Yann Pellequer

The targeting of surface modified silica nanoparticles to inflamed tissue in experimental colitis.

One aspect in the emerging field of nanomedicine is site specific drug delivery via nanoparticles. The use of nanoparticles allows for increased therapeutic efficiency with a lowered risk for and extent of adverse reactions resulting from systemic drug absorption. 5-Amino salicylic acid (5ASA) loaded silica nanoparticles (SiNP) are proposed here as drug delivery system for specific accumulation in inflamed colonic tissues allowing for selective medication delivery to such inflammation sites. The drug was covalently bound to SiNP by a four-step reaction process. In-vitro toxicity of modified SiNP was tested in appropriate cell culture systems, while targeting index and therapeutic efficiency were evaluated in a pre-existing colitis in mice. Particle diameter was around 140 nm after final surface modification. In-vitro drug release demonstrated significant drug retention inside the NP formulation. Toxicity of the different formulations was evaluated in-vitro cell culture exhibiting a lowered toxicity for 5ASA when bound to SiNP. In-vivo, oral SiNP were found to accumulate selectively in the inflamed tissues allowing for significant amounts of drug load. SiNP demonstrated their therapeutic potential by significantly lowering the therapeutically necessary drug dose when evaluating clinical activity score and myeloperoxidase activity (untreated control: 28.0+/-5.0 U/mg; 5ASA-solution (100mg/kg): 8.2+/-3.4 U/mg 5ASA-SiNP (25mg/kg): 5.2+/-2.4 U/mg). SiNP allow to combine advantages from selective drug targeting and prodrugs appearing to be a promising therapeutic approach for clinical testing in the therapy of inflammatory bowel disease.

Delivery of P-glycoprotein substrates using chemosensitizers and nanotechnology for selective and efficient therapeutic outcomes

As a result of its broad substrate specificity and critical localization in excretory and barrier function tissues, P-glycoprotein (P-gp) plays major roles in the pharmacokinetics, safety and efficacy profiles of numerous drugs. P-gp is often responsible for the failure of many chemical treatments against cancer, immunosuppressive, infectious and neurodegenerative diseases. Among the therapeutic approaches to circumvent P-gp function, advances in the design of new chemical P-gp modulators to interact specifically with P-gp have yielded few clinical successful reports. Members of a class of components that were initially developed as surface active agents showed promising results with regard to the modulation of P-gp. These components include surfactants and amphiphilic co-polymers. Alternatively, colloidal systems were developed to facilitate drug uptake in resistant cells. This approach is based on the encapsulation of drugs, which masks them from the biological environment and prevents their transport by P-gp using the surfactants released from the nanocarrier. Likewise, a novel and synergistic strategy is currently being explored and involves nanocarrier-mediated transport and controlled release of both P-gp substrates and P-gp modulators. In this review, we discuss recent results obtained by direct modulation with chemosensitizers and the available nanotechnology to modulate P-gp function. In this manuscript, we also discuss unexplored pathways for future therapies.

A tunable Caco-2/HT29-MTX co-culture model mimicking variable permeabilities of the human intestine obtained by an original seeding procedure

Standard monoculture models utilizing Caco-2 monolayers were extensively used to mimic the permeability of the human intestinal barrier. However, they exhibit numerous limitations such as the lack of mucus layer, an overestimation of the P-gp-mediated efflux and a low paracellular permeability. Here, we suggest a new procedure to set up an in vitro model of intestinal barrier to adjust gradually the properties of the absorption barrier. Mucin-secreting HT29-MTX cells were added to Caco-2 absorptive cells in a Transwell® at different time intervals. Effects of seeding day of HT29-MTX on the paracellular permeability of lucifer yellow (LY) and on the P-gp-mediated efflux of rhodamine 123 were investigated. Apparent permeability of the rhodamine 123 in the secretory direction was highly dependent on the seeding day of goblet cells. Transepithelial electrical resistance values and LY transport across the co-cultures in the apical-to-basolateral direction were intermediary between single Caco-2 and HT29-MTX models. Early seeding days of HT29-MTX allowed increasing the fraction of goblet cells in the co-culture. Co-culture permeability was unchanged between 21 and 30 days after Caco-2 seeding, corresponding to the period of use for Caco-2-based cell models. Thus, the HT29-MTX seeding day was a key factor to set up an in vitro intestinal model with tailor-made barrier properties in terms of P-gp expression and paracellular permeability.

Nanoparticles enhance therapeutic outcome in inflamed skin therapy

Inflammatory reactions of the skin are a major therapeutic field; however, drug delivery is nowadays only related to the use of classical formulations like ointments and creams. Here, we report the behaviour of polymeric submicron particles (NP) for selective drug delivery to the inflamed skin. NPs of nominal diameters from 50 to 1000 nm were administered to an experimental dithranol-induced dermatitis inflammation model in mice ears. The results revealed that smaller particles had an around 3-fold stronger and deeper penetration tendency with a preferential accumulation in inflamed skin hair follicles and sebaceous glands (2.8 ± 0.6% and 2.3 ± 0.4% for NP100 and NP50 compared to 0.84 ± 0.04% and 0.92 ± 0.02% for the same sizes on healthy skin). Betamethasone loaded NP confirmed the size dependency by being therapeutically more efficient from histological examination and measurement of different inflammatory markers in the skin (myeloperoxidase activity of untreated control, 1.2 ± 0.4; NP1000, 1.0 ± 0.4; NP100, 0.5 ± 0.2, all U/mg). This approach holds a high potential for a selective therapy to the inflamed skin by increasing the local intradermal availability with simultaneous reduction in systemic adverse effects.

Surfactant dependent toxicity of lipid nanocapsules in HaCaT cells.

Lipid nanocapsules (LNC) have been suggested for a variety of pharmaceutical applications. Among them approaches for drug delivery to the skin appear particularly interesting. The current standard composition has been modified to better understand their properties by selecting a variety of different surfactants. LNC have been prepared using different non-ionic surfactants (Solutol(®) HS15: Polyoxyl 15 Hydroxystearate; Cremophor(®) EL: Polyoxyl 35 Castor Oil; Simulsol(®) 4000: Polyoxyl 40 Hydrogenated Castor Oil; Vitamin E TPGS(®): alpha-tocopheryl poly(ethylene glycol) succinate; Polysorbate 20 and 80) and analysed for their size, stability, drug release and toxicity on keratinocytes in cell culture. The feasibility of LNC using different surfactant was surprisingly easy and led to a variety of stable formulations that were selected for further investigations. Surfactants led to a variability of the release kinetics (t50% release varied from Polysorbate 20: 2.5h to Simulsol(®) 4000: 5.0h), however different formulations from the same surfactant did not differ significantly. In vitro toxicity of LNC was surfactant type dependent and a correlation between LNC and the pure respective surfactant was found. This toxicity was found to be mainly independent from the surface active properties. The surfactant type in LNC is easily interchangeable from formulation point of view. LNC appear to be appropriate as carrier for cutaneous delivery however toxicity can vary distinctly depending on the surfactant used for the preparation.

Epithelial Heparin Delivery via Microspheres Mitigates Experimental Colitis in Mice

Low-molecular-weight heparins (LMWH) have been shown to be efficient in the treatment of inflammatory bowel disease (IBD). Parenteral heparin therapy, however, may cause hemorrhagic adverse effects. To reduce this risk, epithelial LMWH delivery in combination with a system ensuring selective drug release to the inflamed tissue was tested here. Enoxaparin loaded microspheres (MS) were administered orally to male BALB mice suffering from a pre-existing experimental colitis, whereas control groups received subcutaneous or rectal LMWH solution. Colon weight/length index and alkaline phosphatase and myeloperoxidase activities were assessed to determine the inflammation. Tissue penetration experiments elucidated the processes involved in the proposed new therapeutic approach. Oral LMWH-MS proved to be equally efficient in mitigating experimental colitis as rectally administered LMWH solution when quantified by myeloperoxidase activity (MS, 10.2 ± 1.5 U/mg tissue; rectal, 9.2 ± 1.6 U/mg) and to be superior to subcutaneous LMWH (s.c., 21.6 ± 5.6 U/mg; untreated colitis control, 30.0 ± 3.8 U/mg). Pharmacokinetic studies found a notably low systemic availability of oral LMWH delivered from MS (<5%) indicating a low potential for adverse effects. The tissue permeability was selectively enhanced in the inflamed regions where a 9-fold higher LMWH penetration was found compared with healthy tissue. Epithelial LMWH delivery has been found a promising anti-inflammatory therapeutic approach. The use of LMWH-MS in this context offers a promising tool for IBD therapy by enhancing specifically drug availability at inflamed tissue sites while reducing the risk for systemic adverse effects to a negligibly low level.

Nanoparticle-based clodronate delivery mitigates murine experimental colitis.

In inflammatory bowel disease (IBD) the disruption of the intestinal barrier function and the strong presence of immune-related cells like macrophages in inflamed tissue allow the selective accumulation of particulate carrier systems at the site of action. We developed clodronate loaded nanoparticles (ClNP) based on a cationic polymethacrylate (Eudragit RL) using a modified solvent displacement method. Particle diameter of ClNP was around 120nm and dissolution experiments showed that ionic interactions with either the dissolution medium or mucin have to take place to enable complete drug release. In murine experimental colitis in-vivo, myeloperoxidase activity decreased significantly in 2,4,6-trinitrobenzenesulfonic acid (TNBS)-colitis and oxazolone (OXA)-colitis models after treatment with ClNP while free clodronate did not show a mitigating effect. Similarly, alkaline phosphatase could be lowered significantly from 12.5±1.9 to 6.8±2.2ng/mg tissue in TNBS-colitis and from 16.6±6.2 to 11.8±2.7ng/mg tissue in OXA-colitis. In cultured RAW 264.7 cells, only ClNP but not clodronate alone led to a decrease in tumor necrosis factor-alpha and interleukin-6 secretion of the activated macrophages. The therapeutic benefit of ClNP was confirmed in-vivo although it is limited compared to data with other drugs. Cell culture experiments indicated that intracellular delivery of clodronate was necessary to obtain an anti-inflammatory effect.

Zinc-pectinate beads as an in vivo self-assembling system for pulsatile drug delivery

Zinc-pectinate beads are interesting drug carriers for oral delivery. In order to investigate their in vitro and in vivo release behaviour, ionotropic gelation was used to entrap theophylline into calcium- or zinc-pectinate beads. Beads were investigated in vitro for their particle properties, especially the release kinetic in different media, and their in vivo pharmacokinetic parameters were tested in rats. Particle size varied between 1.8 and 2.8mm and encapsulation rates between 27 and 30% for Ca- and Zn-pectinate beads, respectively. While Ca-pectinate beads revealed a relative fast disintegration, drug release profiles from Zn-pectinate beads were very much release medium-dependent. Especially, in the presence of phosphate ions, the release from Zn-pectinate beads was blocked at 20% and 40% of the total drug load when tested in phosphate buffer or simulated colonic medium. In vivo Zn-pectinate beads (t(max): 12.0 ± 0.1h) led to a significant lag time for the theophylline absorption compared to Ca-pectinate (t(max): 6.0 ± 2.8h) or free theophylline (t(max): 2.5 ± 2.1h). This delayed release was attributed to the formation of a zinc phosphate coating in vitro and in vivo inducing the retention of theophylline release. Zn-pectinate beads exhibit interesting properties due to its potential as pulsatile delivery system induced by the in situ formation of Zn phosphate, while Ca-pectinate was found to be of limited suitability for controlled release of theophylline.

Coadministration of P-Glycoprotein Modulators on Loperamide Pharmacokinetics and Brain Distribution

The efflux transporter P-glycoprotein, expressed at high levels at the blood-brain barrier, exerts a profound effect on the disposition of various therapeutic compounds in the brain. A rapid and efficient modulation of this efflux transporter could enhance the distribution of its substrates and thereby improve central nervous system pharmacotherapies. This study explored the impact of the intravenous coadministration of two P-glycoprotein modulators, tariquidar and elacridar, on the pharmacokinetics and brain distribution of loperamide, a P-glycoprotein substrate probe, in rats. After 1 hour postdosing, tariquidar and elacridar, both at a dose of 1.0 mg/kg, increased loperamide levels in the brain by 2.3- and 3.5-fold, respectively. However, the concurrent administration of both P-glycoprotein modulators, each at a dose of 0.5 mg/kg, increased loperamide levels in the brain by 5.8-fold and resulted in the most pronounced opioid-induced clinical signs. This phenomenon may be the result of a combined noncompetitive modulation by tariquidar and elacridar. Besides, the simultaneous administration of elacridar and tariquidar did not significantly modify the pharmacokinetic parameters of loperamide. This observation potentially allows the concurrent use of low but therapeutic doses of P-gp modulators to achieve full inhibitory effects.

Potentiation of the bactericidal activity of Harungana madagascariensis Lam. ex Poir. (Hypericaceae) leaf extract against oral bacteria using poly (D, L-lactide-co-glycolide) nanoparticles: in vitro study.

Harungana madagascariensis Lam. ex Poir. (Hypericaceae) is known to have biological properties with mainly antibacterial, antifungal, and antiviral effects. The objective of this study was to investigate the in vitro bactericidal activity of the ethyl acetate H. madagascariensis leaf extract (HLE) on the main oral bacterial strains largely implicated in dental caries and gingivitis infections, and the possibility of potentialization of HLE antibacterial effects using the poly (D,L-lactide-co-glycolide) nanoparticles (PLG-NP). The microdilution technique and the interfacial polymer deposition following the solvent diffusion method were used to investigate the in vitro bactericidal activity of ethyl acetate HLE and to prepare nanoparticles, respectively. HLE showed significant bactericidal effects against the bacterial strains tested, with minimal bactericidal concentration (MBC) to 5×102 mg/l or less, except for Lactobacillus casei with 7.5×102 mg/l. With the HLE incorporated into PLG nanoparticles (HLE-PLG-NP), we observed diminution of the bactericidal concentration compared to HLE, the upper MBC being of 1.875×102 mg/l. Incorporation of the HLE into a colloidal carrier optimized its antibacterial performance.