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Biomedical subjects

P Couvreur

Publications and source records attributed to P Couvreur.

At least 19 recordsLinked to original sources

Colloidal carriers and blood-brain barrier (BBB) translocation: a way to deliver drugs to the brain?

The major problem in drug delivery to the brain is the presence of the blood-brain barrier (BBB) which limits drug penetration even if in certain pathological situations the BBB is partly disrupted. Therefore, various strategies have been proposed to improve the delivery of drugs to this tissue. This review presents the status of the BBB in healthy patients and in pathologies like neurodegenerative, cerebrovascular and inflammatory diseases. The second part of this article aims to review the invasive and non-invasive strategies developed to circumvent the BBB and deliver drugs into the brain. The use of nanotechnologies (liposomes, nanoparticles) is especially discussed in the ultimate part of the review evidencing their potentiality as non-invasive technique in the brain delivery of drugs with the possibility to target specific brain tissue thanks to ligand linked to carrier surface.

Animals↗

A relevant in vitro rat model for the evaluation of blood-brain barrier translocation of nanoparticles.

Poly(MePEG2000cyanoacrylate-co-hexadecylcyanoacrylate) (PEG-PHDCA) nanoparticles have demonstrated their capacity to reach the rat central nervous system after intravenous injection. For insight into the transport of colloidal systems across the blood-brain barrier (BBB), we developed a relevant in vitro rat BBB model consisting of a coculture of rat brain endothelial cells (RBECs) and rat astrocytes. The RBECs used in our model displayed and retained structural characteristics of brain endothelial cells, such as expression of P-glycoprotein, occludin and ZO-1, and immunofluorescence studies showed the specific localization of occludin and ZO1. The high values of transendothelial electrical resistance and low permeability coefficients of marker molecules demonstrated the functionality of this model. The comparative passage of polyhexadecylcyanoacrylate and PEG-PHDCA nanoparticles through this model was investigated, showing a higher passage of PEGylated nanoparticles, presumably by endocytosis. This result was confirmed by confocal microscopy. Thanks to a good in vitro/in vivo correlation, this rat BBB model will help in understanding the mechanisms of nanoparticle translocation and in designing new types of colloidal carriers as brain delivery systems.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Puromycin-based purification of rat brain capillary endothelial cell cultures. Effect on the expression of blood-brain barrier-specific properties.

One of the main difficulties with primary rat brain endothelial cell (RBEC) cultures is obtaining pure cultures. The variation in purity limits the achievement of in vitro models of the rat blood-brain barrier. As P-glycoprotein expression is known to be much higher in RBECs than in any contaminating cells, we have tested the effect of five P-glycoprotein substrates (vincristine, vinblastine, colchicine, puromycin and doxorubicin) on RBEC cultures, assuming that RBECs would resist the treatment with these toxic compounds whereas contaminating cells would not. Treatment with either 4 microg/mL puromycin for the first 2 days of culture or 3 microg/mL puromycin for the first 3 days showed the best results without causing toxicity to the cells. Transendothelial electrical resistance was significantly increased in cell monolayers treated with puromycin compared with untreated cell monolayers. When cocultured with astrocytes in the presence of cAMP, the puromycin-treated RBEC monolayer showed a highly reduced permeability to sodium fluorescein (down to 0.75 x 10(-6) cm/s) and a high electrical resistance (up to 500 Omega x cm(2)). In conclusion, this method of RBEC purification will allow the production of in vitro models of the rat blood-brain barrier for cellular and molecular biology studies as well as pharmacological investigations.

Animals↗

A new generation of polymer nanoparticles for drug delivery.

One of the main interests of using polymer nanoparticles as drug carrier systems is to control the delivery of the drugs including their biodistribution. During the last decade, it was clearly demonstrated that surface properties of nanoparticles were the key factor which determined the in vivo fate of such a carrier. Thus, the purpose of this work was to develop a new method which allows the easy fabrication of nanoparticles with versatile surface properties using polysaccharides. This preparation was based on the use of a redox radical polymerization reaction applied for the first time to the emulsion polymerization of alkylcyanoacrylates in aqueous continuous media. The dispersion of nanoparticles was very stable. The nanoparticle surfaces were coated with polysaccharides and their characteristics can be modulated by the type and the molecular weight of the polysaccharides used during the synthesis. Interestingly the biological properties of the polysaccharide immobilized on the nanoparticle surface can be preserved opening very interesting perspectives for such nanoparticles. This method also offers a new strategy for the design of modular biomimetic nanoparticles as drug carrier systems with multiple functions. One of the applications considered in this work was to use these nanoparticles coupled with haemoglobin as an oxygen carrier.

Complement System Proteins↗

Drug delivery to resistant tumors: the potential of poly(alkyl cyanoacrylate) nanoparticles.

Simultaneous cellular resistance to multiple lipophilic drugs represents a major problem in cancer chemotherapy. This drug resistance may appear clinically either as a lack of tumor size reduction or as the occurrence of clinical relapse after an initial positive response to antitumor treatment. The resistance mechanism can have different origins either directly linked to specific mechanisms developed by the tumor tissue or connected to the more general problem of distribution of a drug towards its targeted tissue. The purpose of this paper is to summarize the results of the use of poly(alkyl cyanoacrylate) nanoparticles to overcome multidrug resistance (MDR) phenomena at both the cellular and the non-cellular level.

Animals↗

Novel core(polyester)-shell(polysaccharide) nanoparticles: protein loading and surface modification with lectins.

This study describes new lectin-decorated or protein-loaded nanoparticles with a hydrophobic poly(epsilon-caprolactone) (PCL) core and a hydrophilic dextran (Dex) corona. In this view, a family of block Dex-PCLn copolymers was first synthesized, consisting of a Dex backbone to which n preformed PCL blocks were grafted. The ability of these new copolymers to form nanoparticles was evaluated in comparison with a series of PCL homopolymers of various molecular weights (2000, 10,000 and 40,000 g/mole). Two different nanoparticle preparation methods have been developed and tested for their efficacy to incorporate proteins. For this, three proteins were used: a model protein, bovine serum albumin (BSA), a lectin from leaves of Bauhinia monandra (BmoLL) and Lens culinaris (LC) lectin. All these proteins were successfully incorporated in nanoparticles with a mean diameter around 200 nm. Lectins could also be adsorbed onto the surface of Dex-PCLn nanoparticles. Surface-bound BmoLL conserved its hemagglutinating activity, suggesting the possible application of this type of surface-modified nanoparticles for targeted oral administration. Caco-2 cellular viability was higher than 70% when put in contact with Dex-PCLn nanoparticles, even at concentrations as high as 660 microg/ml.

Animals↗

Cationic emulsions improves the delivery of oligonucleotides to leukemic P388/ADR cells in ascite.

The aim of this study was to investigate the in vivo ability of O/W cationic emulsions to deliver oligonucleotides (ON) in leukemic P388/ADR cells in ascite, after intraperitoneal (IP) administration in mice. Cationic emulsions were prepared by microfluidization as previously described by Teixeira et al. [Pharm. Res 16 (1999) 30]. The formulations consisted mainly of medium chain triglycerides, phosphatidylcholine (PC), poloxamer, and either a monocationic lipid stearylamine (PC/SA-emulsion) or a polycationic lipid RPRC(18) (PC/RPRC(18)-emulsion). A model ON (33P-pdT(16)) was associated with cationic emulsions by single addition at the end of the manufacturing process. Seven days after P388/ADR inoculation IP to mice, ON free or associated with PC/SA or PC/RPRC(18) emulsions was injected IP at a dose of 0.5 mg/kg. At different interval times, ascite including cells, blood and the main organs were collected and the radioactivity counted by liquid scintillation. The overall results showed significantly high amounts of ON in the leukemic cell pellet, 24 h after administration of ON associated to either PC/SA (AUC(0-24 h)=13634, %injected dose/min) or PC/RPRC(18) (AUC(0-24 h)=22592, % injected dose/min), contrary to the free ON solution (AUC(0-24 h)=3095, %injected dose/min), which displayed only reduced capture by cancer cells. In conclusion, complexation of ON with cationic emulsions had a beneficial effect in increasing tumor cells uptake in vivo (up to sevenfold for PC/RPRC(18)-emulsion) after IP administration. This could open interesting prospects for the treatment of ovarian cancers.

Animals↗

N-methylation of anthracyclines modulates their cytotoxicity and pharmacokinetic in wild type and multidrug resistant cells.

Anthracyclines are the most commonly used classes of anticancer agents in chemotherapy. Development of resistance to these molecules is one of the major reasons for treatment failure. The overexpression of the membrane transporter P-glycoprotein (P-gp) is among the principal mechanisms involved in this phenomenon. This pump, which is responsible for the multidrug resistance (MDR) phenotype, decreases the toxicity of a wide range of unrelated anticancer drugs by increasing their cellular efflux. Structure-activity relationship experiments have shown that the positively charged amino group of the anthracyclines could be responsible for their transport by P-gp. Here, we used three new anthracyclines that shared the same chromophore but differed by the degree of N-methylation of their sugar moiety. Oxaunomycin (OXN) possessed a non-methylated amino group, while LB-1 was monomethylated and beta-clamycin T (BCT) was dimethylated. In sensitive cells (FLC), reduced cytotoxicity was related to the level of N-methylation; whereas in resistant cells (DOX-RFLC(1) and DOX-RFLC(2)) overexpressing different levels of P-gp, increased N-methylation enhanced anthracycline cytotoxicity. Decreased resistance in DOX-RFLCs was associated with an increased drug accumulation due to a reduced cellular efflux. As expected, the MDR modulator verapamil decreased resistance to these anthracyclines by increasing the cellular accumulation. These results suggest that N-methylation of anthracyclines circumvents resistance by diminishing drug transport by P-gp in MDR-positive cells. These observations could be the consequence of the steric hindrance created by the methyl group(s) which may impair the interaction between the positively charged amino group and the active site of P-gp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Therapeutic potentialities of EWS-Fli-1 mRNA-targeted vectorized antisense oligonucleotides.

We have used structured antisense oligonucleotides (AON), which are protected against extra and intracellular degradation by their internal structure. We have shown that if correctly designed this structure does not prevent them from hybridizing to the mRNA target. This concept allows reducing the number of thioate groups in the oligonucleotide and therefore the potential toxicity. Junction oncogenes are found in cancers such as certain leukemias, Ewing sarcoma, and thyroid papillary carcinomas. Ewing sarcoma is a cancer of children and young adults with bone metastasis. It is caused by a chromosomic translocation t(11;22) (q24;q12) creating a fusion gene between the genes EWS and Fli-1 giving rise to a chimeric protein which is an unnatural transcription factor. Immortalized NIH/3T3 cells transfected by the EWS-Fli-1 cDNA under the control of the LTR retroviral promoter--which do not undergo apoptosis and which became tumoral--were used for this study. As a model of Ewing sarcoma in nude mice, we have used permanently expressing human EWS-Fli-1 cells grafted to nude mice. The nanospheres or nanocapsules have been used to deliver two different AON: a phosphorothioate, and a structured chimeric AON, both targeted toward the junction area of EWS-Fli-1. Both types of AON-loaded nanoparticles inhibited the growth of the xenografted tumor after intratumoral injections into nude mice, whereas similar nanoparticles with control oligonucleotides had no effect. With AON in nanospheres, we have shown after 24 hours that the mRNA of EWS-Fli-1 was specifically down-regulated, confirming the antisense activity of the targeted AON.

Animals↗

Polyunsaturated fatty acids (PUFA) and eicosanoids in human health and pathologies.

Linoleic and alpha-linolenic acids, obtained from plant material in the diet are the precursors in tissues of two families with opposing effects which are referred to as "essential fatty acids" (EFA): arachidonic acid (AA) and pentaene (eicosapentaenoic acid: EPA) and hexaene (docosahexaenoic acid: DHA) acids. The role of EFA is crucial, without a source of AA or compounds which can be converted into AA, synthesis of prostaglandins (PGs) by a cyclooxygenase (COX) enzyme would be compromised, and this would seriously affect many normal metabolic processes. COX, also known as prostaglandin endoperoxide synthase (Pghs) or as prostaglandin G/H synthase, is a key membrane bound enzyme responsible for the oxidation of AA to PGs. Two COX isoforms have been identified, COX-1 and COX-2 that form PGH2, a common precursor for the biosynthesis of thromboxane A2 (TxA2), prostacyclin (PGI2) and PGs (PGD2, PGE2, PGF2alpha. COX-1 enzyme is expressed constitutively in most cells and tissues. Its expression remains constant under either physiological or pathological conditions controlling synthesis of those PGs primarily involved in the regulation of homeostatic functions. In contrast, COX-2 is an intermediate response gene that encodes a 71-kDa protein. COX-2 is normally absent from most cells but highly inducible in certain cells in response to inflammatory stimuli resulting in enhanced PG release. PGs formed by COX-2 primarily mediate pain and inflammation but have multiple effects that can favour tumorigenesis. They are more abundant in cancers than in normal tissues from which the cancers arise. COX-2 is a participant in the pathway of colon carcinogenesis, especially when mutation of the APC (Adenomatous Polyposis Coli) tumour suppressor gene is the initiating event. In addition, COX-2 up-regulation and elevated PGE2 levels are involved in breast carcinogenesis. It seems that there is a correlation between COX-2 level of expression and the size of the tumours and their propensity to invade underlying tissue. Inhibition by non-steroidal anti-inflammatory drugs (NSAIDs) of COX enzymes which significantly suppress PGE2 levels, reduced breast cancer incidence and protected against colorectal cancer. Therefore it is suggested that consumption of a diet enriched in n-3 PUFA (specifically EPA and DHA) and inhibition of COX-2 by NSAIDs may confer cardioprotective effects and provide a significant mechanism for the prevention and treatment of human cancers.

Cardiovascular Diseases↗

I. Arginine.

L-Arginine (Arg) is classified as an essential amino acid for birds, carnivores and young mammals and a conditionally essential amino acid for adults. It is converted by arginase to L-ornithine, a precursor of polyamines and urea, which is important in the urea cycle. Arg serves as a precursor for creatine, which plays an essential role in the energy metabolism of muscle, nerve and testis and accounts for Arg catabolism and for the synthesis of agmatine and proteins. Via its ability to increase growth hormone secretion it influences immune function. Depending on nutritional status and developmental stage, normal plasma Arg concentrations in humans and animals range from 95 to 250 micromol/l. Systemic or oral Arg administration has been shown to improve cardiovascular function and reduce myocardial ischemia in coronary artery disease patients. It reduces blood pressure and renal vascular resistance in essential hypertensive patients with normal or insufficient renal function. Although Arg plasma concentrations are not altered in hypercholesterolemic individuals, oral or intravenous Arg administration can reverse endothelial dysfunction in hypercholesterolemic patients and in cigarette smokers. The main importance of Arg is attributed to its role as a precursor for the synthesis of nitric oxide (NO), a free radical molecule that is synthesized in all mammalian cells from L-Arg by NO synthase (NOS). NO appears to be a major form of the endothelium-derived relaxing factor (EDRF). NO and EDRF share similar chemical and pharmacological properties and are derived from the oxidation of a terminal guanidine group of L-Arg. Various mechanisms have been implicated in the defect in vascular relaxation. These include, increased diffusional barrier for NO, L-Arg depletion, altered levels of reactive oxygen, inactivation of NO by superoxide anions (O2-). The independent reactions of O2-, NO and their reaction yielding peroxynitrite are critical in the initiation and maintenance of the atherosclerotic state and contribute to the defect in vasorelaxation. NO also plays a role as a neurotransmitter, mediator of immune response and as signaling molecule. The NO synthesized by iNOS in macrophages contributes to their cytotoxic activity against tumor cells, bacteria and protozoa. Our aim here is to review on some amino acids with high functional priority such as Arg and to define their effective activity in human health and pathologies.

Animals↗

II. Glutamine and glutamate.

Glutamine and glutamate with proline, histidine, arginine and ornithine, comprise 25% of the dietary amino acid intake and constitute the "glutamate family" of amino acids, which are disposed of through conversion to glutamate. Although glutamine has been classified as a nonessential amino acid, in major trauma, major surgery, sepsis, bone marrow transplantation, intense chemotherapy and radiotherapy, when its consumption exceeds its synthesis, it becomes a conditionally essential amino acid. In mammals the physiological levels of glutamine is 650 micromol/l and it is one of the most important substrate for ammoniagenesis in the gut and in the kidney due to its important role in the regulation of acid-base homeostasis. In cells, glutamine is a key link between carbon metabolism of carbohydrates and proteins and plays an important role in the growth of fibroblasts, lymphocytes and enterocytes. It improves nitrogen balance and preserves the concentration of glutamine in skeletal muscle. Deamidation of glutamine via glutaminase produces glutamate a precursor of gamma-amino butyric acid, a neurotransmission inhibitor. L-Glutamic acid is a ubiquitous amino acid present in many foods either in free form or in peptides and proteins. Animal protein may contain from 11 to 22% and plants protein as much as 40% glutamate by weight. The sodium salt of glutamic acid is added to several foods to enhance flavor. L-Glutamate is the most abundant free amino acid in brain and it is the major excitatory neurotransmitter of the vertebrate central nervous system. Most free L-glutamic acid in brain is derived from local synthesis from L-glutamine and Kreb's cycle intermediates. It clearly plays an important role in neuronal differentiation, migration and survival in the developing brain via facilitated Ca++ transport. Glutamate also plays a critical role in synaptic maintenance and plasticity. It contributes to learning and memory through use-dependent changes in synaptic efficacy and plays a role in the formation and function of the cytoskeleton. Glutamine via glutamate is converted to alpha-ketoglutarate, an integral component of the citric acid cycle. It is a component of the antioxidant glutathione and of the polyglutamated folic acid. The cyclization of glutamate produces proline, an amino acid important for synthesis of collagen and connective tissue. Our aim here is to review on some amino acids with high functional priority such as glutamine and to define their effective activity in human health and pathologies.

Animals↗

[Oral tolerance induced by poly (lactide-co-glycolide) containing B lactoglobulin].

Allergies to milk proteins are frequently encountered in the new born population. In order to prevent this allergy by inducing oral tolerance, one of the major allergenic milk protein, B lactoglobulin (BLG) was entrapped into biodegradable Poly (lactide-co-glycolide) microspheres and was then orally given to mice. Microspheres are able to protect proteins against degradation by intestinal proteolytic enzymes and to target the Peyers patches which are one important priming site of the mucosal immune system. Microspheres were prepared by the multiple emulsion solvent evaporation method. The goal of the formulation study was to associate large amounts of proteins to the smallest amount of polymer so that a minimal quantity of microspheres would be administered. It was shown that introducing tween 20 in the formulation was able to increase the encapsulation efficiency and to better control protein release reducing the burst release effect. Moreover, Oral administration of microspheres containing BLG reduced significantly (by 10.000) the amount of protein necessary to decrease both specific anti BLG IgE and DTH response. In conclusion, microspheres appear to be optimal systems to induce oral tolerance.

Drug Carriers↗

PEGylated polycyanoacrylate nanoparticles as vector for drug delivery in prion diseases.

PEGylated polymeric nanoparticles are hereby presented as a potential efficient drug carrier for the delivery of active therapeutic molecules in prion experimental diseases. Based on their blood long-circulating characteristics, these PEGylated particles made by the amphiphilic copolymer poly [methoxy poly(ethylene glycol) cyanoacrylate-co-hexadecyl cyanoacrylate] (PEG-PHDCA), showed comparatively conventional non-PEGylated nanoparticles, a higher uptake by the spleen and the brain which are both the target tissues of PrPres accumulation in scrapie infected animals.

Animals↗

Characterization of oligonucleotide/lipid interactions in submicron cationic emulsions: influence of the cationic lipid structure and the presence of PEG-lipids.

We have recently described how oligonucleotide (ON) stability and release from O/W cationic emulsions are governed by the lipid composition. The aim of the present paper was to investigate the properties of the ON/lipid complexes through fluorescence resonance energy transfer (FRET), size, surface tension measurements and cryomicroscopy. Starting from a typical emulsion containing stearylamine as a cationic lipid, the influence of the lipid structure (monocationic molecules bearing mono or diacyl chains, or polycations) as well as of the presence of PEGylated lipids, were studied. The presence of a positive charge on the droplet surface clearly contributed to enhance the ON interaction with lipid monolayers and to bring the ON molecules closer to the interface. Hydrophobic interactions through the acyl chains were shown to further enhance the anchorage of the ON/lipid complexes. In contrast, the incorporation of PEGylated lipids acted as a barrier against the establishment of electrostatic bindings, the polyethyleneglycol chains acting themselves as interaction sites for the ON leading to hydrophilic complexes. Similar features were observed for the polycationic lipid, and cryomicroscopy revealed the existence of bridges of various intensities between the droplets of the emulsion containing either PEG or the polycation, probably because of the configuration of the ON at the interface.

Algorithms↗

Nanoparticulate systems for the delivery of antisense oligonucleotides.

Antisense oligonucleotides are molecules that are able to inhibit gene expression being therefore potentially active for the treatment of viral infections or cancer. However, because of their poor stability in biological medium and their weak intracellular penetration, colloidal drugs carriers such as nanoparticles were developed for the delivery of oligonucleotides (ODN). ODN associated to nanoparticles were shown to be protected against degradation and to penetrate more easily into different types of cells. As a consequence, nanoparticles were shown to improve the efficiency of ODNs for the inhibition of the proliferation of cells expressing the point mutated Ha-ras gene. In vivo, polyalkylcyanoacrylate (PACA) nanoparticles were able to efficiently distribute the ODNs to the liver whereas the alginate nanosponges could concentrate the ODNs in the lungs. Finally, ODN loaded to PACA nanoparticles were able to improve in mice, the treatment of RAS cells expressing the point mutated Ha-ras gene.

Animals↗

Polyisobutylcyanoacrylate nanocapsules containing an aqueous core for the delivery of oligonucleotides.

Antisense oligonucleotides (ODNs) with base sequences complementary to a specific RNA can, after binding to intracellular mRNA, selectively modulate the expression of a gene. However, these molecules are poorly stable in biological fluids and are characterized by a low intracellular penetration. In view of using ODNs as active molecules, the development of nanocapsules containing ODNs in their aqueous core was considered. Nanocapsules were prepared by interfacial polymerization of isobutylcyanoacrylate (IBCA) in a W/O emulsion. After ultracentrifugation and re-suspension in water, the nanocapsules displayed a size of 350+/-100 nm. Oligonucleotide loading did not significantly influence the zeta potential, suggesting that they were located within the core of the nanocapsules. Fluorescence quenching assays confirmed this localization. When encapsulated in the nanocapsules and incubated in the presence of serum, the ODNs were efficiently protected from degradation by nucleases, whereas ODNs adsorbed onto nanospheres were less efficiently protected. This paper describes, for the first time, a nanotechnology able to encapsulate ODNs, rather than adsorbing them at the surface of a solid support. Such a formulation has great potential for oligonucleotide delivery.

Cyanoacrylates↗

Tamoxifen encapsulation within polyethylene glycol-coated nanospheres. A new antiestrogen formulation.

When dealing with solid tumors in vivo, pegylated long-circulating carrier systems show, after intravenous administration, an attractive extravasation profile with an enhanced localization in the tumoral interstitium. These systems could be of help for the delivery of cancer fighting drugs, such as Tamoxifen, a well known antiestrogen used in breast cancer therapy that possesses an extended biodistribution in vivo. This work aimed at encapsulating Tamoxifen in long-circulating poly(MePEGcyanoacrylate-co-hexadecylcyanoacrylate) 1:4 nanospheres. Tamoxifen-loaded poly(MePEGcyanoacrylate-co-hexadecylcyanoacrylate) nanospheres were successfully synthesized and characterized in terms of hydrophilicity/hydrophobicity by a model made up from near infrared spectra using principal component analysis. Zeta potential, drug loading, encapsulation efficiency, as well as biological effect, in vitro release and nanospheres integrity were also investigated. Even though near infrared spectroscopy could not detect Tamoxifen, it revealed that Pluronic F68 was associated with the pegylated nanospheres. HPLC measurements demonstrated that Tamoxifen was encapsulated in the pegylated nanospheres following a partition equilibrium between the polymeric and the aqueous phases. The Tamoxifen encapsulated in the nanospheres still showed a transcription inhibitory activity in ex vivo experiments. However, zeta potential and in vitro release suggested that Tamoxifen was essentially localized at the nanoparticles surface, resulting in an important and immediate drug release.

Drug Delivery Systems↗