Jean-Marc Muller

Anticancer Drug Delivery with Transferrin Targeted Polymeric Chitosan Vesicles

AbstractPurpose. The study reports the initial biological evaluation of targeted polymeric glycol chitosan vesicles as carrier systems for doxorubicin (Dox). Methods. Transferrin (Tf) was covalently bound to the Dox-loaded palmitoylated glycol chitosan (GCP) vesicles using dimethylsuber- imidate (DMSI). For comparison, glucose targeted niosomes were prepared using N-palmitoyl glucosamine. Biological properties were studied using confocal microscopy, flow cytometry, and cytotoxicity assays as well as a mouse xenograft model. Results. Tf vesicles were taken up rapidly with a plateau after 1-2 h and Dox reached the nucleus after 60-90 min. Uptake was not increased with the use of glucose ligands, but higher uptake and increased cytotoxicity were observed for Tf targeted as compared to GCP Dox alone. In the drug-resistant A2780AD cells and in A431 cells, the relative increase in activity was significantly higher for the Tf-GCP vesicles than would have been expected from the uptake studies. All vesicle formulations had a superior in vivo safety profile compared to the free drug. Conclusions. The in vitro advantage of targeted Tf vesicles did not translate into a therapeutic advantage in vivo. All vesicles reduced tumor size on day 2 but were overall less active than the free drug.

Brain delivery of vasoactive intestinal peptide (VIP) following nasal administration to rats

The aim of this work was to study in rats the nasal route for the brain delivery of the vasoactive intestinal peptide (VIP) neuropeptide. After evaluating VIP stability in solutions obtained from nasal washes, the effect of formulation parameters (pH 4-9, 0-1% (w/v) lauroylcarnitine (LC), hypo- or isoosmolality) on the brain uptake of intranasally administered VIP (10(-8)M)/125I-VIP (300,000 cpm/ml) was studied, using an in situ perfusion technique. Brain radioactivity distribution was assessed by quantitative autoradiographic analysis. Results were compared to intravenously administered VIP. With a hypotonic formulation at pH 4 containing 0.1% LC and 1% bovine serum albumin, VIP stability was satisfactory and loss by adsorption was minimal. Using this formulation, around 0.11% of initial radioactivity was found in the brain after 30 min perfusion and was located in the olfactory bulbs, the midbrain and the cerebellum. HPLC analysis of brain and blood extracts demonstrated the presence of intact VIP in brain and its complete degradation in the blood compartment. By intravenous administration, no intact VIP was found either in brain or in blood. In conclusion, intact VIP could be delivered successfully to the brain using the intranasal route for administration.

Disruption of POF1B Binding to Nonmuscle Actin Filaments Is Associated with Premature Ovarian Failure

Premature ovarian failure (POF) is characterized by elevated gonadotropins and amenorrhea in women aged <40 years. In a Lebanese family with five sisters who received the diagnosis of POF, we established linkage to the long arm of the X chromosome (between Xq21.1 and Xq21.3.3), using whole-genome SNP typing and homozygosity-by-descent mapping. By sequencing one candidate gene within that region, POF1B, we identified a point mutation localized in exon 10. This substitution of a nucleotide (G-->A), at position 1123, results in an arginine-->glutamine mutation of the protein sequence at position 329 (mutation R329Q). All the affected family members were homozygous for the mutation, whereas the unaffected members were heterozygous. Because POF1B shares high homology with the tail portion of the human myosin, we assessed the ability of both wild-type and mutant POF1B proteins to bind nonmuscle actin filaments in vitro. We found that the capacity of the mutant protein to bind nonmuscle actin filaments was diminished fourfold compared with the wild type, suggesting a function of POF1B in germ-cell division. Our study suggests that a homozygous point mutation in POF1B influences the pathogenesis of POF by altering POF1B binding to nonmuscle actin filaments.

VIP as a cell-growth and differentiation neuromodulator role in neurodevelopment

In addition to its commonly recognized status as a neuromodulator of virtually all vital functions, including neurobiological., the neuropeptide VIP plays a role in the control of cell growth and differentiation and of neuronal survival. Through, these actions, VIP, whose impact appears early in ontogeny, may possess developmental functions. VIP can be stimulatory or inhibitory on cell growth in function of the model considered. The growth regulatory actions of VIP, which are often independent of cAMP, are most likely significant when mitogenic or trophic factors, eventually released by nontarget cells, are simultaneously present in the extracellular medium. The intracellular mechanisms that mediate these actions of VIP may involve different transduction cascades triggered by subsets of VIP binding sites that may coexist in the same tissue.

HT 29, a model cell line: stimulation by the vasoactive intestinal peptide (VIP); VIP receptor structure and metabolism

HT 29, a cell line derived from a human colonic adenocarcinoma, is highly responsive to the vasoactive intestinal peptide (VIP) as shown by a more than 100-fold intracellular cAMP increase (Ka = 0.3 nM), the stimulations of protein kinase A (Ka = 0.1 nM) and the low-Km cAMP phosphodiesterase (Ka = 40 nM). Remarkably, adenylate cyclase, cAMP-dependent kinase and cAMP-specific phosphodiesterase are activated in a sequential manner. Binding studies with [125I]-labeled VIP indicate a high affinity site with a Kd value (0.5 nM) close to the activation constant value (Ka) of the three enzymes. The molecular structure of the VIP receptor was studied by immunological and chemical approaches. A monoclonal antibody (mAb 109-10-16) which partially decreased the binding of VIP to its receptor allowed the characterization of Mr = 53,000 and Mr = 48-49,000 polypeptides. More precise identification of protein components of the VIP receptor resulted from covalent cross-linking on intact HT 29 cells by four bifunctional reagents: dithiobis-(succinimidyl propionate) and its non-cleavable analog disuccinimidyl suberate, the photoactivable azido phenyl glyoxal and dimethylpimelimidate. Analysis by SDS-polyacrylamide gel electrophoresis demonstrated a major band of Mr = 67,000 regardless of which cross-linker was used. The same band and an Mr = 49,000 species were found in experiments using a crude membrane fraction of HT 29 cells. Assuming one molecule of VIP (Mr = 3326) linked per polypeptide, these observations suggest that an Mr = 64,000 species belongs to the VIP specific plasma membrane receptor. This protein contains an Mr = 20,000 N-linked sialic acid rich oligosaccharidic moiety.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of VIP-Induced Neuroprotection against Neonatal Excitotoxicity

Abstract:  Two VIP receptors, shared with a similar affinity by pituitary adenylate cyclase‐activating polypeptide (PACAP), have been cloned: VPAC1 and VPAC2. PHI binds to these receptors with a lower affinity. We previously showed that VIP protects against excitotoxic white matter damage in newborn mice. This article aimed to determine the receptor involved in VIP‐induced neuroprotection. VIP effects were mimicked with a similar potency by VPAC2 agonists and PHI but not by VPAC1 agonists, PACAP 27 or PACAP 38. VIP neuroprotective effects were lost in mice lacking VPAC2 receptor. In situ hybridization confirmed the presence of VPAC2 mRNA. These data suggest that, in this model, VIP‐induced neuroprotection is mediated by VPAC2 receptors. The pharmacology of this VPAC2 receptor seems unconventional as PACAP does not mimic VIP effects and PHI acts with a comparable potency.

Potential Inhibition of the Neuro-Neoplastic Interactions: The Clue of a GPCR-Targeted Therapy

Other sections of this monograph, dedicated to neuronal activities in tumor tissue, have highlight the chief influence of neurotrophins, neurotransmitters, adhesion, guidance molecules and different nerve cell markers in the progression, but also for the prognostic, therapy and survey of cancers. The G-protein-coupled receptors (GPCR) are among the most successful and promising target proteins for drug discovery and therapeutic research. GPCR are frequently overexpressed in cancer cells, an interesting property for tumor imaging or for a targeted radiotherapy, using radiolabeled ligand derivatives. The tumor microenvironment contains a number of GPCR ligands (e.g., bioactive peptides, biogenic amines, purins, chemokines), known to regulate the proliferation, migration or survival of both tumoral and neural cells and that may be key actors of the neuro-neoplastic interactions. Here will be reviewed the potential utilization of substances that target a selected choice of GPCR, especially neuropeptide receptors, for a novel concept of therapy, concerning the numerous types of cancers where neurons infiltrate the tumoral mass or those where the malignant cells invade nerve branches (perineural invasion). Some molecular mechanisms linked to these GPCR (or linking GPCR to other types of membrane receptors or co-receptors), involved in these processes, will also be considered.

Expression and GTP Sensitivity of Peptide Histidine Isoleucine High-Affinity-Binding Sites in Rat

Abstract:  High‐affinity‐binding sites for the vasoactive intestinal peptide (VIP) analogs peptide histidine/isoleucine‐amide (PHI)/carboxyterminal methionine instead of isoleucine (PHM) are expressed in numerous tissues in the body but the nature of their receptors remains to be elucidated. The data presented indicate that PHI discriminated a high‐affinity guanosine 5′‐triphosphate (GTP)‐insensitive‐binding subtype that represented the totality of the PHI‐binding sites in newborn rat tissues but was differentially expressed in adult animals. The GTP‐insensitive PHI/PHM‐binding sites were also observed in CHO cells over expressing the VPAC2 but not the VPAC1 VIP receptor.

Modification of HT 29 cell response to the vasoactive intestinal peptide (VIP) by membrane fluidization

We used liposomes made with phospholipids of fatty acid chain length ranging from C12:0 to C16:0 to modify the cAMP dependent protein kinase (PK) activity of HT 29 cells induced by VIP or forskolin. Both VIP and forskolin effects were inhibited in dilauroylphosphatidylcholine (DLPC) treated cells. PK activity was slightly lowered when cells were treated by dimyristoylphosphatidylcholine (DMPC) liposomes. However neither VIP nor forskolin-induced PK activities were affected with dipalmitoylphosphatidylcholine (DPPC) liposomes. Furthermore, the binding of [125I]VIP to DLPC treated cells was drastically lowered whereas no change was observed when cells were incubated with DMPC or DPPC liposomes. On the other hand, the interaction of HT 29 cells with DLPC vesicles provoked a decrease in membrane cholesterol content with subsequent increase in membrane fluidity. These findings provide evidence that, in HT 29 cells, the mechanisms of VIP-receptor interaction and of adenylate cyclase activation is lipid dependent and is regulated by membrane fluidity.