Ludovic Bourre

Hydroporphyrins as tumour photosensitizers: synthesis and photophysical studies of 2,3-Dihydro-5,15-di(3,5-dihydroxyphenyl) porphyrin

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.

Improved peptide prodrugs of 5-ALA for PDT: rationalization of cellular accumulation and protoporphyrin IX production by direct determination of cellular prodrug uptake and prodrug metabolization.

Twenty-seven dipeptide derivatives of general structure Ac-Xaa-ALA-OR were synthesized as potential prodrugs for 5-aminolaevulinic acid-based photodynamic therapy (ALA-PDT). Xaa is an alpha-amino acid, chosen to provide a prodrug with appropriately tailored lipophilicity and water solubility. Although no simple correlation is observed between downstream production of protoporphyrin IX (PpIX) in PAM212 keratinocytes and HPLC-derived descriptors of compound lipophilicity, quantification of prodrug uptake reveals that most of the dipeptides are actually more efficiently accumulated than ALA in PAM212 and also A549 and Caco-2 cell lines. Subsequent ALA release is the limiting factor, which emphasizes the importance of decoupling prodrug uptake and intracellular metabolization when assessing the efficacy of ALA derivatives for PDT. In agreement with PpIX fluorescence studies, at a concentration of 0.1 mM, l-Phe derivatives 4m and 4o, and l-Leu, l-Met, and l-Glu derivatives 4f, 4k, and 4u, exhibit significantly enhanced photoxicity in PAM212 cells compared to ALA.

5-Aminolaevulinic acid peptide prodrugs enhance photosensitization for photodynamic therapy

Intracellular porphyrin generation following administration of 5-aminolaevulinic acid (ALA) has been widely used in photodynamic therapy for a range of malignant and nonmalignant lesions. However, ALA is relatively hydrophilic and lacks stability at physiologic pH, limiting its bioavailability. We have investigated more lipophilic, uncharged ALA-peptide prodrugs based on phenylalanyl-ALA conjugates, which are water soluble and chemically stable for improving ALA delivery. Pharmacokinetics of the induced protoporphyrin IX (PpIX) were studied in transformed PAM212 keratinocyte cells and pig skin explants. The intracellular porphyrin production was substantially increased with Ac-l-Phe-ALA-Me (compound 1) and Ac-l-Phe-ALA (compound 3) compared with equimolar ALA: after 6-h incubation, the PpIX fluorescence measured using 0.01 mmol/L of compound 1 was enhanced by a factor of 5 compared with ALA. Phototoxicity results showed good correlation with PpIX levels, giving a LD50 (2.5 J/cm2) of 25 μmol/L for ALA, 6 μmol/L for 5-aminolaevulinic hexyl ester, and 2.6 μmol/L for compound 1, which exhibited the highest phototoxicity. However, these results were stereospecific because the corresponding d-enantiomer, Ac-d-Phe-ALA-Me (compound 2), induced neither porphyrin synthesis nor phototoxicity. PpIX levels were considerably reduced when cells were incubated with compound 1 at low temperatures, consistent with active transport. In pig skin explants, compound 1 induced higher porphyrin fluorescence than ALA by a factor of 3. These results show that water-soluble peptide prodrugs of ALA can greatly increase its cellular uptake, generating more intracellular PpIX and improved tumor cell photosensitization. The derivatives are comparable in efficacy with 5-aminolaevulinic hexyl ester but less toxic and more stable at physiologic pH. [Mol Cancer Ther 2008;7(6):1720–9]

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.

Quantitative determination of 5-aminolaevulinic acid and its esters in cell lysates by HPLC-fluorescence

The development of a reliable sensitive method for the HPLC determination of 5-aminolaevulinic acid (ALA) and ALA esters in cell lysates is described. The method relies on the quantification of a fluorescent derivative of ALA following its derivatisation with acetylacetone and formaldehyde. Following this procedure it is possible to quantify ALA in cell lysates with no need for pre-purification of the sample. The method has been validated in two ranges of concentration (0.6-65 microM, 0.1-10 microg/mL, and 30-600 microM, 5-100 microg/mL), and has also been extended and validated for the determination of ALA released from ALA prodrugs after acidic hydrolysis.

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.

The influence of storage conditions on delta amino levulinic acid induced toxicity and phototoxicity in vitro

Delta amino levulinic acid photodynamic therapy (ALA-PDT) represents one of the most prominent advances in PDT. ALA itself or its derivatives are marketed for a variety of clinical indications. Despite the development of clinical applications, experimental ALA results are very heterogeneous and experimentally used parameters are still not standardized. This suggests that some problems remain unsolved that are likely to impair experiments to be performed but also that clinical results obtained could be greatly improved. Frequently unmentioned or imprecise data concern solvents, pH of ALA solutions, storage time, ALA degradation or ALA efficacy. In addition, diversity of experimental model is huge while capabilities of ALA transformation into PpIX are known to vary from one cell to the other. Thus, the aim of the present paper was to quantify the level of ALA degradation or changes in ALA efficacy using one single cell line without presuming of the mechanisms and determine the conditions of storage inducing the best transformation into PpIX and/or cell phototoxicity. We added ALA diluted in water, PBS or RPMI to C6 cells, a murine brain tumour cell line that can be used in vivo as an orthotopic graft. We measured in cells used as tools for final bio efficacy estimation, both the induced fluorescence and phototoxicity in various conditions of storage before use chosen to be as close as possible to the real lab conditions. Water had been found to better preserve ALA than, respectively, PBS and RPMI and this for any temperature or storage durations. The lowest temperature and the shortest duration for storage used had also been shown to better preserve ALA-induced fluorescence and phototoxicity. The fact that these properties were found to be better preserved in 7.4 buffered solvent could be in relationship with a fast ALA condensation occurring at neutral or lightly acidic pH modifying its availability for an optimal transformation into PpIX.

DNA damages after SIM01 photodynamic treatment

Photodynamic therapy (PDT) is a new approach to cancer treatment for a variety of malignant tumors. A new photosensitizer, 2,3-dihydro-5,15-di(3,5-dihydroxyphenyl)porphyrin (SIM01), had been evaluated for its genotoxic effects on glioma cells (C6). Comet assay had been used to evaluate the potential genotoxic effect induced by SIM01 on the C6 cells. When SIM01 had been shown to be a powerful sensitizer no DNA strand break was detected in the absence of light. SIM01 localized in cytoplasm but not in the nucleus of the tumors cells, which supported the finding of undetectable DNA damage under darkness and low photodynamic dose. Cell exposure to 20Jcm(-2) after an incubation time of 2h with 0, 0.25, 0.5, 2 or 4μgmL(-1) induced less than 25% of cell death but significant Tail Moment changes. If DNA damage intensity increased according to SIM01 doses under light exposure, importance of repair seemed to increase proportionally to PDT-induced damage. Positive controls consisted of doxorubicin-treated C6 cells this mutagen being known to induce genetic damage. Whatever the conditions used SIM01 appeared to be less deleterious than doxorubicin. As the comet assay can not give us the certitude that no mutation, photoadducts or oxidative damage had been developed under light exposure this point will have to be verified with another mutagenicity assay. SIM01 appears to be safe from a mutagenic point of view something of importance as tumors of small volume in patients with a long lifespan are at first indicated for PDT.

Abstract B164: A platform of metastatic bioluminescent PDX: Focus on HBCx14-Luc1 metastatic bioluminescent breast tumorgaft model for preclinical studies.

Background: The ability of tumor cells to spread and form metastases is one of the key parameters used to classify tumor aggressiveness. Metastasis detection at diagnosis or during followup post-surgery is associated with poor prognosis. The paucity of pertinent models to investigate the biology of metastasis renders problematic the set-up of anti-metastasis therapies. We took advantage of our human patient-derived xenograft (PDX) collection to develop models suitable to assess anti-metastatic therapy. To enable evaluation of metastasis response to treatment, we labeled tumors with lentivirus-mediated luciferase insertion, allowing longitudinal followup of metastasis formation by non-invasive imaging. A review of our metastatic bioluminescent PDX panel is described here with a particular focus on our most advanced bioluminescent breast model HBCx-14-Luc1. Methods: Tumor cells from freshly excised PDXs were dissociated and transduced with a defective lentivirus bearing the firefly luciferase gene. One million transduced cells were injected subcutaneously into immunocompromised mice (Nude or SCID), tumor growth and metastasis formation were monitored throughout passages by whole body visualization and ex vivo organ imaging at the end point using the Xenogen IVIS® Lumina. Bioluminescence was measured every two weeks, over 8 to 20 weeks, to assess metastasis appearance and progression. The presence of metastases was confirmed by histological analysis. Results: Lung metastases developed in a variety of PDX models (breast, prostate, pancreas, and melanoma) with different output according to the model and the mouse strain used. SCID mice were more permissive to metastasis development than nude mice. In our most advanced model, HBCx-14-Luc1, the influence of primary tumor resection was assessed on both lung metastasis apparition delay and frequency of metastasis positive mice. Regarding metastasis positive mice at the end point, no difference was observed between resected (7/8 positive at D88) and non-resected mice (11/13 at D93). However, while the first day of metastasis apparition was similar between non-resected (D48) and resected mice (D53), the number of metastasis positive mice at this early time-point was higher for the non-resected (7/8) compared to resected mice (3/18). Histological examination confirmed the adenocarcinomatous origin of metastases. Another breast PDX model, HBCx-5-Luc2, was metastatic for pancreas, brain, spleen, liver, lung, and bones as assessed by ex vivo screening. Additional PDX models were screened similarly to get a metastasis organ mapping. Conclusions and Perspectives: Bioluminescent metastatic PDX models of various tumor histotypes were successfully established by growing luciferase-engineered PDXs subcutaneously into appropriate mouse strains. These models will provide a useful tool to explore the biology of metastasis and evaluate anti-metastatic therapies in a selection of organs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B164. Citation Format: Ludovic Bourre, Mylene Bernes, Enora Leven, Vanessa Yvonnet, Olivier Deas, Myriam Lassalle, Jean-Gabriel Judde. A platform of metastatic bioluminescent PDX: Focus on HBCx14-Luc1 metastatic bioluminescent breast tumorgaft model for preclinical studies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B164.

Abstract C47: A platform of metastatic bioluminescent tumorgafts for preclinical studies.

Background: The ability of tumor cells to spread and form metastasis is one of the key parameters used to classify tumor aggressiveness. Metastasis detection at diagnosis or during follow-up post-surgery is associated with poor prognosis. The lack of pertinent models to investigate the biology of metastasis renders problematic the set-up of anti-metastasis therapies. We took advantage of our tumorgraft collection to develop models suitable to assess anti-metastatic therapy. Furthermore, to enable measurement of metastasis response to treatment, we labeled tumors with lentivirus-mediated luciferase insertion into tumor cells, allowing the follow up of metastasis formation by non invasive imaging. Methods: In order to obtain a bioluminescent model, upon tumor resection, HBCx-12B tumor cells were dissociated and transduced with a defective lentivirus bearing the firefly luciferase gene. One million cells were injected subcutaneously in different strains of immunocompromised mice (RAG γC−/−; RAG γC−/− FLK; Nude; SCID; NOD SCID), tumor growth and metastasis formation were monitored throughout passages by whole body visualization using the Xenogen IVIS® Lumina. Mice were imaged over 10 to 24 weeks, and in order to prolong the follow-up window for metastasis detection, tumor resection was performed when primary tumor size reached 500 mm3. During this period, bioluminescence was measured every two weeks to assess metastasis appearance and progression. Bio-imaging allowed metastasis detection with a threshold of 100 to 400 cells per metastatic focus. The presence of metastases was confirmed by both histological analysis, and ex vivo organ fluorescence imaging. The pro-drug 5-aminolevulinic acid was injected intravenously, transformed into protoporphyrin IX, a fluorescent compound accumulated into tumor cells. Results: Metastases developped in all tested strains with different efficiency. RAG γC −/− and SCID strains had the highest susceptibility, with fast and high metastasis incidence. RAG γC (9/9), RAG γC−/− FLK (2/2) and SCID (4/5) mice presented lung metastases after an average of 50-day independently of the passage, while metastases developed in 1/7 in nude mice after 90 days. Histological examination confirmed the adenocarcinomatous origin of metastases, extravading into the lung arteriolar vessels. Conclusions and perspectives: Bioluminescent metastasis models could be successfully established by growing luciferase-enginered tumorgrafts into appropriate strains of immunocompromised mice. At present, other bioluminescent models of melanoma, pancreatic, and breast cancer tumorgrafts are being validated as metastatic. The establishment of bioluminescent metastasis models of different types of cancer will provide an useful tool to explore the biology of metastasis development and anti-metastatic therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C47.