target as previously defined by CT and MRI sections, and taking into account the position of vessels given by angiography. The principal options we selected were: transfer onto video tapes of teleangiographic films and introduction of CT and MRI images obtained either from films or directly in digits on cartridges or networks. The system includes an advanced PC-type microcomputer with 8 memories of 1,024/1,024 images: 4 mega octets random access memories and a hard disc of 150 mega octets; 2 high-resolution screens to present the images, dialogue tools (alphanumeric screen, keyboard, trackball) and the usual peripherals. Our system performs three main functions: it acquires images to create or complete the patient's records; it exploits the images by calculating the parameters required for operations; it handles the records through the various peripherals of the system. Our system can retrace the proportional 3D squaring according to the CA-CP distance calculated from ventriculography or MRI, and calculates the position of the SEEG electrodes. The stereoscopic effect is obtained by means of glasses with liquid crystal obturator from two angiographic series: one orthogonal, the other 6 degrees out of phase. The daily use of this system on more than 50 patients has shown that it is accurate and reliable, irrespective of the makes of CT and MRI machines.
Explore the source record for details and available documents.
A number of chronic pain syndromes in the perineal area can be related to pudendal nerves suffering. The constancy of symptoms among various patients, and in duration for a particular one, alterations revealed by electrophysiologic studies, pain relief by diagnostic blocks, data from anatomic studies, preliminary results of medical and surgical applied therapies, give consistent arguments for possible organic lesions of pudendal nerves.