Alain Croisy

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).

Diphenyl quinolines and isoquinolines: synthesis and primary biological evaluation.

The synthesis of a series of 35 substituted 3,4-diphenyl quinolines and isoquinolines is described. The majority of these molecules differ from all other triphenylethylene based antiestrogens by a different spatial location of the aminoalkyl side chain. The binding affinity of the most representative molecules (8, 9, 19, 20, 21, 23 and 25), including analogues 8 and 21 without the side chain, for the estrogen receptor alpha (ER) was determined. The ability of these molecules to induce the progesterone receptor was also studied. Antiproliferative activity was evaluated on MCF-7 human breast cancer cells, while intrinsic cytotoxic/cytostatic properties resulting from interaction with other targets than ER were assayed on L1210 murine leukemia cells. Introduction of an aminoalkylamino side chain at carbon 2 confers strong cytotoxic properties to diphenylquinolines 9 and 10 as well as pure antiestrogenic activities. However, cytotoxicity is so high with respect to antiestrogenicity that the latter was clearly observable only in one case (9b). The structure of compound 9b was determined by X-ray crystallography. Molecular modeling of its docking within the hormone-binding domain of the receptor was subsequently undertaken. According to our results, the design of molecules with the side chain bound to the ethylene part of the triphenyl ethylene skeleton might generate compounds of potential pharmacological interest.

Intracellular precipitation of hydroxyapatite mineral and implications for pathologic calcification.

In contrast to physiologic biomineralization occurring in bones, teeth and otoconia in vertebrates, calcification of soft tissues occurs in many pathologic conditions. Although similarities have been noted between the two processes, and despite the important clinical consequences of ectopic calcification, the molecular mechanisms regulating ectopic calcification are poorly understood. Although calcification is mainly extracellular, intracellular calcification has been reported and might indeed contribute to pathologic calcification of soft tissues. To better understand the process of intracellular calcification as a potential origin for pathologic calcification, and to examine the role of proteoglycans in this process, we investigated a pattern of intracellular nucleation and growth of hydroxyapatite in Madin-Darby Canine Kidney (MDCK) epithelial cells using electron microscopy, secondary ion mass spectroscopy (NanoSIMS), cytochemical staining, immunolabeling and biochemical analysis. We report here that under mineralizing cell culture conditions where beta-glycerophosphate (betaGP) was added as an exogenous organic source of phosphate, betaGP-cleaving alkaline phosphatase activity increased and hydroxyapatite crystals subsequently nucleated within intracellular, membrane-bounded compartments. The small, leucine-rich proteoglycan decorin was also upregulated and associated with mineral in these cultures. Such information provides insight into the mechanisms leading to pathologic calcification and describes a process whereby hydroxyapatite deposition in cells might lead to ectopic calcification.

23Na MRI longitudinal follow-up of PDT in a xenograft model of human retinoblastoma

BACKGROUND Photodynamic therapy is an established cancer treatment in which a photosensitizing agent is activated by exposure to light thus generating cytotoxic reactive oxygen species that cause cellular damage. METHODS A new photosensitizer synthesized at Curie Institute was used to treat retinoblastoma xenografts in mice, a glycoconjugated meso substituted porphyrin derivative, that showed some retinoblastoma cell affinity. The longitudinal follow-up of the tumors was carried out by (23)Na MRI (without adding exogenous contrast agents) to map the extracellular compartment and to characterize cell packing. Two regimens were followed to target either blood vessels alone or blood vessels and cancer cells simultaneously. RESULTS AND CONCLUSIONS Only the protocol targeting both cancer cells and blood vessels effectively induces cellular death, confirmed by histology at the end of the experiment. Sodium MRI evidences a huge change in the cellular density of tumors only 24h after a double targeting (vascular and cellular) PDT treatment. We suggest that this change was possibly due to a bystander effect that can be promoted by the intercellular signaling favored by the high cellular density of retinoblastoma. These results indicate that non-invasive (23)Na imaging (which detects the tumor response to treatment from very early stages) in association with non-mutagenic therapies represents an effective option for tailored and individualized clinical treatments.

TETRAPYRROLIC GLYCOSYLATED MACROCYCLES FOR AN APPLICATION IN PDT

The synthesis and characterization of amphiphilic glycoconjugated porphyrins, benzochlorin, and azaporphyrins were reported. Among these molecules, several were found to be efficient photosensitizers in an in vitro assay using the human tumoral cell line HT29. Moreover, glycosylated benzochlorin and azaporphyrins, whose absorption bands in the red region of the visible spectrum are substantially increased as compared to porphyrins, display a good photocytotoxicity on tumor cells after irradiation with wavelength above 590 nm.

Transforming growth factor-β1 enhances the lethal effects of DNA-damaging agents in a human lung-cancer cell line

In tissue culture conditions, exogeneous active transforming growth factor‐β1 (TGF‐β1) enhances the lethal effect of DNA‐damaging agents (UV‐C, gamma rays, cisplatin, methotrexate and 5‐fluorouracil) toward human A549 cells and mink Mv1Lu cells, as detected by the loss of their capacity to give rise to colonies; both these cell lines harbor a wild‐type p53, as determined by immunoprecipitation. Contrastingly, the sole effect of the cytokine used alone is to inhibit reversibly the multiplication of the same cells without further impairing, once withdrawn from their environment, their capacity to divide and give rise to colonies. The lethal synergy between TGF‐β1 and UV‐C was studied on mink and human cell lines, and the biomodulation by TGF‐β1 of cell killing by cisplatin, gamma rays, 5‐fluorouracil or methotrexate was tested only on human cells. As investigated with UV‐C‐irradiated human A549 cells, TGF‐β1 appears to enhance apoptosis rather than to disturb the repair of DNA photolesions (mainly pyrimidine dimers) by the nucleotidic excision repair pathway according to results of nucleosomal ladder and comet tests. Our data raise the possibility that, in vivo, TGF‐β1 might affect the curative and/or undesirable secondary side effects of cancer therapy. Int. J. Cancer 72:356–361, 1997.

Retrograde Delivery of Photosensitizer (TPPp-O-β-GluOH)3 Selectively Potentiates Its Photodynamic Activity

Photodynamic therapy involves administration of a photosensitizing drug and its subsequent activation by visible light of the appropriate wavelength. Several approaches to increasing the specificity of photosensitizers for cancerous tissues and, in particular, through their conjugation to ligands that are directed against tumor-associated antigens have been investigated. Here, we have studied the delivery of the photocytotoxic porphyrin compound TPP(p-O-beta-D-GluOH)3 into tumor cells that overexpress the glycosphingolipid Gb3, using the Gb3-binding nontoxic B-subunit of Shiga toxin (STxB) as a vector. To allow for site-directed chemical coupling, an STxB variant carrying a free sulfhydryl moiety at its C-terminal end has been used. Binding affinity, cellular uptake, singlet oxygen quantum yield, and phototoxicity of the conjugate have been examined. Despite some effect of coupling on both the photophysical properties of TPP(p-O-beta-D-GluOH)3 and the affinity of STxB for its receptor, the conjugate exhibited a higher photocytotoxic activity than the photosensitizer alone and was exquisitely selective for Gb3-expressing tumor cells. Furthermore, our data strongly suggest that STxB-mediated retrograde delivery of the photosensitizer to the biosynthetic/secretory pathway is critical for optimal cytotoxic activity. In conclusion, a strong rationale for using retrograde delivery tools such as STxB in combination with photosensitizing agents for the photodynamic therapy of tumors is presented.

Cytostatic Activity of 1,10-Phenanthroline Derivatives Generated by the Clip-Phen Strategy

The cytostatic activities of a series of twelve 1,10‐phenanthroline (Phen) derivatives and of their copper complexes were studied on L1210 murine leukemia cells. Large increases in the biological activity were observed for compounds of the 3‐Clip‐Phen series, in which two Phen moieties were bridged at their C3 positions by an alkoxy linker, the 3‐pentyl‐Clip‐Phen derivative showing an IC50 value of 130 nM while Phen shows an IC50 value of 2500 nM under the same conditions. IC50 values seemed to be modulated not only by the position, the nature, and the length of the linker of Clip‐Phen but also by hydrophobicity. Since copper complexes of Phen are chemical nucleases and nucleic acids are thus a potential target for these compounds, the corresponding copper complexes were also studied. Copper complexation of the 3‐Clip‐Phen ligands did not increase their biological activities. Attempts to vectorize 3‐Clip‐Phen derivatives with a DNA binder such as spermine or with a cell‐penetration peptide failed to increase their biological activity relative to the original 3‐Clip‐Phen series.

Direct in vivo observation of 5-fluorouracil release from a prodrug in human tumors heterotransplanted in nude mice: a magnetic resonance study

A glucuro‐conjugated carbamate derivative of 5‐fluorouracil (5‐FU), originally designed as a prodrug for antibody‐directed enzyme prodrug therapy (ADEPT) application, has been used for direct in vivo observation of in situ 5‐FU generation in two human colon tumors heterotransplanted in nude mice. Because of the very fast elimination of glucuro‐conjugated drugs, this observation required intratumoral injection. These tumors, when becoming necrotic, are rich enough in β‐glucuronidase to allow 19F magnetic resonance spectroscopy monitoring, at the tumor level, of both prodrug elimination and 5‐FU liberation without preliminary treatment by a specifically targeted enzyme conjugate. Convenient tumors have been selected by magnetic resonance imaging (MRI) on the basis of a correlative study between MRI and conventional histology. This contribution is the first report evidencing such a direct intra‐tumoral conversion of a glucuro‐conjugated prodrug into the expected active drug. This method, which should allow overall estimation of the β‐glucuronidase content of tumors, might also be helpful for selecting tumors as specific targets for non‐toxic glucuro‐conjugated prodrugs without prior treatment with a fusion protein. Copyright

In vitro photobiological activity of a new series of photosensitizers: the glycoconjugated porphyrins

The in vitro photodynamic inactivation of a KB cell line is studied using a large series of neutral water-soluble glycosylated porphyrins. These compounds in which hydrophilic glycosyl moieties and hydrophobic substituents are linked at the meso positions, generate singlet oxygen in roughly 70%. Results reveal that the phototoxicity is markedly dependent on the nature, the number, and the linking position of glycosyl moieties controlling their amphiphilic characters.