Maelle Monteil

Superparamagnetic nanovector with anti-cancer properties: γFe2O3@Zoledronate

We elaborate a magnetic nanovector to vectorize Zoledronate, an anti-cancer interest molecule of the hydroxmethylenebisphosphonates family. In fact, Zoledronate is a powerful adjuvant in the treatment of bone diseases such as osteoporosis and Pagets disease. But, recent studies have shown that in addition to anti-osteoclastic properties, it presents antitumour properties notably in the case of breast and prostate cancer. However, these properties cannot be exploited due to their very high affinity to divalent cations and their preferentially accumulation in bone. To overcome this problem, one strategy is the vectorization trough maghemite nanocrystal functionalization. The specific surface coating permits to consider gamma Fe(2)O(3)@Zoledronate as a drug delivery vehicle for therapeutic activity. The anchoring to the nanoparticles surface allowed to increase their hydrophobicity and also to change the therapeutic target, increasing the Zoledronate intestinal absorption instead of their accumulation in bone. We show that Zoledronate link the nanoparticle surface through phosphonate groups. The biological in vitro tests performed on breast cancer cell line, MDA-MB 231, showed that gamma Fe(2)O(3)@Zoledronate have antiproliferative activity. In addition, the gamma Fe(2)O(3) core could be used as MRI contrast agent for a good therapeutic evaluation.

Structural requirements for bisphosphonate binding on hydroxyapatite: NMR study of bisphosphonate partial esters.

Eighteen different bisphosphonates, including four clinically used bisphosphonate acids and their phosphoesters, were studied to evaluate how the bisphosphonate structure affects binding to bone. Bisphosphonates with weak bone affinity, such as clodronate, could not bind to hydroxyapatite after the addition of one ester group. Medronate retained its ability to bind after the addition of one ester group, and hydroxy-bisphosphonates could bind even after the addition of two ester groups. Thus, several bisphosphonate esters are clearly bone binding compounds. The following conclusions about bisphosphonate binding emerge: (1) a hydroxyl group in the geminal carbon takes part in the binding process and increases the bisphosphonates ability to bind to bone; (2) the bisphosphonates ability to bind decreases when the amount of ester groups increases; and (3) the location of the ester groups affects the bisphosphonates binding ability.

Cyclodextrins: A promising drug delivery vehicle for bisphosphonate

Bisphosphonates are well established pharmaceutical drugs with wide applications in medicine. Nevertheless, the side chain and the nature of phosphorous groups could induce a poor aqueous solubility and act on their bioavailability. At the same time, cyclodextrins are cage molecules that facilitate transport of hydrophobic molecules to enhance the intestinal drug absorption of these molecules by forming inclusion complexes. Here we demonstrate that cyclodextrins could be used as a bisphosphonate carrier. The formation of cyclodextrins-bisphosphonate complexes was characterized by 1D and 2D NMR spectroscopy, Isothermal Titration Calorimetry and UV-vis spectroscopy. The results revealed that only the side chain of bisphosphonate was involved in the inclusion phenomenon and its length was a crucial parameter in the control of affinity. Findings from this study suggest that cyclodextrin will be a useful carrier for bisphosphonates.

Targeting VEGF-A with a vaccine decreases inflammation and joint destruction in experimental arthritis

AbstractObjectivesInflammation and angiogenesis are two tightly linked processes in arthritis, and therapeutic targeting of pro-angiogenic factors may contribute to control joint inflammation and synovitis progression. In this work, we explored whether vaccination against vascular endothelial growth factor (VEGF) ameliorates collagen-induced arthritis (CIA).Methods Anti-VEGF vaccines were heterocomplexes consisting of the entire VEGF cytokine (or a VEGF-derived peptide) linked to the carrier protein keyhole limpet hemocyanin (KLH). Two kinds of vaccines were separately tested in two independent experiments of CIA. In the first, we tested a kinoid of the murine cytokine VEGF (VEGF-K), obtained by conjugating VEGF-A to KLH. For the second, we selected two VEGF-A-derived peptide sequences to produce heterocomplexes (Vpep1-K and Vpep2-K). DBA/1 mice were immunized with either VEGF-K, Vpep1-K, or Vpep2-K, before CIA induction. Clinical and histological scores of arthritis, anti-VEGF, anti-Vpep Ab titers, and anti-VEGF Abs neutralizing capacity were determined.ResultsBoth VEGF-K and Vpep1-K significantly ameliorated clinical arthritis scores and reduced synovial inflammation and joint destruction at histology. VEGF-K significantly reduced synovial vascularization. None of the vaccines reduced anti-collagen Ab response in mice. Both VEGF-K and Vpep1-K induced persistently high titers of anti-VEGF Abs capable of inhibiting VEGF-A bioactivity.ConclusionVaccination against the pro-angiogenic factor VEGF-A leads to the production of anti-VEGF polyclonal Abs and has a significant anti-inflammatory effect in CIA. Restraining Ab response to a single peptide sequence (Vpep1) with a peptide vaccine effectively protects immunized mice from joint inflammation and destruction.

Synthesis and biological evaluation of new bisphosphonate-dextran conjugates targeting breast primary tumor.

Bisphosphonates (BPs) have interesting antitumor effects as well in vitro as in vivo, despite their poor bioavailability in the organism after oral ingestion. To overcome this problem and reduce drug doses and secondary effects, we report the chemical synthesis of new bioconjugates. They were built with a nitrogen-containing BP as the drug covalently coupled to the carboxymethyldextran. This polysaccharide was used as a carrier, in order to increase BP lifetime in bloodstream and to target tumor cells which have a strong affinity with dextran. The efficiency of our vectorization system was biologically proved in vitro and in vivo on mammalian carcinoma models in mice.

Fragmentation patterns of new esterified and unesterified aromatic 1-hydroxymethylene-1, 1-bisphosphonic acids by ESI-MSn

1-Hydroxymethylene-1,1-bisphosphonic acids (HMBPs) are compounds that have interesting pharmacological applications. Unfortunately few studies exist on their analyses by mass spectrometry (MS). In this work, we have analyzed new aromatic HMBPs and their prodrugs with electrospray tandem mass spectrometry (ESI-MS(n)). We describe, for the first time, a complete study of fragmentation patterns, in both positive and negative-ion modes. In positive mode, the cation dissociations are mainly elimination of water and phosphorus fragments. In negative mode, losses of ROH (R==H, C(6)H(5), CH(3)OC(6)H(5)) and HPO(2) were observed. The results have revealed specific structural fingerprints for the screening of these compounds in complex biological mixtures.

Towards potential nanoparticle contrast agents: Synthesis of new functionalized PEG bisphosphonates

Summary The use of nanotechnologies for biomedical applications took a real development during these last years. To allow an effective targeting for biomedical imaging applications, the adsorption of plasmatic proteins on the surface of nanoparticles must be prevented to reduce the hepatic capture and increase the plasmatic time life. In biologic media, metal oxide nanoparticles are not stable and must be coated by biocompatible organic ligands. The use of phosphonate ligands to modify the nanoparticle surface drew a lot of attention in the last years for the design of highly functional hybrid materials. Here, we report a methodology to synthesize bisphosphonates having functionalized PEG side chains with different lengths. The key step is a procedure developed in our laboratory to introduce the bisphosphonate from acyl chloride and tris(trimethylsilyl)phosphite in one step.

Polyphosphonate ligands: From synthesis to design of hybrid PEGylated nanoparticles toward phototherapy studies

The use of phosphonate ligands to modify the nanoparticle (NPs) surface has attracted a strong interest in the last years for the design of highly functional hybrid materials. Here, we applied a methodology to synthesize bisphosphonates having functionalized PEG side chains with a specific length in order to design a novel class of hybrid nanomaterials composed by tetraphosphonate-complex-gold COOH-terminated PEG-coated NPs (Bis-PO-PEG-AuNPs). The synthetic approach consist in three steps: (1) Complexation between new phosphonate ligands (Bis PO) and tetrachloroauric acid (HAuCl4) to form gold clusters; (2) adsorption of COOH-terminated PEG molecules (PEG) onto Bis PO-Au complex; (3) reduction of metal ions in that vicinity, growth of gold particles and colloidal stabilization. The obtained snow-shape-like hybrid nanoparticles, have been characterized by ultra-violet/visible, Raman spectroscopies, and electron microscopy imaging, involving their optical properties and photothermal activity in pancreatic adenocarcinoma cancer cells (PDAC).

A1.36 Active immunisation against peptides of vegf improves joint inflammation and destruction in collagen-induced arthritis

Background Angiogenesis is a key pathophysiological process in rheumatoid arthritis (RA). VEGF-A is considered the major player in both physiological and pathological angiogenesis. VEGF–VEGFR system blockade delays synovitis onset and ameliorates collagen-induced arthritis (CIA). We previously demonstrated the effectiveness of active anti-cytokine immunisation in RA models, with a class of therapeutics called kinoids. The kinoids (heterocomplexes consisting of a cytokine conjugated to a carrier protein, the keyhole limpet hemocyanin, KLH) induce the endogenous production of polyclonal anti-cytokine Abs. Here we tested the effectiveness in CIA of two distinct peptidic vaccines obtained by conjugation of one of two different VEGF-derived peptides to KLH. Objectives To demonstrate the inhibitory effect on CIA of sustained VEGF blockade by vaccines based on VEGF-derived peptides linked to KLH (Vpep-K). Materials and Methods Two peptides were chosen in the sequence of the VEGF murine isoformVEGF-A164 (90% identity and 93% homology with the human VEGF-A165): Vpep1 (16 aminoacids (aa), position 98-113) localised in the region cleaved by the plasmin between the exons 4 and 5, and Vpep2 (77 carboxy-terminal aa) which comprises the Arginine 164, essential for binding of VEGF to Neuropilin-1 (NRP1). Each peptide was linked to keyhole limpet hemocyanin (KLH) to form Vpep1-kinoid (Vpep1-K) and Vpep2-kinoid (Vpep2-K), respectively. CIA was induced in 48 DBA/1 male mice with two injections of 100µg of bovine collagen type II (day 0 and 21). Mice were divided in 4 groups to receive one of the following: Vpep1-K, Vpep2-K, KLH or PBS emulsified in Incomplete Freund Adjuvant (IFA, 5 intra muscular injections at days -36, -22, -8, 7 and 37). Sera were sampled from each mouse three times during the experiment (days -44, -2, 48). Results Vpep1-K group showed lower arthritic scores compared to KLH and PBS groups (p<0.05). At histological analysis, inflammation and destruction scores of the paws were lower in Vpep1-K group versus KLH and PBS group (p<0.005). Vpep1-K and Vpep2-K groups showed anti-VEGF Ab production as assessed by ELISA at day -2 and sacrifice. Conclusions Active immunotherapy with the kinoid of a single VEGF-derived peptide (Vpep1-K) induces anti-VEGF antibodies and ameliorates collagen-induced arthritis. Active anti-angiogenic immunotherapy based on peptidic vaccines might be a potential strategy to control chronic inflammation.

Bifunctional Tripeptide with a Phosphonic Acid as a Bronsted Acid for Michael Addition: Mechanistic Insights

Enamine catalysis is a widespread activation mode in the field of organocatalysis and is often encountered in bifunctional organocatalysts. We previously described H-Pro-Pro-pAla-OMe as a bifunctional catalyst for Michael addition between aldehydes and aromatic nitroalkenes. Considering that opposite selectivities were observed when compared to H-Pro-Pro-Glu-NH2 , an analogue described by Wennemers, the activation mode of H-Pro-Pro-pAla-OMe was investigated through kinetic, linear effect studies, NMR analyses, and structural modifications. It appeared that only one bifunctional catalyst was involved in the catalytic cycle, by activating aldehyde through an (E)-enamine and nitroalkene through an acidic interaction. A restrained tripeptide structure was optimal in terms of distance and rigidity for better selectivities and fast reaction rates. Transition-state modeling unveiled the particular selectivity of this phosphonopeptide.