Protoporphyrin IX enhancement by 5-aminolaevulinic acid peptide derivatives and the effect of RNA silencing on intracellular metabolism.

Intracellular generation of the photosensitiser, protoporphyrin IX, from a series of dipeptide derivatives of the haem precursor, 5-aminolaevulinic acid (ALA), was investigated in transformed PAM212 murine keratinocytes, together with studies of their intracellular metabolism. Porphyrin production was substantially increased compared with equimolar ALA using N-acetyl terminated phenylalanyl, leucinyl and methionyl ALA methyl ester derivatives in the following order: Ac-L-phenylalanyl-ALA-Me, Ac-L-methionyl-ALA-Me and Ac-L-leucinyl-ALA-Me. The enhanced porphyrin production was in good correlation with improved photocytotoxicity, with no intrinsic dark toxicity apparent. However, phenylalanyl derivatives without the acetyl/acyl group at the N terminus induced significantly less porphyrin, and the replacement of the acetyl group by a benzyloxycarbonyl group resulted in no porphyrin production. Porphyrin production was reduced in the presence of class-specific protease inhibitors, namely serine protease inhibitors. Using siRNA knockdown of acylpeptide hydrolase (ACPH) protein expression, we showed the involvement of ACPH, a member of the prolyl oligopeptidase family of serine peptidases, in the hydrolytic cleavage of ALA from the peptide derivatives. In conclusion, ALA peptide derivatives are capable of delivering ALA efficiently to cells and enhancing porphyrin synthesis and photocytotoxicity; however, the N-terminus state, whether free or substituted, plays an important role in determining the biological efficacy of ALA peptide derivatives.

In vitro fluorescence, toxicity and phototoxicity induced by delta-aminolevulinic acid (ALA) or ALA-esters.

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.

Enhancement of delta aminolevulinic acid-photodynamic therapy in vivo by decreasing tumor pH with glucose and amiloride.

OBJECTIVES/HYPOTHESIS: Delta aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) is a fluorescent sensitizer that permits detection and treatment of squamous cell carcinoma of the oral cavity. An exogenously induced decrease in tissue pH was evaluated for its effect in enhancing cellular uptake of ALA and facilitating its transformation into PpIX. STUDY DESIGN: Mice grafted with HT29 colonic cancers had been given glucose and amiloride to modify the pH of tissues. Influence of pH changes has been evaluated on ALA-induced PPIX fluorescence by optic fiber spectrofluorimetry as well as on tumor growth. RESULTS: The pH in HT 29 tumor decreased from 7.1 to 6.67 (P < .05) after intraperitoneal injection of glucose and amiloride. The PpIX fluorescence ratios in tumor or muscle before, between, and 2 hours after glucose and amiloride injection were not higher than control ratios. Aminolevulinic acid-photodynamic therapy was more efficient on HT 29 tumor-bearing mice when the pH value was decreased with glucose and amiloride, showing a difference in the tumor growth index ratio from the 1st to 14th day of 22% between amiloride-glucose aminolevulinic acid-photodynamic therapy and aminolevulinic acid-photodynamic therapy alone (P < .05). CONCLUSIONS: Glucose and amiloride did not change PpIX fluorescence in HT 29 tumor after intraperitoneal injection of aminolevulinic acid but enhanced aminolevulinic acid-photodynamic therapy efficacy. This was probably a result of mechanisms other than an increase in aminolevulinic acid cellular penetration and PpIX production, such as susceptibility to free radical toxicity or alteration of cellular repair enzymes under acidotic conditions. If a decrease of pH induces a more efficient photodynamic therapy as suggested by our results, an easier way to obtain this decrease than glucose and amiloride would be necessary for clinical applications.

Tissue detection of diphenylchlorin sensitizer (SIM01) by fluorescence and high-performance liquid chromatography.

Cancer is today a major problem of public health. Unfortunately, the current treatments remain still too often impotent or too heavy compared to the gross national product of many countries. The use of PDT in the treatment of the malignant tumours currently raises great hopes. This physicochemical method is based on the combined action of a nontoxic drug given systematically to the patient and of the visible light delivered locally to the tumour using optical fibres. The radiation will activate the significant substance preferentially fixed on cancerous cells and will cause the death of the tumoral cells while releasing from the toxic ridicalizing species which then will deteriorate vital cellular targets. Tissue distribution and elimination kinetics of the SIM01 were analysed in biological samples from mice tissues by spectrofluorometry and HPLC. Measurements were performed 4, 6, 12, 24 and 48h after an intraperitoneal injection for SIM01 doses of 2, 5 and 15mgkg(-1). Elimination seemed to concern essentially gallbladder, liver and stools, where maximum fluorescence reached, respectively, 20,000, 2800 and 15,000cps for 5mgkg(-1), 6h after injection. Among the tissues examined with HPLC, the highest SIM01 levels were found in stools, urine, liver, gallbladder and spleen. Liver, gallbladder, and stool homogenates from drug-treated animals contained an additional peak (16, 7min) detectable only after injection of at least 15mgkg(-1). Our HPLC determinations and in vivo fluorescence detection of SIM01 gave comparable kinetic profiles. These techniques should be considered as complementary rather than exclusive for kinetic profiles determination.

Synthesis, and in vitro and in vivo evaluation of a diphenylchlorin sensitizer for 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.

Gene silencing of TNF-alpha in a murine model of acute colitis using a modified cyclodextrin delivery system.

Inflammatory bowel disease (IBD) is a chronic relapsing inflammation of the gastrointestinal tract. The cytokine TNF-alpha (TNF-α) plays a pivotal role in mediating this inflammatory response. RNA interference (RNAi) holds great promise for the specific and selective silencing of aberrantly expressed genes, such as TNF-α in IBD. The aim of this study was to investigate the efficacy of an amphiphilic cationic cyclodextrin (CD) vector for effective TNF-α siRNA delivery to macrophage cells and to mice with induced acute-colitis. The stability of CD.siRNA was examined by gel electrophoresis in biorelevant media reflecting colonic fluids. RAW264.7 cells were transfected with CD.TNF-α siRNA, stimulated with lipopolysaccharide (LPS) and TNF-α and IL-6 responses were measured by PCR and ELISA. Female C57BL/6 mice were exposed to dextran sodium sulphate (DSS) and treated by intrarectal administration with either CD.siRNA TNF-α or a control solution. In vitro, siRNA in CD nanocomplexes remained intact and stable in both fed and fasted simulated colonic fluids. RAW264.7 cells transfected with CD.TNF-α siRNA and stimulated with LPS displayed a significant reduction in both gene and protein levels of TNF-α and IL-6. CD.TNF-α siRNA-treated mice revealed a mild amelioration in clinical signs of colitis, but significant reductions in total colon weight and colonic mRNA expression of TNF-α and IL-6 compared to DSS-control mice were detected. This data indicates the clinical potential of a local CD-based TNF-α siRNA delivery system for the treatment of IBD.

Effects of BAPTA-AM, Forskolin, DSF and Z.VAD.fmk on PDT-induced apoptosis and m-THPC phototoxicity on B16 cells.

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.

Cellular distribution and phototoxicity of benzoporphyrin derivative and Photofrin.

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.

PDT effects of m-THPC and ALA, phototoxicity and apoptosis.

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.