European Journal of Pharmaceutics and Biopharmaceutics (v.63, #1)

APV Diary (S1).

On the role of methacrylic acid copolymers in the intracellular delivery of antisense oligonucleotides by Marie-Andrée Yessine; Christian Meier; Hans-Ulrich Petereit; Jean-Christophe Leroux (1-10).
The delivery of active biomacromolecules to the cytoplasm is a major challenge as it is generally hindered by the endosomal/lysosomal barrier. Synthetic titratable polyanions can overcome this barrier by destabilizing membrane bilayers at pH values typically found in endosomes. This study investigates how anionic polyelectrolytes can enhance the cytoplasmic delivery of an antisense oligonucleotide (ODN). Novel methacrylic acid (MAA) copolymers were examined for their pH-sensitive properties and ability to destabilize cell membranes in a pH-dependent manner. Ternary complex formulations prepared with the ODN, a cationic lipid and a MAA copolymer were systematically characterized with respect to their size, zeta potential, antisense activity, cytotoxicity and cellular uptake using the A549 human lung carcinoma cell line. The MAA copolymer substantially increased the activity of the antisense ODN in inhibiting the expression of protein kinase C-alpha. Uptake, cytotoxicity and antisense activity were strongly dependent on copolymer concentration. Metabolic inhibitors demonstrated that endocytosis was the major internalization pathway of the complexes, and that endosomal acidification was essential for ODN activity. Confocal microscopy analysis of cells incubated with fluorescently-labeled complexes revealed selective delivery of the ODN, but not of the copolymer, to the cytoplasm/nucleus. This study provides new insight into the mechanisms of intracellular delivery of macromolecular drugs, using synthetic anionic polyelectrolytes.
Keywords: Methacrylic acid copolymers; Oligonucleotides; Intracellular delivery; Endosomal release; Drug delivery;

We developed novel acrylic-based polymers that can be used as mucoadhesive delivery systems. Poly(acrylic acid) hydrogels were modified by grafting adhesion promoter chains such as poly(ethylene glycol) (PEG) onto their back-bone chains, thus promoting the adhesive process by interpenetration. The copolymers synthesized were designated as P(AA-g-EG). Hydrogels were synthesized using PEG of two different molecular weights, 1000 and 2000, and with varying molar feed ratio of AA–EG (20:80, 40:60, 60:40, 80:20, 12:88, 25:75, 44:56, 67:33). The copolymers were synthesized by using free radical solution UV-polymerization. The effects of different PEG-tethered structures on mucoadhesion were studied using a tensiometric testing and the work of adhesion was calculated. Preswollen P(AA-g-EG) copolymer films composed of 40% acrylic acid (AA) and 60% ethylene glycol (EG), containing PEG 1000 tethers, exhibited the highest value for the work of mucoadhesion, 130×10−3±27×10−3  mJ, that is five times higher than the formulation composed of pure PAA. Based on these results and associated molecular analysis, we conclude that the higher mucoadhesive properties of this specific copolymer were the result of the synergistic effects of both monomers. AA functional groups allowed the polymer to form multiple hydrogen bonds with the glycoproteins present in the mucus. PEG tethers possibly acted as mucoadhesive promoters, enhancing interpenetration of polymer chains into the mucus.
Keywords: Mucoadhesive hydrogels; Poly(acrylic acid); Poly(ethylene glycol)-tethered structures; pH-Sensitive copolymers; Drug delivery;

This paper describes and interprets the coherence and the tensile strength of bi-component compacted tablets, composing a mixture of a poorly compactable drug, paracetamol and a very cohesive and ductile carrier, microcrystalline cellulose (MCC), Avicel® PH 102, using the concepts of the stored elastic strain in conjunction with the particle size and the relative volume fraction of the powders. Cylindrical compacts of the bi-component tablets, at various compositions formed at a common ultimate stress of 99 MPa, were subsequently fractured using the indirect tensile test method (Brazilian test method) to obtain a measure of their tensile strength. Various inter-relations between the compaction and tensile rupture characteristics are described. A simple model, which may predict the required volume fraction of MCC to produce a cohesively viable tablet is suggested, and applied to the current system. The results show to some extent the consistency of the suggested model with the experimental results.
Keywords: Coherence; Tensile strength; Paracetamol; Microcrystalline cellulose; Avicel®; Brazilian test;

The formation of interpolyelectrolyte complexes (IPEC) between Eudragit® E PO (EE) and Eudragit® L 100-55 (EL) was investigated, using turbidimetry, apparent viscosity measurements, elementary analysis and MT-DSC. The structure of the synthesized IPEC was investigated using FT-IR spectroscopy. The binding ratio of a unit molecule of EL with EE was found to be approximately 1:1 at pH 5.5. Based on the results of elementary analysis and FT-IR, the binding ratio of each component in the solid complexes was very close to that observed in turbidity and apparent viscosity measurements and indicate that the synthesized products can be considered as IPEC. As a result of electrostatic interaction between the polymer chains, the glass transition temperature of the IPEC increased significantly. Due to the structure of the IPEC, two maxima were observed in the swelling behavior as a function of pH. The release of the model drug ibuprofen (IBF) was significantly retarded from tablets made up of the IPEC as compared with individual copolymers, its physical mixture and Eudragit® RL PO (RL), RS PO (RS).
Keywords: Interpolyelectrolyte complex; Eudragit® E PO; Eudragit® L 100-55; Eudragit® RL PO; Eudragit® RS PO; Glass transition temperature; pH-dependent swelling behavior; Ibuprofen; Controlled release;

Many new drugs exhibit poor wetting behaviour and low aqueous solubility. This is particularly an issue for preclinical studies like toxicological trials, in which considerably higher doses and volumes are being administered compared to clinical studies. Preclinical vehicles typically contain high levels of surfactants that can exert biological effects. However, the biological inertness of vehicles is pivotal for the application in preclinical studies stressing the need in finding new excipients to solve formulation problems of today's drug discovery. The present study investigated the technical feasibility of surfactant-free suspensions using a new poorly soluble drug as model. It was shown that octenyl succinate-modified starches adequately wetted the drug and homogenous tasteless suspensions were obtained. The polymer xanthan gum was identified as macroscopically compatible gelling agent. Concentration effects of xanthan, drug and different modified starches were studied in a D-optimal design with respect to rheological properties. The suspensions were also tested in an analytical centrifuge using NIR transmission profiles to obtain a measure of sedimentation stability under accelerated conditions. The modified starches exhibited only little influence on the viscosity as well as on the yield point in contrast to the rheological effects of xanthan gum. This gelling agent was the main stabilising excipient as the modified starches hindered to a lesser extent sedimentation. The most stable suspensions displayed convenient flow properties. The viscosity at 100 s−1 and 25 °C was in technically acceptable range of 120–140 mPa s in view of a application via gavage or a syringe in animal studies.The results demonstrated that surfactant-free drug suspensions with excellent technical performance can be obtained using octenyl succinate-modified starches. The vehicles were tasteless and based on the experience of modified starches in the food industry, the vehicles should exhibit good tolerability. The future use of such surfactant-free drug suspensions in toxicological, pharmacokinetic and pharmacodynamic studies will have to determine their advantage in terms of biological inertness.
Keywords: Octenyl succinate; Modified starch; Surfactant-free; Suspension stability; D-optimal design;

The suitability of transepidermal water loss (TEWL) measurements in vitro as a barrier integrity test for human heat separated epidermis (HSE) was investigated. A model system consisting of a Teflon membrane mounted in Franz diffusion cells (FDC) filled with phosphate buffer saline (PBS) was set up. The membrane was used intact and punctured with a needle (up to five holes). After each puncturing the TEWL was measured. Only the TEWL of intact and punctured membrane differed significantly regardless of the number of holes. From three donators intact human HSE and punctured HSE were compared and no significant difference of the TEWL was found. Permeation experiments with flufenamic acid (FFA) showed a significantly higher diffusion rate through punctured HSE. TEWL and drug permeation were compared for skin stripped three, seven and 15 times prior to heat separation to an intact control group. Only the TEWL values of intact HSE and HSE stripped 15 times differed significantly. However, seven and 15 times stripping resulted in significantly higher diffusion rate. In conclusion, TEWL measurements can detect severe damage of the stratum corneum (SC) but not small changes, which nevertheless may already influence drug diffusion. Therefore, TEWL measurements appears to be of limited use as a barrier integrity test for human HSE in in vitro test systems.
Keywords: Tewameter; TEWL; Barrier integrity test; Human skin; Heat separated epidermis; Permeation data;

The present study relates to compositions of solid lipidic microparticles (SLmP), composed of biocompatible phospholipids and cholesterol, and their use as carriers or as fillers delivering drugs directly to the lungs via a dry powder inhaler (DPI). SLmP were obtained by spray-drying and were formulated as lipidic matrices entrapping budesonide or as physical blends (drug carrier). They were developed in order to improve the delivery of the active drug by the pulmonary route. The SLmP were evaluated for their physical characteristics and in vitro deposition measurements were performed using the Multi-stage Liquid Impinger (MsLI). The Pulmicort Turbuhaler® DPI (AstraZeneca) was used as a comparator product.The SLmP appeared to be spherical low-density material characterized by a smooth surface. The mass median diameters (D(0,5)), and the volume mean diameters (D[4,3]) were tiny and ranged from 1,7 to 3,1 μm and from 2,0 to 3,9 μm, respectively. The SLmP formulations, delivered by the Cyclohaler® inhaler, were found to emit a fine particle dose (FPD) of 93–113 μg, which is very promising comparing to the FPD (68 μg) delivered by the Pulmicort Turbuhaler®.
Keywords: Inhalation; Lipid particles; Excipients; Dry powder inhaler; Drug deposition;

Microcrystalline cellulose (MCC) is commonly used as an excipient in extrusion/spheronisation process. However, MCC owns several disadvantages as lack of disintegration and drug adsorption. Therefore, κ-carrageenan was investigated to substitute MCC in pelletising processes. Formulations with 20% of pelletisation aid (κ-carrageenan or MCC) and acetaminophen as a model drug have been produced. Different fillers (lactose, mannitol, maize starch and dicalciumphosphate dihydrate) at fractions of 0, 20, 40 and 80% were evaluated and the properties of the resulted pellets were determined (e.g. yield, aspect ratio, mean Feret diameter, 10% interval fraction, tensile strength and release profile). κ-Carrageenan has proven to be a suitable substitute as pellets with sufficient quality were produced. The pellet batches of different formulations were characterised by high yield, spherical pellet shape and narrow pellet size distribution. The distinguished behaviour between κ-carrageenan and MCC pellets was the lower tensile strength and the faster release of κ-carrageenan pellets. For the various types and fractions of fillers only minor effects to the pelletisation process and pellet properties were noticed. From the practical view these effects are neglectable indicating a robust formulation and process.
Keywords: Pellets; Extrusion/spheronisation; Pelletisation aid; Carrageenan; Microcrystalline cellulose; Dissolution; Tensile strength;

The extrusion/spheronisation process is an established technique to produce pellets for pharmaceutical applications. Microcrystalline cellulose (MCC) is being usually used as a pelletisation excipient in the extrusion process. However, MCC has some disadvantages, e.g. lack of disintegration and prolonged drug dissolution. Therefore, κ-carrageenan was investigated as a substitute for MCC to overcome such disadvantages. A fixed ratio of κ-carrageenan (20%) was combined with different fillers (lactose, mannitol, maize starch and dicalciumphosphate dihydrate) and different drugs (acetaminophen, theophylline, mesalamine and hydrochlorothiazide) in several formulations. Some pellet properties (yield, aspect ratio, mean Feret diameter, 10% interval fraction, tensile strength and dissolution profile) were determined. Most formulations resulted in pellets of a sufficient quality with respect to size, size distribution and shape independent of the incorporated fillers and drugs. In contrast to MCC pellets, the release profile of κ-carrageenan pellets was much less affected by the solubility of the drug. Generally, κ-carrageenan pellets owned fast disintegration and fast drug release in contrast to MCC pellets.
Keywords: Pellets; Extrusion/spheronisation; Pelletisation aid; Carrageenan; Microcrystalline cellulose; Dissolution; Tensile strength;