Enhanced drug loading in polymerized micellar cargo.

A new drug carrier system based on self-assembly and polymerization of polydiacetylenic amphiphiles is described. Although classical amphiphiles can help in solubilizing hydrophobic molecules upon self-arrangement into a variety of nanometric structures, a greater effect on drug loading was observed for our polymerized micelles as compared to the non-polymerized analogues. This permitted higher aqueous solubilization of lipophilic drugs with low micelle concentration. (14)C labeling of a model drug on one side and of the amphiphile on the other side permitted assessment, after intravenous injection, of biodistribution and excretion profiles of the drug cargo.

Biodistribution and anticancer activity of a new Vinca alkaloid encapsulated into long-circulating liposomes.

S12363 is a potent therapeutic agent with a strong in vitro activity against a variety of tumor types but also a high in vivo toxicity. Loading of this drug into long-circulating liposomes is expected to enhance its therapeutic index. Pharmacokinetics of liposomal S12363 showed that circulating S12363 was entrapped into liposomes until 24 hours after intravenous injection in mice. The liposomal formulation significantly increased the plasma concentration, half-life, and AUC and decreased the plasma clearance rates and volume of distribution of S12363. Liposome extravasation was evaluated with two tumor models by both microscopic analysis and liposome radiolabeling. Liposome accumulation was much more important in the case of B16 melanoma, compared to H460 tumor, with both inoculated subcutaneously and with comparable size. H460 tumor was also inoculated into the lung. The tumor localization did not influence liposome accumulation into the tissue. The liposomal formulation injected into mice bearing B16 melanoma allowed a 10-fold accumulation of S12363 into the tumor interstitium, as compared to the solution. Bioluminescence data, supported by the survival curves of the animals, showed that S12363-liposomes were able to significantly restrict B16 melanoma progression and increase mice survival.

Influence of PLA-PEG nanoparticles manufacturing process on intestinal transporter PepT1 targeting and oxytocin transport.

Oral administration of peptides still remains a challenging issue. We previously pointed out the possibility to target intestinal PepT1 transporter with functionalized PLA-PEG nanoparticles (NPs) formulated by nanoprecipitation, and to improve drug-loaded intestinal permeability. Nevertheless, alternative manufacturing processes exist and the impact on the intestinal transporter targeting could be interesting to study. Our objective is consequently to assess the ability of functionalized NPs to target PepT1 according to the manufacturing process, and the possibility to improve peptide absorption. PLA-PEG-Valine NPs were formulated by nanoprecipitation, double and simple emulsion with median particle size <200 nm. Using Caco-2 cells, the competition between PLA-PEG-Val NPs formulated by the different manufacturing processes, and [3H]Glycylsarcosine, a well-known substrate of PepT1, was observed to evaluate the impact of the process on the intestinal transporter PepT1 targeting. Simultaneously, PLA-PEG-Val NPs were labeled with fluorescein (FITC) to evaluate PepT1 targeting and to observe the behavior of the NPs close to the cell according to the manufacturing process by confocal imaging. Finally, oxytocin peptide (OXY) was encapsulated in Val-NPs according to the most relevant process and the transport of the drug was assessed in vitro and in vivo, and compared to free drug. It was possible to observe by TEM imaging a better organization and expression of the ligand at the surface for NPs formulated by emulsion processes. Furthermore, the competition between functionalized NPs and [3H]Glycylsarcosine revealed a better transport inhibition of [3H]Glycylsarcosine for NPs formulated by double emulsion (≈ 67%). These results were confirmed by fluorescence measurements, comparing the amount of fluorescence linked to the cells after incubation with fluorescent Val-NPs for the 3 processes (≈ 39% for double emulsion). Additionally, confocal microscopy confirmed the ability of Val-NPs prepared by double emulsion to target the cell membrane and even to reach the intracellular space. OXY was then encapsulated by double emulsion in Val-NPs with a drug load of ≈ 4%. It was thus shown in vitro that drug transport was doubled compared to free drug. In vivo, OXY plasma concentration after oral administration were significantly increased when encapsulated in Val-NPS obtained by double emulsion compared to free drug. These results demonstrated that NPs prepared by double emulsion allowed a better PepT1 targeting and is a promising approach for oral peptide delivery.

Optimization of magnetic retention in the gastrointestinal tract: Enhanced bioavailability of poorly permeable drug.

Oral administration of low permeable drugs remains a challenge as they do not cross biological membrane efficiently and therefore exhibit a poor bioavailability. Herein, the effect of magnetic retention on the circulation and bioavailability of magnetic beads in the gastrointestinal tract in the presence of an external magnetic field is evaluated. Retention efficiency is imaged using magnetic resonance and near infrared techniques. The effect on bioavailability is then evaluated in a pharmacokinetic study. Iron oxide nanoparticles, the drug (dipeptidyl peptidase-IV inhibitor) and a fluorophore (Alexa Fluor-750) are co-encapsulated in chitosan-alginate core-shell beads. Retention of these beads is induced by the presence of an external permanent magnet on the abdomen of rats. After single administration of magnetic beads containing 20mg/kg of drug to fasted rats, a 2.5-fold increase in drug's bioavailability is observed in the presence of an external magnetic field, significantly higher than the same dose administered to rats without the field or for the drug in aqueous solution. Retention of the magnetic carriers in the presence of an external magnet proves to accumulate these carriers in a specific localization of the intestine leading to a significant improve in the drug's bioavailability.

Mechanistic characterization of S 38093, a novel inverse agonist at histamine H3 receptors.

Histaminergic H3 inverse agonists, by stimulating central histamine release, represent attractive drug candidates to treat cognitive disorders. The present studies aimed to describe the mechanistic profile of S 38093 a novel H3 receptors inverse agonist. S 38093 displays a moderate affinity for rat, mouse and human H3 receptors (Ki=8.8, 1.44 and 1.2µM, respectively) with no affinity for other histaminergic receptors. In cellular models, the compound was able to antagonize mice H3 receptors (KB=0.65µM) and to suppress cAMP decrease induced by an H3 agonist via human H3 receptors (KB=0.11µM). The antagonism properties of the compound were confirmed by electrophysiological studies on rat hippocampal slices (from 0.1µM). In cells expressing a high H3 density, S 38093 behaved as a moderate inverse agonist at rat and human H3 receptors (EC50=9 and 1.7µM, respectively). S 38093 was rapidly absorbed in mouse and rat (Tmax=0.25-0.5h), slowly in monkey (2h), with a bioavailability ranging from 20% to 60% and t1/2 ranging from 1.5 to 7.4h. The compound was widely distributed with a moderate volume of distribution and low protein binding. The brain distribution of S 38093 was rapid and high. In mice, S 38093 significantly increased ex vivo N-tele-Methylhistamine cerebral levels from 3mg/kg p.o. and antagonized R-α-Methylhistamine-induced dipsogenia from 10mg/kg i.p. Taken together, these data suggest that S 38093, a novel H3 inverse agonist, is a good candidate for further in vivo evaluations, in particular in animal models of cognition.

Localisation of drug permeability along the rat small intestine, using markers of the paracellular, transcellular and some transporter routes.

The small intestine is the major site of drug absorption. Some reports in the literature have evoked the concept of "absorption windows" in the small intestine: are there specific regions where drug absorption is significantly higher than others? To investigate this question, we used an everted gut sac method to study the permeability of drugs and markers every 3-4cm down the entire small intestine in rat. These markers were chosen to be representative of the mechanisms by which drugs cross the small intestinal mucosa: paracellular and transcellular passive diffusion, via influx transporters, and a drug (digoxin) that is effluxed from cells by P-glycoprotein (P-gp). The passive diffusion and influx transporter markers gave similar profiles with a plateau of permeability along the jejunum, and with the exception of L-Dopa, lower permeability in the ileum. Digoxin showed a linear decrease in the profile from the proximal jejunum to the ileum. Permeability in the duodenum was two to three times lower than the jejunum for all compounds. There were no narrow specific regions of high permeability and so the concept of discrete "absorption windows" along the small intestine as suggested from some pharmacokinetic studies may be related to other effects such as pH and/or solubility.

Quantitative mass spectrometry imaging of propranolol and olanzapine using tissue extinction calculation as normalization factor.

In order to quantify small molecules at the early stage of drug discovery, we developed a quantitation approach based on mass spectrometry imaging (MSI) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) without the use of a labeled compound. We describe a method intended to respond to the main challenges encountered in quantification through MALDI imaging dedicated to whole-body or single heterogeneous organ samples (brain, eye, liver). These include the high dependence of the detected signal on the matrix deposition, the MALDI ionization yield of specific target molecules, and lastly, the ion suppression effect on the tissue. To address these challenges, we based our approach on the use of a normalization factor called the TEC (Tissue Extinction Coefficient). This factor takes into account the ion suppression effect that is both tissue- and drug-specific. Through this protocol, the amount of drug per gram of tissue was determined, which in turn, was compared with other analytical techniques such as Liquid Chromatography-Mass spectrometry (LC-MS/MS).

Development of an in vitro rat intestine segmental perfusion model to investigate permeability and predict oral fraction absorbed.

PURPOSE: The aims of the study are to develop and evaluate an in vitro rat intestine segmental perfusion model for the prediction of the oral fraction absorbed of compounds and to assess the ability of the model to study intestinal metabolism. METHODS: The system consisted of a perfusion cell with a rat intestinal segment and three perfusion circulations (donor, receiver, and rinsing circulation). Lucifer yellow (LY) was applied as internal standard together with test compounds in the donor circulation. To validate the model, the permeability of eight noncongeneric passively absorbed drugs was determined. Intestinal N-demethylation of verapamil into norverapamil was followed in the donor and receiver circulations by high-performance liquid chromatography analysis. RESULTS: The in vitro model allowed ranking of the tested compounds according to their in vivo absorption potential. The Spearman's correlation coefficient between the oral fraction absorbed in humans and the ratio of permeation coefficient of test compound to the permeation coefficient of LY within the same experiment was 0.98 (P < 0.01). Moreover, intestinal N-demethylation of verapamil, its permeation, and the permeation of its metabolite norverapamil could be assessed in parallel. CONCLUSIONS: Up to six permeation kinetics can be obtained per rat, and the method has shown to be a valuable tool to estimate human oral absorption.