Double or Simple Emulsion Process to Encapsulate Hydrophilic Oxytocin Peptide in PLA-PEG Nanoparticles.

PURPOSE: Oral drug delivery using NPs is a current strategy for poorly absorbed molecules. It offers significant improvement in terms of bioavailability. However, the encapsulation of proteins and peptides in polymeric NPs is a challenge. Firstly, the present study focused on the double emulsion process in order to encapsulate the OXY peptide. Then the technique was challenged by a one-step simplified process, the simple emulsion. METHODS: In order to study the influence of formulation and process parameters, factorial experimental designs were carried on. The responses observed were the NP size (<200 nm in order to penetrate the intestinal mucus layer), the suspension stability (ZP < |30| mV) and the OXY loading. RESULTS: It was thus found that the amount and the nature of surfactant, the ratio between the phases, the amount of PLA-PEG polymer and OXY, the presence of a viscosifying agent, and the duration of the sonication could significantly influence the responses. Finally, OXY-loaded NPs from both processes were obtained with NP size of 195 and 226 nm and OXY loading of 4 and 3.3% for double and simple emulsions, respectively. CONCLUSION: The two processes appeared to be suitable for OXY encapsulation and comparable in term of NP size, peptide drug load and release obtained.

Novel surfactants with diglutamic acid polar head group: drug solubilization and toxicity studies.

PURPOSE: Novel surfactants made of diglutamic acid (DG) polar head linked to lithocholic, arachidonic, linoleic or stearic acids were designed for drug solubilization. METHODS: Surfactants 3-D conformer and packing parameter were determined by molecular modelling and self-assembling properties by pyrene fluorescence measurements. Cytotoxicity was assessed on Human Umbilical Vein Endothelial Cells (HUVEC) and haemolyitic activity on rat red blood cells. Drug solubilization was quantified and its interaction with hydrophobic moieties was characterized using differential scanning calorimetry and X-ray diffraction. Self organisation of stearoyl-DG was observed by cryogenic transmission electron microscopy. Toxicity after repeated injections of stearoyl-DG was investigated in Wistar rats. RESULTS: DG-based surfactants self-assemble into water and their critical micellar concentrations are comprised between 200 and 920 µg/mL. Cytotoxicity and haemolysis were lower than for polysorbate 80. At best, stearoyl-DG solubilized the drug up to 22% (w/w). Solid-state characterization evidenced drug/lipid interactions leading to the formation of a new complex. Stearoyl-DG formed spherical micelles of 20 nm, as predicted by packing parameter calculation. However, it induced a possible liver toxicity after intravenous administration in rats. CONCLUSIONS: Among the surfactants tested, stearoyl-DG is the more efficient for drug solubilization but its use is limited by its possible liver toxicity.

Supramolecular organization of S12363-liposomes prepared with two different remote loading processes.

The S12363 anticancer drug was encapsulated into liposomes in an attempt to increase its therapeutic index. Loading of S12363 was achieved using two different processes based on the formation of either a pH gradient or an ammonium gradient between the acidic inner liposomal compartment and the basic outer phase. High encapsulation yields (>90%) were obtained using both processes for sphingomyelin/cholesterol/cholesterol-PEG vesicles. Spectrofluorimetry measurements have shown that liposomes were characterized by an internal pH around 4 for both loading processes. This internal pH was stable over a period of at least 20 days. Differential scanning calorimetry coupled with time-resolved synchrotron X-ray diffraction was used to study the drug/carrier supramolecular organization. In ammonium sulfate, S12363 was inserted into the bilayer in the vicinity of the polar headgroup. In citrate buffer, S12363 was mainly adsorbed at the water-lipid interface. The drug partitioning into the membrane was inhomogeneous and led to the formation of drug-rich and drug-poor domains. This effect was enhanced in the presence of cholesterol, especially in ammonium sulfate. To conclude, for both processes, the encapsulated drug was found inside the liposome aqueous core but strongly interacting with the membrane.

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.

Contribution of Intrinsic Fluorescence to the Design of a New 3D-Printed Implant for Releasing SDABS.

Single-domain antibodies (sdAbs) offer great features such as increased stability but are hampered by a limited serum half-life. Many strategies have been developed to improve the sdAb half-life, such as protein engineering and controlled release systems (CRS). In our study, we designed a new product that combined a hydrogel with a 3D-printed implant. The results demonstrate the implant's ability to sustain sdAb release up to 13 days through a reduced initial burst release followed by a continuous release. Furthermore, formulation screening helped to identify the best sdAb formulation conditions and improved our understanding of our CRS. Through the screening step, we gained knowledge about the influence of the choice of polymer and about potential interactions between the sdAb and the polymer. To conclude, this feasibility study confirmed the ability of our CRS to extend sdAb release and established the fundamental role of formulation screening for maximizing knowledge about our CRS.

Consequences of ions and pH on the supramolecular organization of sphingomyelin and sphingomyelin/cholesterol bilayers.

For drug delivery purpose the anticancer drug S12363 was loaded into ESM/Chol-liposomes using either a pH or an ammonium gradient. Association between the drug and the liposome depends markedly on the liposome membrane structure. Thus, ESM and ESM/Chol bilayer organization had been characterized by coupled DSC and XRDT as a function of both cholesterol concentration and aqueous medium composition. ESM bilayers exhibited a ripple lamellar gel phase P(beta') below the melting temperature and adopted a L(beta)-like gel phase upon Chol insertion. Supramolecular organization of ESM and ESM/Chol bilayers was not modified by citrate buffer or ammonium sulfate solution whatever the pH (3< or = pH < or =7). Nevertheless, in ESM bilayer, ammonium sulfate salt induced a peculiar organization of head groups, leading to irregular d-spacing and weakly correlated bilayers. Moreover, in the presence of salts, a weakening of van der Waals attraction forces was seen and led to a swelling of the water layer.

[Oral bioavailability and drug/carrier particulate systems]. / Biodisponibilité et vecteurs particulaires pour la voie orale.

The oral route remains the preferred route of administration to ensure patient satisfaction and compliance. However, new chemical entities may exhibit low bioavailability after oral administration because of poor stability within the gastrointestinal tract, poor solubility in gastrointestinal fluids, low mucosal permeability, and/or extensive first-pass metabolism. Consequently, these new drug substances cannot be further developed using conventional oral formulations. This issue is addressed by an innovative approach based on the entrapment of drug molecules in drug/carrier assembling systems. The carrier materials are lipids, naturally occurring polymers or synthetic polymers, which are considered as nontoxic and biocompatible materials. Drug entrapment is intended to protect drug substances against degradation by gastrointestinal fluids. Fine drug/carrier particle size ensures increased drug dissolution rates. Carriers and particle supramolecular organization can be designed to enhance drug absorption through the intestinal epithelium and lymphatic transport. Promising preclinical results have been obtained with model drugs like paclitaxel, insulin, calcitonin, or cyclosporin. Attention has focused on mucoadhesive carriers like chitosan that favor an intimate and extended contact between drugs and intestinal cells, thus enhancing absorption. Addition of ligands such as lectins improves intestinal drug absorption through specific binding of the carrier to intestinal cell carbohydrates. In conclusion, drug/carrier particulate systems are an attractive and exciting drug delivery strategy for highly potent drug substances unsuitable for oral use. Further evidence will determine whether this approach has marked therapeutic benefits over conventional drug formulations and is compatible with large-scale industrial production and stringent registration requirements. Producing highly effective particulate systems requiring low-complexity manufacturing processes is therefore an ongoing challenge.

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.

Functionalized PLA-PEG nanoparticles targeting intestinal transporter PepT1 for oral delivery of acyclovir.

Targeting intestinal di- and tri-peptide transporter PepT1 with prodrugs is a successful strategy to improve oral drug bioavailability, as demonstrated with valacyclovir, a prodrug of acyclovir. The aim of this new drug delivery strategy is to over-concentrate a poorly absorbed drug on the intestinal membrane surface by targeting PepT1 with functionalized polymer nanoparticles. In the present study, poly(lactic acid)-poly(ethylene glycol)-ligand (PLA-PEG-ligand) nanoparticles were obtained by nanoprecipitation. A factorial experimental design allowed us to identify size-influent parameters and to obtain optimized ≈30nm nanoparticles. Valine, Glycylsarcosine, Valine-Glycine, and Tyrosine-Valine were chemically linked to PLA-PEG. In Caco-2 cell monolayer model, competition between functionalized nanoparticles and [3H]Glycylsarcosine, a strong substrate of PepT1, reduced [3H]Glycylsarcosine transport from 22 to 46%. Acyclovir was encapsulated with a drug load of ≈10% in valine-functionalized nanoparticles, resulting in a 2.7-fold increase in permeability as compared to the free drug. An in vivo pharmacokinetic study in mice compared oral absorption of acyclovir after administration of 25mg/kg of valacyclovir, free or encapsulated acyclovir in functionalized nanoparticles. Acyclovir encapsulation did not statistically modify AUC or Cmax, but increased t1/2 and MRT 1.3-fold as compared to free acyclovir. This new strategy is promising for poorly absorbed drugs by oral administration.

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.

Performance of magnetic chitosan-alginate core-shell beads for increasing the bioavailability of a low permeable drug.

This work reports the synthesis and performance of magnetic chitosan-alginate core-shell beads for oral administration of small molecules in order to increase their bioavailability. For this purpose, we designed magnetic core-shell beads suitable for oral delivery that are resistant in acidic media (stomach pH), mucoadhesive, exhibit a superparamagnetic behavior and a very high entrapment efficiency. Ex vivo experiments were performed in Ussing chambers, to emphasize the effect of magnetic accumulation. The amount of drug permeated through the membrane exhibited a threefold increase with our novel drug delivery system. According to a correlation law, our ex vivo model showed that the adsorbed fraction (FA) in human is expected to reach 70% when using the magnetic retention system which is a great improvement when compared to the controls (FA=20%).

Comparative static curing versus dynamic curing on tablet coating structures.

Curing is generally required to stabilize film coating from aqueous polymer dispersion. This post-coating drying step is traditionally carried out in static conditions, requiring the transfer of solid dosage forms to an oven. But, curing operation performed directly inside the coating equipment stands for an attractive industrial application. Recently, the use of various advanced physico-chemical characterization techniques i.e., X-ray micro-computed tomography, vibrational spectroscopies (near infrared and Raman) and X-ray microdiffraction, allowed new insights into the film-coating structures of dynamically cured tablets. Dynamic curing end-point was efficiently determined after 4h. The aim of the present work was to elucidate the influence of curing conditions on film-coating structures. Results demonstrated that 24h of static curing and 4h of dynamic curing, both performed at 60°C and ambient relative humidity, led to similar coating layers in terms of drug release properties, porosity, water content, structural rearrangement of polymer chains and crystalline distribution. Furthermore, X-ray microdiffraction measurements pointed out different crystalline coating compositions depending on sample storage time. An aging mechanism might have occur during storage, resulting in the crystallization and the upward migration of cetyl alcohol, coupled to the downward migration of crystalline sodium lauryl sulfate within the coating layer. Interestingly, this new study clearly provided further knowledge into film-coating structures after a curing step and confirmed that curing operation could be performed in dynamic conditions.

Drug solubilization and in vitro toxicity evaluation of lipoamino acid surfactants.

To improve solubilization of a water insoluble anticancer drug, novel surfactants were synthesized. All surfactants derived from lysine, with a so-called nitrilo triacetic acid (NTA) polar head, and differed from the length and saturation degree of their hydrophobic moieties: C19:0-NTA, C20:4-NTA, C25:0-NTA and C25:4-NTA. Self-assembling properties and critical micellar concentration (CMC) values were determined using pyrene fluorescence and cytotoxicity using MTT and LDH assays on endothelial cells. Surfactant haemolytic activity and drug solubilization capacity were also evaluated. All surfactants self-assemble with low CMC values from 0.012 to 0.430 mg/mL. Cytotoxicity assays showed that C20:4-NTA and C25:0-NTA were less cytotoxic than polysorbate 80. Unsaturations and alkane chain length have a marked influence on toxicity. Saturated surfactants had a similar haemolytic activity, explained by their low CMC values and the linear configuration of their hydrophobic tail. C20:4-NTA and C25:4-NTA were less haemolytic than polysorbate 80. Furthermore, C19:0-NTA, C25:0-NTA and C25:4-NTA increased drug solubility from <0.15 µg/mL up to 7 mg/mL, with 46% (w/w) drug loading, due to their linear and flexible hydrophobic chain configuration, as evidenced by molecular modelling. Although these solubilizers are promising, a compromise between drug solubilization and toxicity remains to be found.

Comprehensive study of dynamic curing effect on tablet coating structure.

The dissolution method is still widely used to determine curing end-points to ensure long-term stability of film coatings. Nevertheless, the process of curing has not yet been fully investigated. For the first time, joint techniques were used to elucidate the mechanisms of dynamic curing over time from ethylcellulose (Aquacoat)-based coated tablets. X-ray micro-computed tomography (XµCT), Near Infrared (NIR), and Raman spectroscopies as well as X-ray microdiffraction were employed as non-destructive techniques to perform direct measurements on tablets. All techniques indicated that after a dynamic curing period of 4h, reproducible drug release can be achieved and no changes in the microstructure of the coating were any longer detected. XµCT analysis highlighted the reduced internal porosity, while both NIR and Raman measurements showed that spectral information remained unaltered after further curing. X-ray microdiffraction revealed densification of the coating layer with a decrease in the overall coating thickness of about 10 µm as a result of curing. In addition, coating heterogeneity attributed to cetyl alcohol was observed from microscopic images and Raman analysis. This observation was confirmed by X-ray microdiffraction that showed that crystalline cetyl alcohol melted and spread over the coating surface with curing. Prior to curing, X-ray microdiffraction also revealed the existence of two coating zones differing in crystalline cetyl alcohol and sodium lauryl sulfate concentrations which could be explained by migration of these constituents within the coating layer. Therefore, the use of non-destructive techniques allowed new insights into tablet coating structures and provided precise determination of the curing end-point compared to traditional dissolution testing. This thorough study may open up new possibilities for process and formulation control.

Real-time predictions of drug release and end point detection of a coating operation by in-line near infrared measurements.

The aim of this work was to carry out real-time near infrared (NIR) predictions of drug release from sustained release coated tablets and to determine end point of coating operation. In-line measurements were ensured by implementation of a NIR probe inside a pan coater. Tablets were coated using a functional aqueous dispersion of ethylcellulose blended with PVA-PEG graft copolymer to obtain a controlled drug release dosage form over 16h. Samples were collected at regular intervals and subjected to a standardized curing step. Percentages of released drug at 4h, 8h and 12h were selected to describe the controlled drug release of cured tablets. These dissolution criteria were used as reference values to calibrate NIR spectral information and to develop three partial least squares regressions. Low predictive errors of 1.7%, 1.9% and 1.5%, respectively, were obtained. The coating operation was stopped while desired dissolution criteria were achieved, corresponding to a coating level around 10%. The present study demonstrated that real-time NIR measurements could be performed on non-finished drug products to predict dissolution properties of cured coated tablets. This novel and innovative approach fulfils the expectations of ICH Q8 guideline on pharmaceutical development, in terms of process understanding and process analytical technology (PAT) control strategy. This approach should be however adapted to curing operation to allow a real-time release testing.

Physicochemical characterization and toxicity evaluation of steroid-based surfactants designed for solubilization of poorly soluble drugs.

To overcome poor water-solubility of new drug candidates, four innovative surfactants based on naturally-occuring hydrophilic and hydrophobic moities were designed and synthesized: cholesteryl-glutamic acid, cholesteryl-poly[N-2-hydroxyethyl-l-glutamine] (PHEG), ursodeoxycholanyl-PHEG (UDCA-PHEG) and ursodeoxycholanyl-poly-l-glutamic acid (UDCA-PGA). Their self-assembling capacity was evaluated using pyrene fluorescence measurements which allow to determine their critical aggregation concentration (CAC). Size measurements were carried out using dynamic light scattering (DLS). Surfactant cytotoxicity was investigated on human umbilical vein endothelial cells (HUVEC) by determining tetrazolium salt (MTT) activity and lactate dehydrogenase (LDH) release. In addition, surfactant haemolytic activity was assessed using rat red blood cells (RBCs). Finally, the ability of these surfactants to solubilize a model poorly soluble drug was quantified. Surfactant self-assembly, cytotoxicity and solubilization properties were compared to those obtained with polysorbate 80, a model solubilizer. Except for cholesteryl-glutamic acid, surfactants were water-soluble. UDCA-PGA was not able to self-assemble or to increase significantly drug solubility. Results showed that cholesteryl-PHEG and UDCA-PHEG were self-assembling with low CAC values (17 and 120µg/ml) into nano-structures with mean diameters of 13 and 250nm, respectively. Cholesteryl-PHEG was the most efficient surfactant in increasing drug solubility (2mg/ml) but exhibited a similar or higher toxicity than polysorbate 80. UDCA-PHEG did not present any cytotoxicity but was far less efficient to solubilize the drug (0.2mg/ml). These results evidence interesting properties of cholesteryl-PHEG and UDCA-PHEG as novel solubilizers.

Development of a Process Analytical Technology (PAT) for in-line monitoring of film thickness and mass of coating materials during a pan coating operation.

The aim of this study was to perform in-line Near Infrared (NIR) measurements inside a pan coater to monitor a coating operation in real-time, by predicting the increases in mass of coating materials and coating thickness. A polymer combination of ethylcellulose/poly(vinyl-alcohol)-poly(ethylene-glycol) graft copolymer was used as functional aqueous coating. Coated tablets were sampled at regular intervals during the coating operation, then subjected to either simple and fast weighing (n=50) or accurate and non-destructive Terahertz Pulsed Imaging (TPI) measurements (n=3). Off-line NIR spectra analysis revealed that the coating operation could efficiently be controlled by focusing on two distinct NIR regions, related to absorption bands of ethylcellulose. Principal component analysis of in-line NIR spectra gave a clear classification of the collected coated tablets. Real-time quantitative monitoring of the coating operation was successfully performed from partial least square calibration models built using either TPI or weighing as reference method. Coating thicknesses as well as mass of coating materials used as primary values provided accurate NIR predictions. A comparison study demonstrated that both reference methods led to reliable and accurate real-time monitoring of the coating operation. This work demonstrated that in-line NIR measurements associated with multivariate analyses can be implemented to monitor in real-time a pan coating operation in order to fulfil the expectations of ICH Q8 guideline on pharmaceutical development, especially in terms of PAT control strategy and reduced end-product testing.