Publications by authors named "Pierre Vierling"

29 Publications

  • Page 1 of 1

Formulation of highly functionalizable DNA nanoparticles based on 1,2-dithiolane derivatives.

Chembiochem 2015 Mar 16;16(5):792-804. Epub 2015 Feb 16.

Institut de Chimie de Nice, équipe Molécules Bioactives (Vectorisation & Diagnostic), CNRS UMR 7272, Université de Nice-Sophia Antipolis, Faculté des Sciences, 06108 Nice Cedex 2 (France).

We describe the formulation of synthetic virus models based on ionic compounds bearing the polymerizable 1,2-dithiolane moiety. First, cationic amphiphiles containing the polymeric inducer were prepared and used to efficiently condense a DNA plasmid (pDNA) into a highly monodisperse population of small polymeric cationic DNA nanoparticles (NPs; Dh ∼100 nm). These nonspecific cationic particles were then functionalized with anionic PEGylated conjugates, also based on the 1,2-dithiolane motifs, in order to produce stable and fully dispersible stealth DNA nanoparticles. Our results show that both ionic interactions and polymerization based on the 1,2-dithiolane pattern occur and that they produce highly functionalizable nonviral DNA NPs.
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http://dx.doi.org/10.1002/cbic.201402657DOI Listing
March 2015

In vivo characterization of dynein-driven nanovectors using Drosophila oocytes.

PLoS One 2013 12;8(12):e82908. Epub 2013 Dec 12.

University of Nice Sophia-Antipolis, Institute of Biology Valrose (iBV), UMR 7277-CNRS, UMR 1091 INSERM, Nice, France.

Molecular motors transport various cargoes including vesicles, proteins and mRNAs, to distinct intracellular compartments. A significant challenge in the field of nanotechnology is to improve drug nuclear delivery by engineering nanocarriers transported by cytoskeletal motors. However, suitable in vivo models to assay transport and delivery efficiency remain very limited. Here, we develop a fast and genetically tractable assay to test the efficiency and dynamics of fluospheres (FS) using microinjection into Drosophila oocytes coupled with time-lapse microscopy. We designed dynein motor driven FS using a collection of dynein light chain 8 (LC8) peptide binding motifs as molecular linkers and characterized in real time the efficiency of the FS movement according to its linker's sequence. Results show that the conserved LC8 binding motif allows fast perinuclear nanoparticle's accumulation in a microtubule and dynein dependent mechanism. These data reveal the Drosophila oocyte as a new valuable tool for the design of motor driven nanovectors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0082908PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3861458PMC
March 2015

Amphiphilic oligoethyleneimine-β-cyclodextrin "click" clusters for enhanced DNA delivery.

J Org Chem 2013 Aug 1;78(16):8143-8. Epub 2013 Aug 1.

Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Profesor García González 1, E-41012 Sevilla, Spain.

Monodisperse amphiphilic oligoethyleneimine (OEI)-β-cyclodextrin (βCD) clusters have been prepared, and their potential as gene delivery systems has been evaluated in comparison with a nonamphiphilic congener. The general prototype incorporates tetraethyleneimine segments linked to the primary rim of βCD through either triazolyl or thioureidocysteaminyl connectors. Transfection efficiency data for the corresponding CD:pDNA nanocomplexes (CDplexes) in BNL-CL2 murine hepatocytes evidenced the strong beneficial effect of facial amphiphilicity.
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http://dx.doi.org/10.1021/jo400993yDOI Listing
August 2013

A general approach for the microrheology of cancer cells by atomic force microscopy.

Micron 2013 Jan 7;44:287-97. Epub 2012 Aug 7.

Laboratoire de Physique de la Matière Condensée (LPMC), CNRS UMR 7336, Université de Nice-Sophia Antipolis, Parc Valrose, 06108 Nice Cedex 2, France.

The determination of the viscoelastic properties of cells by atomic force microscopy (AFM) is mainly realized by looking at the relaxation of the force when a constant position of the AFM head is maintained or at the evolution of the indentation when a constant force is maintained. In both cases the analysis rests on the hypothesis that the motion of the probe before the relaxation step is realized in a time which is much smaller than the characteristic relaxation time of the material. In this paper we carry out a more general analysis of the probe motion which contains both the indentation and relaxation steps, allowing a better determination of the rheological parameters. This analysis contains a correction of the Hertz model for large indentation and also the correction due to the finite thickness of the biological material; it can be applied to determine the parameters representing any kind of linear viscoelastic model. This approach is then used to model the rheological behavior of one kind of cancer cell called Hep-G2. For this kind of cell, a power law model does not well describe the low and high frequency modulus contrary to a generalized Maxwell model.
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http://dx.doi.org/10.1016/j.micron.2012.07.006DOI Listing
January 2013

Polycationic amphiphilic cyclodextrins as gene vectors: effect of the macrocyclic ring size on the DNA complexing and delivery properties.

Org Biomol Chem 2012 Aug 26;10(29):5570-81. Epub 2012 Jun 26.

Institut de Chimie de Nice, UMR 7272, Université de Nice Sophia Antipolis - CNRS, 28, Avenue de Valrose, F-06100 Nice, France.

A collection of homologous monodisperse facial amphiphiles consisting of an α-, β- or γ-cyclodextrin (α, β or γCD) platform exposing a multivalent display of cationic groups at the primary rim and bearing hexanoyl chains at the secondary hydroxyls have been prepared to assess the influence of the cyclooligosaccharide core size in their ability to complex, compact and protect pDNA and in the efficiency of the resulting nanocondensates (CDplexes) to deliver DNA into cells and promote transfection in the presence of serum. All the polycationic amphiphilic CDs (paCDs) were able to self-assemble in the presence of the plasmid and produce transfectious nanoparticles at nitrogen/phosphorous ratios ≥5. CDplexes obtained from βCD derivatives generally exhibited higher transfection capabilities, which can be ascribed to their ability to form inclusion complexes with cholesterol, thereby enhancing biological membrane permeability. The presence of thiourea moieties as well as increasing the number of primary amino groups then favour cooperative complexation of the polyphosphate chain, enhancing the stability of the complex and improving transfection. In the α and γCD series, however, only the presence of tertiary amino groups in the cationic clusters translates into a significant improvement of the transfection efficiency, probably by activating endosome escape by the proton sponge mechanism. This set of results illustrates the potential of this strategy for the rational design and optimisation of nonviral gene vectors.
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http://dx.doi.org/10.1039/c2ob25786fDOI Listing
August 2012

Mannosyl-coated nanocomplexes from amphiphilic cyclodextrins and pDNA for site-specific gene delivery.

Biomaterials 2011 Oct 7;32(29):7263-73. Epub 2011 Jul 7.

Instituto de Investigaciones Químicas, CSIC-Universidad de Sevilla, Sevilla, Spain.

Fully homogeneous facial amphiphiles consisting in a cyclodextrin (CD) platform onto which a polycationic cluster and a multi-tail hydrophobic moiety have been installed (polycationic amphiphilic CDs; paCDs) self-organized in the presence of plasmid DNA to form nanometric complexes (CDplexes) which exhibit broad-range transfection capabilities. We hypothesized that biorecognizable moieties located at the hydrophilic rim in the CD scaffold would be exposed at the surface of the corresponding nanoparticles after DNA-promoted aggregation, endowing the system with molecular recognition abilities towards cell receptors. This concept has been demonstrated by developing an efficient synthetic strategy for the preparation of multivalent polycationic glyco-amphiphilic CDs (pGaCDs). Self-assembled nanoparticles obtained from mannosylated pGaCDs and pDNA (average hydrodynamic diameter 80 nm) have been shown to be specifically recognized by mannose-specific lectins, including concanavalin A (Con A) and the human macrophage mannose receptor (MMR). Further macrophage adhesion studies indicated that unspecific binding, probably due to electrostatic interactions with negatively charged cell membrane components, can also operate. The relative specific versus non-specific internalization is dependent on the pGaCD:pDNA proportion, being optimal at a protonable nitrogen/phosphate (N/P) ratio of 5. The resulting GlycoCDplexes were shown to specifically mediate transfection in Raw 264.7 (murine macrophage) cells expressing the mannose-fucose receptor in vitro. FACS experiments confirmed that transfection using these nanoparticles is mannose-dependent, supporting the potential of the approach towards vectorized gene delivery.
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http://dx.doi.org/10.1016/j.biomaterials.2011.06.025DOI Listing
October 2011

β-Cyclodextrin-based polycationic amphiphilic "click" clusters: effect of structural modifications in their DNA complexing and delivery properties.

J Org Chem 2011 Aug 6;76(15):5882-94. Epub 2011 Jul 6.

Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Profesor García González 1, E-41012 Sevilla, Spain.

Monodisperse facial amphiphiles consisting of a β-cyclodextrin (βCD) platform exposing a multivalent display of cationic groups at the primary rim and bearing hydrophobic chains at the secondary oxygens have been prepared by implementing two very robust "click" methodologies, namely cuprous cation-catalyzed azide-alkyne cycloaddition (CuAAC) and thiourea-forming reaction. Most interestingly, the use of solid-supported Cu(I) catalysts was found to be very well suited for multiple CuAAC while facilitating purification of the C(7)-symmetric macromolecular triazole adducts. The strategy is compatible with molecular diversity-oriented approaches, which has been exploited to generate a small library of click polycationic amphiphilic CDs (paCDs) for assessing the influence of structural modifications in the ability to complex, compact, and protect pDNA and the efficiency of the resulting paCD:pDNA nanocomplexes (CDplexes) to deliver DNA into cells and promote transfection. The results indicate that fine-tuning the hydrophilic/hydrophobic balance is critical to achieve optimal self-assembling properties and stability of the resulting CDplexes in saline- and serum-containing media. Triazole-type paCDs were, in general, less efficient in promoting gene transfection than thiourea-type derivatives. Nevertheless, the current body of results support that the "dual click" approach implying sequential CuAAC and thiourea-forming reactions represents a versatile strategy to optimize the gene delivery capabilities of cyclodextrin-based facial amphiphiles.
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http://dx.doi.org/10.1021/jo2007785DOI Listing
August 2011

On the origin of primitive cells: from nutrient intake to elongation of encapsulated nucleotides.

Angew Chem Int Ed Engl 2010 May;49(22):3738-50

LCMBA UMR 6001 CNRS, Institut de Chimie de Nice, Université de Nice-Sophia Antipolis, Faculté des Sciences, Parc Valrose, 06108 Nice, France.

Recent major discoveries in membrane biophysics hold the key to a modern understanding of the origin of life on Earth. Membrane bilayer vesicles have been shown to provide a multifaceted microenvironment in which protometabolic reactions could have developed. Cell-membrane-like aggregates of amphiphilic molecules capable of retaining encapsulated oligonucleotides have been successfully created in the laboratory. Sophisticated laboratory studies on the origin of life now show that elongation of the DNA primer takes place inside fatty acid vesicles when activated nucleotide nutrients are added to the external medium. These studies demonstrate that cell-like vesicles can be sufficiently permeable to allow for the intake of charged molecules such as activated nucleotides, which can then take part in copying templates in the protocell interior. In this Review we summarize recent experiments in this area and describe a possible scenario for the origin of primitive cells, with an emphasis on the elongation of encapsulated nucleotides.
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http://dx.doi.org/10.1002/anie.200905465DOI Listing
May 2010

Polycationic amphiphilic cyclodextrins for gene delivery: synthesis and effect of structural modifications on plasmid DNA complex stability, cytotoxicity, and gene expression.

Chemistry 2009 Nov;15(46):12871-88

Instituto de Investigaciones Químicas, CSIC and Universidad de Sevilla, Av. Américo Vespucio 49, Isla de la Cartuja, 41092 Sevilla, Spain.

A molecular-diversity-oriented approach for the preparation of well-defined polycationic amphiphilic cyclodextrins (paCDs) as gene-delivery systems is reported. The synthetic strategy takes advantage of the differential reactivity of primary versus secondary hydroxyl groups on the CD torus to regioselectively decorate each rim with cationic elements and lipophilic tails, respectively. Both the charge density and the hydrophobic-hydrophilic balance can be finely tuned in a highly symmetrical architecture that is reminiscent of both cationic lipids and cationic polymers, the two most prominent types of nonviral gene vectors. The monodisperse nature of paCDs and the modularity of the synthetic scheme are particularly well suited for structure-activity relationship studies. Gel electrophoresis revealed that paCDs self-assemble in the presence of plasmid DNA (pDNA) to provide homogeneous, stable nanoparticles (CDplexes) of 70-150 nm that fully protect pDNA from the environment. The transfection efficiency of the resulting CDplexes has been investigated in vitro on BNL-CL2 and COS-7 cell lines in the absence and presence of serum and found to be intimately dependent on architectural features. Facial amphiphilicity and the presence of a cluster of cationic and hydrogen-bonding centers for cooperative and reversible complexation of the polyanionic DNA chain is crucial to attain high transgene expression levels with very low toxicity profiles. Further enhancement of gene expression, eventually overcoming that of polyplexes from commercial polyethyleneimine (PEI) polymers (22 kDa), is achieved by building up space-oriented dendritic polycationic constructs.
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http://dx.doi.org/10.1002/chem.200901149DOI Listing
November 2009

Tailoring beta-cyclodextrin for DNA complexation and delivery by homogeneous functionalization at the secondary face.

Org Lett 2008 Nov 22;10(22):5143-6. Epub 2008 Oct 22.

Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Sevilla, Spain.

An efficient general strategy for the incorporation of functional elements onto the secondary hydroxyl rim of beta-cyclodextrin has been developed and applied to the synthesis of a novel series of C7-symmetric homogeneous macromolecular polycations with improved DNA complexing and delivery properties.
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http://dx.doi.org/10.1021/ol802081zDOI Listing
November 2008

Rational design of cationic cyclooligosaccharides as efficient gene delivery systems.

Chem Commun (Camb) 2008 May 25(17):2001-3. Epub 2008 Feb 25.

Instituto de Investigaciones Químicas, CSIC, Américo Vespucio 49, Isla de la Cartuja, E-41092, Sevilla, Spain.

Self-assembled cyclodextrin (CD)-DNA nanoparticles (CDplexes) exhibiting transfection efficiencies significantly higher than PEI-based polyplexes have been prepared from homogeneous seven-fold symmetric polyaminothiourea amphiphiles constructed on a beta-cyclodextrin scaffold.
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http://dx.doi.org/10.1039/b718672jDOI Listing
May 2008

Synthesis and in vitro biological evaluation of valine-containing prodrugs derived from clinically used HIV-protease inhibitors.

Eur J Med Chem 2008 Jul 14;43(7):1506-18. Epub 2007 Sep 14.

Laboratoire de Chimie des Molécules Bioactives et des Arômes, UMR 6001, Université de Nice Sophia-Antipolis, CNRS, Faculté des Sciences, Institut de Chimie de Nice, Nice, France.

In an approach to improve the pharmacological properties and pharmacokinetic profiles of the current protease inhibitors (PIs) used in clinics, and consequently, their therapeutic potential, we performed the synthesis of PI-spacer-valine prodrugs (PI=saquinavir, nelfinavir and indinavir; spacer=-C(O)(CH(2))(5)NH-), and evaluated their in vitro stability with respect to hydrolysis, anti-HIV activity, cytotoxicity, and permeation through a monolayer of Caco-2 cells (used as a model of the intestinal barrier), as compared with their parent PI and first generation of valine-PIs (wherein valine was directly connected through its carboxyl to the PIs). The PI-spacer-valine conjugates were prepared in two steps, in good yields, by condensing an acid derivative of the appropriate protected valine-spacer moiety with the PI, followed by deprotection of the valine protecting group. With respect to hydrolysis, we found that the PI-spacer-valine prodrugs were chemically more stable than the first generation of PI-Val prodrugs. Their stabilities correlated with the low to very low in vitro anti-HIV activity measured for those prodrugs wherein the coupling of valine-spacer residue to the PIs was performed onto the peptidomimetic PI's hydroxyl. Prodrugs wherein the coupling of the valine-spacer residue was performed onto the non-peptidomimetic PI hydroxyl displayed a higher antiviral activity, indicating that these prodrugs are also to some extent anti-HIV drugs by themselves. While the direct conjugation of L-valine to the PIs constituted a most appealing alternative, which improved their absorptive diffusion across Caco-2 cell monolayers and reduced their recognition by efflux carriers, its conjugation to the PIs through the -C(O)(CH(2))(5)NH- spacer was found to inhibit their absorptive and secretory transepithelial transport. This was attributable to a drastic reduction of their passive permeation and/or active transport, indicating that the PI-spacer-valine conjugates are poor substrates of the aminoacid carrier system located at the brush border side of the Caco-2 cell monolayer.
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http://dx.doi.org/10.1016/j.ejmech.2007.08.016DOI Listing
July 2008

Synthesis and in vitro biological evaluation of mannose-containing prodrugs derived from clinically used HIV-protease inhibitors with improved transepithelial transport.

Bioconjug Chem 2006 Nov-Dec;17(6):1568-81

Laboratoire de Chimie des Molécules Bioactives et des Arômes, UMR 6001 CNRS, Université de Nice Sophia-Antipolis, Parc Valrose, F-06108 Nice Cédex 2, France.

In an approach to improve the pharmacological properties, safety and pharmacokinetic profiles, and their penetration into HIV reservoirs or sanctuaries, and consequently, the therapeutic potential of the current protease inhibitors (PIs) used in clinics, we investigated the synthesis of various mannose-substituted saquinavir, nelfinavir, and indinavir prodrugs, their in vitro stability with respect to hydrolysis, anti-HIV activity, cytotoxicity, and permeation through a monolayer of Caco-2 cells used as a model of the intestinal barrier. Mannose-derived conjugates were prepared in two steps, in good yields, by condensing an acid derivative of a protected mannose with the PIs, followed by deprotection of the sugar protecting group. With respect to hydrolysis, these PI prodrugs are chemically stable with half-life times in the 50-60 h range that are compatible with an in vivo utilization aimed at improving the absorption/penetration or accumulation of the prodrug in specific cells/tissues and liberation of the active free drug inside HIV-infected cells. These stabilities correlate closely with the low in vitro anti-HIV activity measured for those prodrugs wherein the coupling of mannose to the PIs was performed through the peptidomimetic PI's hydroxyl. Importantly, mannose conjugation to the PIs was further found to improve the absorptive transepithelial transport of saquinavir and indinavir but not of nelfinavir across Caco-2 cell monolayers, by contrast to glucose conjugation which had the opposite effect. The mannose-linked prodrugs of saquinavir and indinavir display therefore a most promising therapeutic potential provided that bioavailability, penetration into the HIV infected macrophages, and HIV-reservoirs of these PIs are improved.
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http://dx.doi.org/10.1021/bc060210mDOI Listing
January 2007

Design, synthesis and activity against Toxoplasma gondii, Plasmodium spp., and Mycobacterium tuberculosis of new 6-fluoroquinolones.

Eur J Med Chem 2006 Dec 25;41(12):1478-93. Epub 2006 Sep 25.

Laboratoire de Chimie Bioorganique UMR-CNRS 6001, Université de Nice-Sophia Antipolis, Parc Valrose, 06108 Nice Cedex 2, France.

This paper reports on the rational design of a series of new 6-fluoroquinolones by QSAR analysis against Toxoplasma (T.) gondii, their synthesis, their biological evaluation against T. gondii and Plasmodium (P.) spp., and their effect on Mycobacterium (M.) tuberculosis DNA gyrase and growth inhibition. Of the 12 computer-designed 8-ethyl(or methoxy)- and 5-ethyl-8-methoxy-6-fluoroquinolones predicted to be active against T. gondii, we succeeded in the synthesis of four 6-fluoro-8-methoxy-quinolones. The four 6-fluoro-8-methoxy-quinolones are active on T. gondii but only one is as active as predicted. One of these four compounds appears to be an antiparasitical drug of great potential with inhibitory activities comparable to or higher than that of trovafloxacin, gatifloxacin, and moxifloxacin. They also inhibit DNA supercoiling by M. tuberculosis gyrase with an efficiency comparable to that of the most active quinolones but are poor inhibitors of M. tuberculosis growth.
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http://dx.doi.org/10.1016/j.ejmech.2006.07.003DOI Listing
December 2006

Synthesis of acridine-nuclear localization signal (NLS) conjugates and evaluation of their impact on lipoplex and polyplex-based transfection.

Eur J Med Chem 2005 Dec 11;40(12):1295-306. Epub 2005 Oct 11.

Laboratoire de Chimie Bioorganique, UMR 6001 CNRS, Faculté des Sciences, Université de Nice Sophia-Antipolis, 06108 Nice cedex 2, France.

We report on the synthesis of various acridine (Acr)-spacer-nuclear localization signal (NLS) peptide conjugates and explore whether their use as NLS-labeling agent of plasmidic DNA could improve gene nuclear import and expression into cells when mediated by synthetic DNA complexes. As the conditions of successful use of the NLS properties to enhance gene transfer are not clear, and with the aim of detecting and defining the requirements of NLS-enhanced transfection, we investigated gene delivery and expression into various cell lines with various DNA complexes (lipoplexes or polyplexes) that were formulated for various N/P ratios from various preformed Acr-spacer-NLS/DNA complexes (1:1, 5:1 and 10:1 molar ratio). For the in vitro transfection assays, the lipoplexes and polyplexes were formulated from the preformed Acr-spacer-NLS/DNA complexes and dioctadecylamidoglycylspermine (DOGS)/dioleylphosphatidylethanolamine (DOPE) 1:1 mol and branched polyethyleneimine (PEI) 25 kDa, respectively, which are very efficient in vitro gene transfer systems. We show by fluorescence experiments that part of the acridine-NLS-conjugates remains intercalated within the plasmid for most of the N/P lipoplexes and polyplexes investigated. We show that, as several other studies performed with NLS-conjugates that are not covalently linked to DNA, the expression of the transgene is in most cases not improved upon complexation of plasmidic DNA with NLS-intercalating conjugates prior to its formulation as lipoplexes or polyplexes.
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http://dx.doi.org/10.1016/j.ejmech.2005.07.015DOI Listing
December 2005

Quinolone-based drugs against Toxoplasma gondii and Plasmodium spp.

Curr Drug Targets Infect Disord 2005 Sep;5(3):227-45

Laboratoire de Chimie Bioorganique, UMR 6001 CNRS, Université de Nice Sophia-Antipolis, Faculté des Sciences, 06108 Nice Cédex 2, France.

Owing to the rapid emergence of multi-resistant strains of Plasmodium spp. (the causative agents of malaria) and the limitations of drugs used against Toxoplasma gondii (an important opportunistic pathogen associated with AIDS and congenital birth defects), the discovery of new therapeutical targets and the development of new drugs are needed. The presence of the prokaryotic-like organelle in apicomplexan parasites (i.e. plastids), which comprise these major human pathogens, may represent a unique target for antibiotics against these protozoa. Quinolones which are known to be highly potent against bacteria were also found to specifically disrupt these parasites. They inhibit DNA replication by interacting with two essential bacterial type II topoisomerases, DNA gyrase and topoisomerase IV. There are some clues that quinolones act on plastids with a similar mechanism of action. After a brief presentation of plasmodium and toxoplasma dedicated to their life cycle, the chemotherapies presently used in clinics to fight against these protozoa and the potential new targets and drugs, we will focus our attention on their plastid which is one of these promising new targets. Then, we will present the various drugs and generations of quinolones, the leading molecules, and their inhibitory effects against these parasites together with their pharmacological properties that have been established from in vitro and in vivo studies. We will also discuss their possible mode of action.
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http://dx.doi.org/10.2174/1568005054880172DOI Listing
September 2005

New perfluorinated polycationic dimerizable detergents for the formulation of monomolecular DNA nanoparticles and their in vitro transfection efficiency.

Biochim Biophys Acta 2005 Jun 2;1724(1-2):203-14. Epub 2005 Apr 2.

Laboratoire de Chimie Bioorganique, UMR 6001 CNRS, Université de Nice Sophia-Antipolis, Faculté des Sciences, 06108 Nice Cédex 2, France.

We describe the synthesis of new perfluorinated dimerizable detergents which contain a tricationic or tetracationic (linear or branched spermine, respectively) polar head, and report on their cmc, their ability to condense DNA into cationic monomolecular DNA nanoparticles as well as on the in vitro transfection efficiency of these nanoparticles. Such cationic nanoparticles were prone to display efficient cell transfection properties as a result of increased contact to the anionic cell surface and internalization by endocytosis, low size compatible with improved intracellular diffusion and nuclear pore crossing, and the presence of amine function of low pK(a) for their endosomal escape. The challenge was to design polymerizable polycationic detergents that display a cmc high enough for the monomer to perform monomolecular DNA condensation (as cationic particles) and low enough for the dimer to form stable nanoparticles capable of efficient cell transfection. Although we succeeded in formulating small-sized cationic monomolecular DNA nanoparticles (<40 nm) with these dimerizable perfluorinated spermine-based detergents for N/P ratios of up to 5 (N=number of detergent amine equivalents/P=number of DNA phosphate equivalents), these small-sized cationic nanoparticles proved to be poor non-specific transfection agents in vitro, even in the presence of chloroquine. Their poor transfection potential could be due more likely to Brownian motion which prevents these very small-sized particles from sedimentation and adsorption onto the adherent cell monolayer, and, consequently, from proteoglycan-triggered endocytosis.
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http://dx.doi.org/10.1016/j.bbagen.2005.03.007DOI Listing
June 2005

Investigation of oral bioavailability and brain distribution of the Ind(8)-Val conjugate of indinavir in rodents.

J Pharm Pharmacol 2005 Apr;57(4):453-8

EA3809 Pharmacologie des Agents Anti-Infectieux, Faculté de Médecine & Pharmacie, 86005 Poitiers Cedex, France.

Protease inhibitors are successfully used for the treatment of acquired immune deficiency syndrome (AIDS) although their biopharmaceutical characteristics are not optimal. Prodrugs have therefore been synthesized to increase protease inhibitor bioavailability and brain distribution. Among several compounds tested, a valine derivative of indinavir (Ind(8)-Val) showed promising characteristics using an in-vitro Caco-2 cell model. The objective of this study was to further investigate this compound using in-situ and in-vivo approaches. The pharmacokinetics of indinavir (Ind) and Ind(8)-Val were investigated in rats after intravenous and oral administration. Free indinavir resulting from in-vivo hydrolysis of Ind(8)-Val could not be detected in the plasma of rats receiving Ind(8)-Val. Furthermore Ind(8)-Val bioavailability was only 32% on average compared with 76% for indinavir, and effective permeability coefficients determined with a single-pass intestinal perfusion method were close to 25x10(6)cms(-1) for the two compounds. Brain-to-plasma concentration ratios in the post equilibrium phase after intravenous administration to mice were 9.7+/-8.1% for indinavir and 2.5+/-2.7% for Ind(8)-Val. In conclusion, the promising biopharmaceutical characteristics of Ind(8)-Val suggested from previous in-vitro experiments with the Caco-2 cell model were not confirmed by in-situ and in-vivo experiments.
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http://dx.doi.org/10.1211/0022357055786DOI Listing
April 2005

Transfection with fluorinated lipoplexes based on new fluorinated cationic lipids and in the presence of a bile salt surfactant.

Bioconjug Chem 2004 Jul-Aug;15(4):901-8

Laboratoire de Chimie Bioorganique, UMR 6001 CNRS, Université de Nice Sophia-Antipolis, Faculté des Sciences, 06108 Cédex 2, France.

The synthesis of two fluorinated cationic lipids, which are analogues of frequently used synthetic gene carrier agents (including the cationic 2,3-dioleoyloxy-N-[2-(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA) component of the commercially available liposomal Lipofectamine), and the disintegration and DNA accessibility (evaluated by the ethidium bromide (BET) intercalation assay) as well as the in vitro transfection efficacy of cationic lipoplexes formulated with these new lipids in conjunction with conventional or fluorinated helper lipids, in the absence or presence of sodium taurocholate (STC), a powerful anionic bile salt detergent, is reported. A higher stability, with respect to the STC lytic activity and DNA accessibility, of the fluorinated cationic lipoplexes as compared with their respective lipofectamine-based ones was demonstrated. Indeed, while the Lipofectamine lipoplexes were fully disintegrated at a [STC]/[lipid] molar ratio of 2000, only 40-60% of the DNA intercalation sites of the lipoplexes based on the fluorinated analogue of DOSPA were accessible to ethidium bromide. A higher transfection potential in the presence of STC was further found for the lipoplexes formulated with the fluorinated analogue of DOSPA as compared with the Lipofectamine preparation. For a STC concentration of 7.5 mM, lipofection mediated with these fluorinated lipoplexes was significantly higher (nearly 30- to 50-fold, p < 0.05) than with the Lipofectamine ones. These results confirm the remarkable transfection potential of fluorinated lipoplexes.
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http://dx.doi.org/10.1021/bc049942+DOI Listing
January 2005

AMD3100 conjugates as components of targeted nonviral gene delivery systems: synthesis and in vitro transfection efficiency of CXCR4-expressing cells.

Bioconjug Chem 2004 Mar-Apr;15(2):413-23

Laboratoire de Chimie Bioorganique, UMR 6001 CNRS, Faculté des Sciences, Université de Nice-Sophia Antipolis, 06108 Nice Cédex 2, France.

We describe the synthesis of a series of AMD3100-lipid and AMD3100-polycationic conjugates which were used as components of targeted lipoplexes (in conjunction with (poly)cationic lipids) and polyplexes, respectively, for mediating specific gene transfer into cells expressing CXCR4 which displays a high affinity for AMD3100. Transfection studies were investigated with suspension CXCR4(+) human lymphoma Jurkat cells and with adherent CXCR4(-) human glioblastoma T98G and human lung carcinoma A549 cells lines in order to demonstrate a receptor-mediated endocytosis pathway and to minimize nonspecific transfection pathways. Altogether, our results show that polyplexes formulated with AMD-labeled polymers constitute, under certain conditions, specific gene transfer systems into suspension CXCR4(+) Jurkat cells. This is more particularly the case when the nonspecific transfection pathways are minimized (i.e. for N/P
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http://dx.doi.org/10.1021/bc034220oDOI Listing
November 2004

Prodrugs of HIV protease inhibitors-saquinavir, indinavir and nelfinavir-derived from diglycerides or amino acids: synthesis, stability and anti-HIV activity.

Org Biomol Chem 2004 Feb 5;2(3):345-57. Epub 2004 Jan 5.

Laboratoire de Chimie Bioorganique, UMR 6001 CNRS, Université de Nice Sophia-Antipolis, Parc Valrose, 06108 Nice Cédex 2, France.

With the aim of improving the pharmacological properties of current protease inhibitors (PIs), the synthesis of various acyl and carbamate amino acid- or diglyceride-containing prodrugs derived from saquinavir, indinavir and nelfinavir, their in vitro stability with respect to hydrolysis and their anti-HIV activity in CEM-SS and MT4 cells have been investigated. l-Leucine (Leu) and l-phenylalanine (Phe) were connected through their carboxyl to the PIs while l-tyrosine (Tyr) was conjugated through its aromatic hydroxyl via various spacer units. Hydrolysis of the prodrug with liberation of the active free drug was crucial for antiviral activity. The Leu- and Phe-PI prodrugs released the active free drug very rapidly (half-lives of hydrolysis in buffer at 37 degree C of 3-4 h). The Tyr-PI conjugates with a -C(O)(CH(2))(4)- linker exhibited half-lives in the 40-70 h range and antiviral activities in the 21-325 nM range (from 2 to 22 nM for the free PIs). The chemically very stable carbamate "peptidomimetic" Tyr-PI prodrugs (no hydrolysis detected after 7 days in buffer) displayed a very low anti-HIV activity or were even inactive (EC(50) from 2300 nM to >10 microM). A very low antiviral activity was measured for the diglyceride-substituted saquinavir and for all of the disubstituted indinavir and nelfinavir prodrugs. All these prodrugs probably released the active parent PI too slowly under the antiviral assay conditions. These results combined with those from transepithelial transport studies (Rouquayrol et al., Pharm. Res., 2002, 19, 1704-1712) indicate that conjugation of amino acids (through their carboxyl) to the PIs constitutes a most appealing alternative which could improve the intestinal absorption of the PIs and reduce their recognition by efflux carriers.
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http://dx.doi.org/10.1039/b313119jDOI Listing
February 2004

New bicyclam-GalCer analogue conjugates: synthesis and in vitro anti-HIV activity.

Bioorg Med Chem Lett 2004 Jan;14(2):495-8

Laboratoire de Chimie Bioorganique UMR-CNRS 6001, Université de Nice-Sophia Antipolis, Parc Valrose, 06108 Cédex 2, Nice, France.

The synthesis of bipharmacophore anti-HIV compounds which, in a single molecule, combine two ligands, that is, the bicyclam AMD3100 and a GalCer analogue, that might inhibit several steps of the complex virus/cell cascade interactions has been performed. The 'double-drug' Gal-AMD3100 conjugates elicited inhibitory effects on T (or X4)-tropic HIV-1 replication in all CXCR4 expressing cell lines with EC(50) values ranging from 0.25 to 6.0 microM which were however approximately 40- to 125-fold lower than that of AMD3100. Concerning the mechanism of inhibition of the Gal-AMD3100 conjugates, experiments performed with X4 or R5HIV-1 strains and GHOST cells genetically modified to express CD4 and CXCR4 or CCR5 indicated clearly that the conjugates interact with CXCR4 and not with CCR5.
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http://dx.doi.org/10.1016/j.bmcl.2003.10.036DOI Listing
January 2004

Synthesis and studies of modified oligonucleotides-directed triple helix formation at the purine-pyrimidine interrupted site.

Nucleosides Nucleotides Nucleic Acids 2003 May-Aug;22(5-8):1277-80

Laboratoire de Chimie des Plantes et de Synthèse Organique et Bioorganique, Universitè des Sciences, Rabat, Morocco.

Triple helix formation is still restricted to oligopurine-oligopyrimidine double stranded DNA target. Herein we focus on our progress achieved in nucleobase and oligonucleotide modifications area to address the chemical challenge to circumvent the recognition of a purine-pyrimidine base pair interruption in an oligopyrimidine-oligopurine DNA sequence.
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http://dx.doi.org/10.1081/NCN-120022945DOI Listing
December 2003

Prodrugs of HIV protease inhibitors.

Curr Pharm Des 2003 ;9(22):1755-70

Laboratoire de Chimie Bioorganique, UMR 6001 CNRS, Université de Nice-Sophia Antipolis, Parc Valrose, 06108 Nice Cédex 2, France.

Despite the efficiency of the present polytherapies against AIDS, HIV replication continues indicating difficulties in drug adherence, drug-drug interactions, resistance issues, and the existence of reservoirs or sanctuaries for the virus. Moreover, most of the current FDA-approved HIV protease inhibitors (PIs) display disadvantageous physicochemical and pharmacological properties such as low water solubility, low oral bioavailability and/or low level of penetration into the HIV sanctuaries resulting from their in vivo binding to the plasma proteins and to the Multi-Drug-Resistant P-glycoprotein, their rapid metabolization and inactivation by the liver cytochrome P450 enzymes. To overcome these suboptimal pharmacokinetics, high daily doses must be ingested, which complicate patient adherence to the prescribed regimen and contribute to the appearance of serious long-term metabolic complications and to the decrease of the viral treatment outcome. Another attractive alternative aimed at improving the safety, pharmacokinetics, and therapeutic potency of the current PIs is to modify these PIs into pharmacologically inactive prodrugs which are converted in vivo into their parent active drug. The present review is dedicated to the different prodrug approaches, including the "lipophilic", "hydrophilic", "active transport" and "double-drug" prodrug strategies, which have been applied more particularly to the current HIV PIs used in clinic. Among the strategies explored up to now, the most successful one was the "hydrophilic" prodrug approach which has led to the discovery of fosamprenavir, a phosphate ester prodrug of amprenavir, which has reached phase III clinical trials. This success gives strong support for the search of PI prodrugs as a therapeutic alternative in addition to the development of new and well-tolerated PIs.
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http://dx.doi.org/10.2174/1381612033454441DOI Listing
May 2004

Novel galactosylated polyamine bolaamphiphiles for gene delivery.

Bioconjug Chem 2003 Mar-Apr;14(2):358-67

Laboratoire de Chimie Bioorganique, UMR 6001 CNRS-Université de Nice Sophia-Antipolis, Faculté des Sciences, 06108 Nice Cédex 2, France.

We describe the synthesis of a series of alpha-galacto-omega-polyamine double-chain bolaamphiphiles (Gal-CL) and report on the gene transfer mediated with lipoplexes they form either when used in conjunction with DOPE or with pcTG90:DOPE. Lipofection was investigated with human HepG2 and murine BNL-CL2 hepatocytes expressing the asialoglycoprotein (ASGP) receptor which displays a high affinity for galactosyl residues, and with A549 cells which do not express ASGP. Our results show that cationic N/P = 5 and 2.5 Gal-CL lipoplexes constitute very efficient nonspecific gene transfer systems. Lipofection experiments performed in the presence of asialofetuin (a high affinity ligand of ASGP) led us to evidence also the involvement of a specific receptor-mediated endocytosis pathway for the transfection of the ASGP(+) HepG2 or BNL-CL2 hepatocytes with some Gal-CL formulations. This work suggests that targetable lipopolyamines presenting a single galactose residue appear as promising synthetic vectors for specific gene delivery to ASGP(+) cells.
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http://dx.doi.org/10.1021/bc025645yDOI Listing
November 2003

Transepithelial transport of prodrugs of the HIV protease inhibitors saquinavir, indinavir, and nelfinavir across Caco-2 cell monolayers.

Pharm Res 2002 Nov;19(11):1704-12

Laboratoire de Chimie Bioorganique, UMR 6001 CNRS, Université de Nice Sophia-Antipolis, Faculté des Sciences, Parc Valrose, 06108 Nice Cedex 2, France.

Purpose: [corrected] This study is dedicated to the permeation of various amino acid-, D-glucose-, and PEG-conjugates of indinavir, saquinavir, and nelfinavir across monolayers of Caco-2 cells as models of the intestinal barrier. This screening is aimed at detecting the most promising prodrugs for improving the intestinal absorption of these protease inhibitors.

Methods: The bidirectional transport of the prodrugs was investigated using P-gp-expressing Caco-2 monolayers grown on membrane inserts using high-performance liquid chromatography for quantitation.

Results: The L-valyl, L-leucyl, and L-phenylalanyl ester conjugates led to an enhancement of the absorptive flux of indinavir or saquinavir. These results are likely attributable to an active transport mechanism and/or to a decrease of their efflux by carriers such as P-gp. Connection of tyrosine through its hydroxyl, of D-glucose, or of polyethylene glycol decreased their absorptive and secretory diffusion.

Conclusions: Conjugation of the protease inhibitors to amino acids constitutes a most appealing alternative that could improve their intestinal absorption and oral bioavailability. Whether it could improve their delivery into the central nervous system remains to be explored. D-Glucose conjugation will most probably not improve their intestinal absorption or their crossing of the blood-brain barrier. If some pharmacologic benefits are to be expected from PEG-protease inhibitor conjugates, they must then be administered intravenously.
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http://dx.doi.org/10.1023/a:1020913631309DOI Listing
November 2002

Polycationic diblock and random polyethylene glycol- or tris(hydroxymethyl)methyl-grafted (co)telomers for gene transfer: synthesis and evaluation of their in vitro transfection efficiency.

Bioconjug Chem 2002 Nov-Dec;13(6):1292-301

Laboratoire de Chimie Bioorganique, UMR 6001 CNRS, Université de Nice Sophia-Antipolis, Faculté des Sciences, 06108 Nice Cédex 2, France.

We report on the synthesis of a series of polycationic telomers, polycationic diblock and random polyethylene glycol (PEG)-grafted (co)telomers, and polycationic random tris(hydroxymethyl)methyl (THM) cotelomers, and on their in vitro gene transfer capability. These compounds were obtained by a telomerization process of various amino-, tetraethylene glycol-, or THM-acrylamide taxogens with thiols which might derive from PEG2000. For N/P ratios [N is the number of (co)telomer amine equivalents; P is the number of DNA phosphate equivalents] from 0.8 to 10, these (co)telomers condensed DNA, forming (co)teloplexes with mean sizes in the 85-330 nm range, even for an N/P ratio of 0.8 or 1.25. Some structure-transfection efficiency relationships were established. Among the new polycationic derivatives that were synthesized and investigated for their transfection efficiency, the (i)Bu-[NH](75) telomers and the diblock polyethylene glycol-conjugated PEG2000-[NH](n) telomers are very promising candidates for gene transfer purposes.
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http://dx.doi.org/10.1021/bc0255440DOI Listing
May 2003

Transfection with fluorinated lipoplexes based on fluorinated analogues of DOTMA, DMRIE and DPPES.

Biochim Biophys Acta 2002 Aug;1564(2):349-58

Laboratoire de Chimie Bio-Organique, UMR 6001 CNRS, Université de Nice Sophia-Antipolis, Faculté des Sciences, B.P. 71, 06108 Nice Cédex 2, France.

Fluorinated double-chain (poly)cationic lipids (one or both of these chains being ended by a highly fluorinated tail) which are close analogues of DOTMA, DMRIE or DPPES were designed as synthetic vectors for gene delivery. For N/P ratios (N=number of amine functions of the lipid; P=number of DNA phosphates) from 0.8 to 5, these fluorinated cationic lipids condensed DNA, with or without the use of DOPE, to form fluorinated lipoplexes. No specific cell toxicity was evidenced for these new fluorinated lipoplexes. The efficiency of some of the fluorinated lipoplexes to transfect lung epithelial A549 cells was comparable to that of the first generation of fluorinated lipoplexes made from fluorinated analogues of DOGS (Transfectam) [Bioconjug. Chem. 12 (2001) 114]. These results, combined with the higher in vivo transfection potential found for fluorinated lipoplexes than for conventional lipoplexes or PEI polyplexes [J. Gene Med. 3 (2001) 109], confirm that fluorinated lipoplexes are very promising gene transfer systems.
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http://dx.doi.org/10.1016/s0005-2736(02)00469-8DOI Listing
August 2002

Polycationic telomers and cotelomers for gene transfer: synthesis and evaluation of their an vitro transfection efficiency.

Bioconjug Chem 2002 Jan-Feb;13(1):59-75

Laboratoire de Chimie Bio-Organique, UMR 6001 CNRS, Université de Nice Sophia-Antipolis, Faculté des Sciences, 06108 Nice Cédex 2, France.

We report on the synthesis of a series of lipopolyamine telomers, [I(Asp)-14,n(A)(NH), I(His)-18,n(A)(NH), I-18,n(B)(NMe), Gal-n(A)(NH)], and random cotelomers, [I-18,n(A)(NH)-n(B)(NMe) and I-18,n(A)(NH)-n(C)(OH)], and on their in vitro gene transfer capability. They were obtained by a telomerization process of various amino-acrylamide taxogens with various lipophilic thiol telogens which might also contain an aspartic or a histidine residue or with a thiogalactosyle derivative. For N/P ratios (N = number of (co)telomer amine equivalents, P = number of DNA phosphates) from 0.8 to 10, these polyamine (co)telomers condensed DNA, with or without the use of DOPE, forming (co)teloplexes of mean sizes less than 200 nm, except for N/P 1.25 for which precipitates were observed. Some trends, structure-transfection efficiency relationships, were established. Thus, aspartic-containing telomers were found to lead to efficient formulations for plasmid delivery to A549 cells and for N/P ratios from 1.25 to 5.
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http://dx.doi.org/10.1021/bc010047pDOI Listing
April 2002
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