European Journal of Pharmaceutics and Biopharmaceutics (v.74, #2)

APV Diary (S1).

Guidelines to cell engineering for monoclonal antibody production by A. Rita Costa; M. Elisa Rodrigues; Mariana Henriques; Joana Azeredo; Rosário Oliveira (127-138).
Monoclonal antibodies (mAbs) are currently used for many diagnostic and therapeutic applications. The high demand for these biopharmaceuticals has led to the development of large-scale manufacturing processes, with productivity improvements being mainly achieved by optimization of bioreactor systems. However, more recently, the early steps of production, previous to bioreactor culture, have been presented as alternative areas where productivity enhancements can be achieved. Thus, this review describes the progress made for the improvement of productivity in mammalian expression systems for the high production of mAbs. Advances in the development of mAb-producing cell lines are being made, particularly regarding expression vector design and methods used for transfection, with the intent to create a reproducible methodology. Selection of the most suitable clones is also a critical step that can be improved, by including variables other than the expression level, which is still the common practice. Furthermore, strategies of cell engineering, although still mostly based on trial-and-error experimentation and not in standard protocols, hold great interest to improve cell growth and productivity, as well as product quality in the future. Improvements of the initial steps of the production process would not only result in cells with higher expression ability, but would also speed-up the process development.
Keywords: Monoclonal antibody; Mammalian cells; Cell engineering; Transfection methods; Vector design; Clone selection; Cell adaptation; Productivity enhancement;

Relevant shaking stress conditions for antibody preformulation development by Annette Eppler; Markus Weigandt; Andrea Hanefeld; Heike Bunjes (139-147).
In protein formulation development, shaking stress is often employed to assess the physical stability of antibody formulations against aggregation. Since there are currently no guidelines describing suitable test conditions, very different shaking stress designs are used. These different designs may influence the resulting stability data. The aim of this study was to establish a shaking stress design within the protein range of 2–5 mg/ml which can rapidly distinguish between antibody formulations of poor stability and those with potential for further development. Small scale shaking stress experiments were performed with different monoclonal IgG antibodies (as buffered solutions or marketed formulations). Variables were the filling degree of the sample containers, the container type and size and the shaking intensity. The stability of the samples was assessed by visual inspection, UV–VIS spectrophotometric turbidity measurements and size exclusion chromatography. All tested parameters had a strong influence on the stability results. The most discriminating conditions were obtained when shaking of the formulations was performed at 200 rpm in a 2 ml injection vial filled with 1 ml protein solution. This experimental setup led to clearly different stability results for buffered solutions and marketed products. Moreover, this setup required only relatively small amounts of protein solution which is advantageous in prefomulation studies.
Keywords: Antibody formulation; Shaking stress; Stress conditions; Physical stability; Mechanical stress; Protein aggregation; Protein formulation; Size exclusion chromatography; Turbidity; Preformulation;

A quantitative evaluation of the molecular binding affinity between a monoclonal antibody conjugated to a nanoparticle and an antigen by surface plasmon resonance by Nir Debotton; Hagit Zer; Marcela Parnes; Oshrat Harush-Frenkel; Jean Kadouche; Simon Benita (148-156).
We have designed a site-specific drug colloidal carrier ultimately for improving pancreatic and lung cancer treatment. It is based on a nanoparticulate drug delivery system that targets tumors overexpressing H-ferritin. A monoclonal antibody, AMB8LK, specifically recognizing H-ferritin was thiolated and conjugated to maleimide-activated polylactide nanoparticles (NPs) resulting in the formation of immunonanoparticles (immunoNPs). The AMB8LK immunoNPs exhibited a mean diameter size of 112 ± 20 nm and a density of 76 antibody molecules per NP. AMB8LK immunoNPs were evaluated for uptake and binding properties on CAPAN-1 and A-549 cell lines, using confocal microscopy. ImmunoNPs demonstrated specific binding and increased uptake of the desired cells by means of monoclonal antibodies (MAbs), compared to nonconjugated NPs. A lipophilic paclitaxel derivative, paclitaxel palmitate (pcpl), was encapsulated within the various NP formulations, and their cytotoxic effect was evaluated on A-549 cells using MTT assay. Pcpl-loaded AMB8LK immunoNPs showed a significantly increased cytotoxic effect when compared to pcpl solution and pcpl NPs. Surface plasmon resonance (SPR) was used to determine quantitatively the affinity constants of native AMB8LK and AMB8LK immunoNPs to gain insight on the affinity of the MAbs following the conjugation process onto NPs. The results of the association/dissociation and affinity kinetics of the interaction between H-ferritin and native AMB8LK or AMB8LK immunoNPs revealed similar constant values, showing that the conjugation process of the MAb to the NPs did not alter the intrinsic specificity and affinity of the MAb to the antigen. In conclusion, at the cellular level, AMB8LK immunoNPs may carry drugs to desired overexpressing antigen cells with adequate affinity properties, potentially leading to improved drug therapy and reduced systemic adverse effects.
Keywords: Affinity constant; Cancer; Ferritin; Immunonanoparticles; Monoclonal antibody; Paclitaxel palmitate; Surface plasmon resonance;

Drug delivery to the brain using surfactant-coated poly(lactide-co-glycolide) nanoparticles: Influence of the formulation parameters by Svetlana Gelperina; Olga Maksimenko; Alexander Khalansky; Lyudmila Vanchugova; Elena Shipulo; Kenul Abbasova; Rustam Berdiev; Stefanie Wohlfart; Nina Chepurnova; Jörg Kreuter (157-163).
Poly(lactide-co-glycolide) (PLGA) nanoparticles coated with poloxamer 188 (Pluronic® F-68) or polysorbate 80 (Tween® 80) enable an efficient brain delivery of the drugs after intravenous injection. This ability was evidenced by two different pharmacological test systems employing as model drugs the anti-tumour antibiotic doxorubicin and the agonist of opioid receptors loperamide, which being P-gp substrates can cross the blood–brain barrier (BBB) only in pharmacologically insignificant amounts: binding of doxorubicin to the surfactant-coated PLGA nanoparticles, however, enabled a high anti-tumour effect against an intracranial 101/8 glioblastoma in rats, and the penetration of nanoparticle-bound loperamide into the brain was demonstrated by the induction of central analgesic effects in mice. Both pharmacological tests could demonstrate that therapeutic amounts of the drugs were delivered to the sites of action in the brain and showed the high efficiency of the surfactant-coated PLGA nanoparticles for brain delivery. The results of the study also demonstrated that the efficacy of brain delivery by nanoparticles not only is influenced by the coating surfactants but also by other formulation parameters such as core polymer, drug, and stabilizer.
Keywords: Blood–brain barrier; Doxorubicin; Glioblastoma; Loperamide; Mice; Nanoparticles; Poly(lactide-co-glycolide); Poloxamer 188; Polysorbate 80; Rats; Tail-flick test;

Preparation and evaluation of tacrolimus-loaded nanoparticles for lymphatic delivery by Sae-Byeok Shin; Hea-Young Cho; Dae-Duk Kim; Han-Gon Choi; Yong-Bok Lee (164-171).
In an effort to improve lymphatic targeting efficiency and reduce the toxicity of tacrolimus, the emulsification–diffusion method was used to load the drug into nanoparticles (NP). Poly(lactide-co-glycolide) (PLGA) and PLGA surface-modified with poly(ethylene glycol) (PEG) were used as polymers. Mean particle size and drug encapsulation efficiency of PLGA were 218 ± 51 nm and 60.0 ± 1.2% and for PEG–PLGA NP were 220 ± 33 nm and 60.3 ± 2.0%. NP were characterized by thermal analyzer and X-ray diffractometry (XRD), and their shapes were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In vitro release profiles were affected by the pH of dissolution media. The prepared NP and commercial product of tacrolimus (Prograf® inj.) were intravenously administered to rats to compare their pharmacokinetic characteristics and lymphatic targeting efficiency. The area under the whole blood concentration–time curve (AUC), mean residence time (MRT), and total clearance (CL t ) of PEG–PLGA NP were significantly different (P  < 0.05) compared with those of Prograf® inj., and lymphatic targeting efficiencies of both NP formulations at the mesenteric lymph node significantly increased (P  < 0.05). These results showed that the prepared tacrolimus-loaded NP are good possible candidates as a lymphatic delivery system of tacrolimus.
Keywords: Tacrolimus; Nanoparticles; Pharmacokinetics; Lymphatic delivery; Emulsification–diffusion method;

The potential of osmotic pressure driven release of proteins from poly(trimethylene carbonate) and poly(trimethylene carbonate-co-d,l-lactide) (poly(TMC-co-DLLA)) elastomers with varying amounts of DLLA was investigated using bovine serum albumin (BSA) as a model protein. The BSA was co-lyophilized with either trehalose or trehalose combined with NaCl as osmotigens to produce particles with sufficient osmotic activity. Elastomers composed solely of TMC were not suitable for osmotically driven release when trehalose was the main osmotigen in the solid particles. Copolymerizing TMC with small amounts of DLLA decreased the tear resistance of the elastomer and consequently increased the rate and the total amount of BSA released. Elongation at break played a significant role in determining the osmotic release behavior; elastomers with comparable Young’s modulus and tensile strength, but smaller elongation at break, provided faster release rates. Elastomer degradation played a minor role in the osmotic release, as the mechanical properties underwent very little change during the investigated period of release. The poly(TMC-co-DLLA)(80:20) elastomer was able to provide near zero order release of BSA for up to 12 days, and the total amount of BSA released was 74 ± 4% after 34 days, when small amounts of NaCl was added to trehalose. No significant reduction in the microenvironmental pH occurred after 17 days of release. TMC elastomers copolymerized with small amounts of DLLA are potential candidates for the localized delivery of acid-sensitive proteins.
Keywords: Biodegradable elastomer; Protein delivery; Osmotic release; Mechanical properties;

Size-dependent release of fluorescent macromolecules and nanoparticles from radically cross-linked hydrogels by Matthias Henke; Ferdinand Brandl; Achim M. Goepferich; Joerg K. Tessmar (184-192).
Hydrogels play an important role in drug delivery and tissue engineering applications due to their excellent biocompatibility and their variable mechanical and physical properties, which allow their optimization for many different aspects of the intended use. In this study, we examined the suitability of poly(ethylene glycol) (PEG)-based hydrogels as release systems for nanometer-sized drugs or drug carriers, like nanoparticles, using the radically cross-linkable oligo(poly(ethylene glycol)fumarate) (OPF) together with two cross-linking agents. Different fluorescent nanoparticulate probes with respect to size and physical structure were incorporated in the cross-linked hydrogels, and the obtained release profiles were correlated with the physical properties and the chemical structure of the gels, indicating a strong dependence of the release on the chosen PEG prepolymers. The prepared hydrogels were characterized by oscillatory rheometry and swelling experiments. Release experiments as well as diffusion measurements using fluorescence recovery after photobleaching showed the great potential of this type of hydrogels for the preparation of adjustable release systems by altering the molecular weights of the used PEG molecules.
Keywords: Thermally cross-linkable; Hydrogel; Nanoparticle; Macromolecular drugs; Characterization; Release; FRAP;

Cyclodextrin-based nanosponges encapsulating camptothecin: Physicochemical characterization, stability and cytotoxicity by Shankar Swaminathan; Linda Pastero; Loredana Serpe; Francesco Trotta; Pradeep Vavia; Dino Aquilano; Michele Trotta; GianPaolo Zara; Roberta Cavalli (193-201).
Camptothecin (CAM), a plant alkaloid and a potent antitumor agent, has a limited therapeutic utility because of its poor aqueous solubility, lactone ring instability and serious side effects. Cyclodextrin-based nanosponges (NS) are a novel class of cross-linked derivatives of cyclodextrins. They have been used to increase the solubility of poorly soluble actives, to protect the labile groups and control the release. This study aimed at formulating complexes of CAM with three types of β-cyclodextrin NS obtained with different cross-linking ratio (viz. 1:2, 1:4 and 1:8 on molar basis with the cross-linker) to protect the lactone ring from hydrolysis and to prolong the release kinetics of CAM. Crystalline (F1:2, F1:4 and F1:8) and paracrystalline NS formulations were prepared. XRPD, DSC and FTIR studies confirmed the interactions of CAM with NS. XRPD showed that the crystallinity of CAM decreased after loading. CAM was loaded as much as 21%, 37% and 13% w/w in F1:2, F1:4 and F1:8, respectively while the paracrystalline NS formulations gave a loading of about 10% w/w or lower. The particle sizes of the loaded NS formulations were between 450 and 600 nm with low polydispersity indices. The zeta potentials were sufficiently high (−20 to −25 mV) to obtain a stable colloidal nanosuspension. The in vitro studies indicated a slow and prolonged CAM release over a period of 24 h. The NS formulations protected the lactone ring of CAM after their incubation in physiological conditions at 37 °C for 24 h with a 80% w/w of intact lactone ring when compared to only around 20% w/w of plain CAM. The cytotoxicity studies on HT-29 cells showed that the CAM formulations were more cytotoxic than plain CAM after 24 h of incubation.
Keywords: Nanosponges; Camptothecin; β-Cyclodextrin; Crystallinity; Stability; Complexation; Cytotoxicity;

Influence of reaction medium during synthesis of Gantrez® AN 119 nanoparticles for oral vaccination by Katrien Vandamme; Vesna Melkebeek; Eric Cox; Dieter Deforce; Joke Lenoir; Els Adriaens; Chris Vervaet; Jean Paul Remon (202-208).
Two synthesis methods of poly(methyl vinyl ether-co-maleic anhydride) (Gantrez® AN 119) nanoparticles (NP) (used for oral vaccination) were compared. Wheat germ agglutinin (WGA) was used as ligand to enhance the bioadhesive properties of NP and β-galactosidase as antigen. The first method encapsulated β-galactosidase in NP by co-precipitation in an acetone/water mixture containing 44% acetone. In the second method, antigen addition occurred in 100% acetone. To improve stability, NP were crosslinked with 1,3-diaminopropane. The stability of WGA-conjugated NP with encapsulated antigen diminished at lower pH and when decreasing the amount of crosslinker. The binding type between WGA and polymer depended on the synthesis method: predominantly ionic bonds were formed using the 44% acetone method, whereas synthesis via the 100% acetone method resulted in covalent bonds. The biological activity of the WGA coating, evaluated via a pig gastric mucin binding test, was lower in NP prepared via the 100% acetone method. No release of native antigen was detected after hydrolysis of NP, due to the covalent antigen binding during antigen encapsulation and the high reactivity of the polymer. Moreover, the mucosal irritation capacity was evaluated upon nanoparticle hydrolysis using a slug mucosal irritation assay. Herein, hydrolysed NP of the 44% acetone method were classified as mild irritative.
Keywords: Poly(methyl vinyl ether-co-maleic anhydride); Gantrez® AN 119; Nanoparticles; Oral antigen delivery; Adjuvant; WGA; Slug mucosal irritation assay;

Surface-functionalized polymethacrylic acid based hydrogel microparticles for oral drug delivery by S. Sajeesh; K. Bouchemal; C.P. Sharma; C. Vauthier (209-218).
Aim of the present work was to develop novel thiol-functionalized hydrogel microparticles based on poly(methacrylic acid)–chitosan–poly(ethylene glycol) (PCP) for oral drug delivery applications.PCP microparticles were prepared by a modified ionic gelation process in aqueous medium. Thiol modification of surface carboxylic acid groups of PCP micro particles was carried out by coupling l-cysteine with a water-soluble carbodiimide. Ellman’s method was adopted to quantify the sulfhydryl groups, and dynamic light-scattering technique was used to measure the average particle size. Cytotoxicity of the modified particles was evaluated on Caco 2 cells by MTT assay. Effect of thiol modification on permeability of paracellular marker fluorescence dextran (FD4) was evaluated on Caco 2 cell monolayers and freshly excised rat intestinal tissue with an Ussing chamber set-up. Mucoadhesion experiments were carried out by an ex vivo bioadhesion method with excised rat intestinal tissue.The average size of the PCP microparticles was increased after thiol modification. Thiolated microparticles significantly improved the paracellular permeability of FD4 across Caco 2 cell monolayers, with no sign of toxicity. However, the efficacy of thiolated system remained low when permeation experiments were carried out across excised intestinal membrane. This was attributed to the high adhesion of the thiolated particles on the gut mucosa. Nevertheless, it can be concluded that surface thiolation is an interesting strategy to improve paracellular permeability of hydrophilic macromolecules.
Keywords: Thiolation; Mucoadhesion; Hydrogels; Microparticles; Intestinal absorption;

Microemulsion and mixed micelle for oral administration as new drug formulations for highly hydrophilic drugs by Yahya Mrestani; Laila Behbood; Albert Härtl; Reinhard H.H. Neubert (219-222).
Microemulsions (MEs) and mixed micelles (MMs) have been used as new drug formulations for high hydrophilic drugs such as cefpirom and cefodizim for oral administration. Cefpirom and cefodizim are neither actively nor passively transported across cell membranes. Up to date, they can be only administrated intravenously (i.v.) or intramuscularly (i.m.). The rabbit (Chinchilla) in vivo model was used in the present work to investigate ways of overcoming the poor oral absorption of these cephalosporins. The cephalosporins at 100 mg/kg were formulated in MEs and MMs and administered intraduodenally (i.d.). Very low bioavailability (2.5–3.0%) was observed, if cefpirom or cefodizim i.d. were applied without colloidal vehicle. However, the addition of the cephalosporins to ME or MM is shown to be highly effective in increasing the bioavailability values (up to 64% absolute bioavailability) of the model drugs. In conclusion, MEs and MMs improve essentially the oral bioavailability of the high hydrophilic drugs.
Keywords: Cefpirom; Cefodizim; Pharmacokinetics;

Our goal was to develop safe and stable multilayer emulsions capable of enhancing intestinal absorption of biopharmaceutics classification system (BCS) class III drugs. First, w/o emulsions were prepared using calcein as a model BCS class III compound and condensed ricinoleic acid tetraglycerin ester as a hydrophobic emulsifier. Then water-in-oil-in-water (w/o/w) emulsions were prepared with shirasu porous glass (SPG) membranes. Particle size analyses and calcein leakage from oil droplets in w/o/w emulsions led us to select stearic acid hexaglycerin esters (HS-11) and Gelucire 44/14 as hydrophilic emulsifiers. Analyses of the absorption-enhancing effects of w/o/w emulsions on intestinal calcein absorption in rats showed that calcein bioavailability after intraduodenal (i.d.) administration of HS-11 or Gelucire 44/14 + polyvinyl alcohol (PVA) w/o/w emulsions prepared with 0.1-μm pore-sized SPGs was significantly higher than that of the calcein control. However, serum calcein concentration vs. time profiles after i.d. administration of w/o/w emulsions prepared with 1.1-μm and 30-μm pore-sized SPGs and an emulsion prepared with a calcein-containing outer water phase were comparable to control profiles. These results suggested that HS-11 or Gelucire 44/14 + PVA are safe outer water phase additives and that 0.1-μm pore-sized SPGs are important for preparing w/o/w emulsions that enhanced intestinal calcein absorption.
Keywords: w/o/w emulsion; Nanoparticle; BCS class III; Emulsifier; Intestinal absorption;

Inulin solid dispersion technology to improve the absorption of the BCS Class IV drug TMC240 by Marinella R. Visser; Lieven Baert; Gerben van ’t Klooster; Laurent Schueller; Marian Geldof; Iris Vanwelkenhuysen; Herman de Kock; Sandra De Meyer; Henderik W. Frijlink; Jan Rosier; Wouter L.J. Hinrichs (233-238).
TMC240 is a very poorly soluble and poorly permeating HIV protease inhibitor. In order to enhance its oral bioavailability, a fast dissolving inulin-based solid dispersion tablet was developed. During the dissolution test in water (0.5% or 1.0% SLS), this tablet released at least 80% of TMC240 within 30 min, while a tablet with the same composition, but manufactured as physical mixture, released only 6% after 2 h. In a subsequent single-dose study in dogs (200 mg of TMC240), plasma concentrations of TMC240 remained below the lower limit of quantification (<1.00 ng/mL) in all animals (n  = 3 per tested formulation), except in one dog receiving the inulin solid dispersion tablet (C max  = 1.8 ng/mL, AUC0-7 h  = 3.0 ng h/mL). In the latter treatment group, ritonavir co-administration (10 mg/kg b.i.d.) increased TMC240 exposure more than 30-fold (mean AUC0-7 h  = 108 ng h/mL; F rel  = 3588%). Exposure was also 16-fold higher than after TMC240 administration as PEG400 suspension in the presence of ritonavir (AUC0-7 h  = 6.7 ng h/mL). The current data demonstrate that a solid dispersion of TMC240 in an inulin matrix allows considerable improvement in the release of poorly water-soluble TMC240, both in vitro in the presence of a surfactant and in vivo upon oral administration.
Keywords: TMC240; Inulin solid dispersion; Pharmacokinetics; Absorption; BCS Class IV; Ritonavir;

Characterization of the copolymer poly(ethyleneglycol-g-vinylalcohol) as a potential carrier in the formulation of solid dispersions by Sandra Guns; Pieterjan Kayaert; Johan A. Martens; Jan Van Humbeeck; Vincent Mathot; Thijs Pijpers; Evgeny Zhuravlev; Christoph Schick; Guy Van den Mooter (239-247).
In order to fully exploit the graft copolymer poly(ethyleneglycol-g-vinylalcohol) (EG/VA) in the formulation of solid dispersions, a characterization of its phase behavior before, during and after spray-drying and hot-melt extrusion is performed. Solid state characterization was performed using MDSC and XRPD. The effect of heating/cooling rate on the degree of crystallinity was studied using HPer DSC and ultra-fast chip calorimetry. EG/VA consists of two semi-crystalline fractions, one corresponding to the polyethyleneglycol (PEG) fraction (T g  = −57 °C, T m  = 15 °C) and one corresponding to the polyvinylalcohol (PVA) fraction (T g  = 45 °C, T m  = 212 °C). XRPD analysis confirmed its semi-crystallinity, and EG/VA showed Bragg reflections comparable to those of PVA. Spray-drying at a temperature lower than 170 °C resulted in amorphization of the PVA fraction, while after hot-melt extrusion at different temperatures, the crystallinity of this fraction increases. In both cases, the PEG fraction is not influenced. Plasticization of the amorphous domains of the PEG or PVA fraction of the copolymer was dependent on the type and concentration of plasticizer, suggesting that also other small organic molecules like drugs may not homogeneously mix with both amorphous domains. A controlled cooling rate of 3000 °C/s was necessary to make the copolymer completely amorphous.
Keywords: Poly(ethyleneglycol-g-vinylalcohol) (EG/VA, Kollicoat® IR); Differential Scanning Calorimetry (DSC); High Performance Differential Scanning Calorimetry (HPer DSC); X-ray powder diffraction (XRPD); Chip calorimetry; Solid dispersion;

Thermally sensitive gels based on chitosan derivatives for the treatment of oral mucositis by S. Rossi; M. Marciello; M.C. Bonferoni; F. Ferrari; G. Sandri; C. Dacarro; P. Grisoli; C. Caramella (248-254).
The aim of the present work was the development of a thermally sensitive mucoadhesive gel based on chitosan derivatives for the treatment of oral mucositis. Trimethyl chitosan (TMC) and methylpyrrolidinone chitosan (MPC) were considered. They were mixed with glycerophosphate (GP) according to different polymer/GP molar ratios and characterized for gelation properties by means of rheological analysis in comparison with chitosan. The influence of molecular weight and substitution degree (SD) of TMC on gelation temperature and time was investigated. The mucoadhesive properties of the mixtures were also assessed using porcine buccal mucosa. The best properties were shown by TMC with high MW and low SD mixed with GP according to 1:2 molar ratio. Such mixture was loaded with benzydamine hydrochloride, an anti-inflammatory drug with antimicrobial properties and subjected to in vitro drug release and wash away test. The formulation based on TMC/GP mixture was able to prolong drug release and to withstand the removal physiological mechanisms. The antimicrobial properties of both vehicle and formulation were investigated. Also in absence of drug, TMC/GP mixture was characterized by antimicrobial properties.
Keywords: Thermally sensitive mucoadhesive gels; Trimethyl chitosan; Methylpyrrolidinone chitosan; Benzydamine hydrochloride; Oral mucositis;

Characterization of a polyurethane-based controlled release system for local delivery of chlorhexidine diacetate by Truc Thanh Ngoc Huynh; Karine Padois; Fabio Sonvico; Alessandra Rossi; Franca Zani; Fabrice Pirot; Jacques Doury; Françoise Falson (255-264).
Conventional formulations of chlorhexidine usually provide short-term efficiency, requiring repeated applications to maintain antibacterial activity. Therefore, appropriate release system of chlorhexidine controlling local drug delivery would reduce the number of applications and enhance patient compliance.The aim of this study was to develop a controlled release system based on medical polyurethane for the local delivery of chlorhexidine diacetate (CDA). CDA-loaded polyurethane films (CDA-Films) and CDA-loaded polyurethane sandwiches (CDA-Sandwiches) were obtained by casting and solvent evaporation.The physico-chemical aspects of CDA-loaded polyurethane systems were investigated, and the crystalline state of CDA in the polymeric system was highlighted. CDA-Films exhibited appropriate mechanical properties for further applications. Drug release was measured in two different media: (i) distilled water and (ii) physiological saline solution to mimic in vivo conditions. Drug release studies were performed up to 11 days on CDA-Films and 29 days for CDA-Sandwiches. Release of CDA depended on drug loading and the structure of the system. In particular, release of CDA from the sandwich system followed zero-order kinetic. The release rate was significantly lower in physiological solution. Antibacterial studies were carried out on CDA-Films against Staphylococcus aureus and Staphylococcus epidermidis showing 35 days persisting antibacterial activity.In conclusion, the polyurethane-based system developed in this study is potentially useful as a local delivery system for CDA and could be used not only in surgery but also in dental and clinical applications.
Keywords: Controlled release system; Local delivery; Chlorhexidine diacetate; Polyurethane; Mechanical properties; Antibacterial activity;

Pharmacodynamics of cisplatin-loaded PLGA nanoparticles administered to tumor-bearing mice by Daniel Moreno; Sara Zalba; Iñigo Navarro; Conchita Tros de Ilarduya; María J. Garrido (265-274).
Biodegradable poly (lactic-co-glycolic) acid (PLGA) nanoparticles incorporating cisplatin have been developed to evaluate its in vivo efficacy in tumor-bearing mice. In vitro study proved two mechanisms of action for cisplatin depending on the dose and the rate at which this dose is delivered. In vivo study, 5 mg/kg of cisplatin nanoparticles administered to mice, exhibited a tumor inhibition similar to free cisplatin, although the area under cisplatin concentration–time curve between 0 and 21 days (AUC0–21) had lower value for the formulation than for drug solution (P  < 0.05). This result was associated with a higher activation of apoptosis in tumor, mediated by caspase-3, after nanoparticles administration. Toxicity measured as the change in body weight, and blood urea nitrogen (BUN) plasma levels showed that cisplatin nanoparticles treatment did not induce significant changes in both parameters compared to control, while for free drug, a statistical (P  < 0.01) increase was observed. In addition, a good correlation was found between time profiles of tumor volume and vascular endothelial growth factor (VEGF) plasma levels, suggesting that its expression could help to follow the efficacy of the treatment. Therefore, the PLGA nanoparticles seem to provide a promising carrier for cisplatin administration avoiding its side effects without a reduction of the efficacy, which was consistent with a higher activation of apoptosis than free drug.
Keywords: Cisplatin; PLGA nanoparticles; Antiproliferative effect in vitro; Apoptosis; Antitumor efficacy in vivo;

Pharmacokinetics evaluation of soft agglomerates for prompt delivery of enteric pantoprazole-loaded microparticles by Renata P. Raffin; Letícia M. Colomé; Cristiane R.D. Hoffmeister; Paolo Colombo; Alessandra Rossi; Fabio Sonvico; Lucas M. Colomé; Claudio C. Natalini; Adriana R. Pohlmann; Teresa Dalla Costa; Silvia S. Guterres (275-280).
Soft agglomerates containing pantoprazole-loaded microparticles were developed with the aim of prompt delivery of gastro-resistant particles. The objective was to evaluate the relative bioavailability in dogs after the oral administration of soft agglomerates. Gastro-resistant pantoprazole-loaded microparticles prepared by spray drying were mixed with mannitol/lecithin spray-dried powder and agglomerated by vibration. One single oral dose (40 mg) was administered to dogs. Each dog received either a reference tablet or hard gelatin capsules containing the agglomerates. The plasma profiles were evaluated by non-compartmental and compartmental approaches, and the pharmacokinetic parameters were determined. The agglomerates presented 100% of drug particle loading and a production yield of 80.5%. The amount of drug absorbed after oral dosing was similar after reference or agglomerate administration, leading to a relative bioavailability of 108%. The absorption lag-time was significantly reduced after agglomerate administration (from 135.5 ± 50.6 to 15.0 ± 2.5 min). The agglomerated gastro-resistant pantoprazole-loaded microparticles reduced time to peak plasma. The agglomerates were equivalent to the reference tablets in terms of extent but not in terms of rate of absorption, showing that this formulation is an alternative to single-unit oral dosing with enteric coating and with the advantage of reducing time to effect.
Keywords: Pantoprazole; Microparticles; Agglomerates; Pharmacokinetics; Fast release;

Transport of phenylethylamine at intestinal epithelial (Caco-2) cells: Mechanism and substrate specificity by Wiebke Fischer; Reinhard H.H. Neubert; Matthias Brandsch (281-289).
This study was performed to characterize the intestinal transport of β-phenylethylamine (PEA). Uptake of [14C]PEA into Caco-2 cells was Na+-independent but strongly stimulated by an outside directed H+ gradient. At extracellular pH 7.5, the concentration-dependent uptake of PEA was saturable with kinetic parameters of 2.6 mM (K t) and 96.2 nmol/min per mg of protein (V max). Several biogenic amines such as harmaline and N-methylphenylethylamine as well as cationic drugs such as phenelzine, tranylcypromine, d,l -amphetamine, methadone, chlorphenamine, diphenhydramine and promethazine strongly inhibited the [14C]PEA uptake with K i values around 1 mM. Tetraethylammonium, N-methyl-4-phenylpyridinium and choline had no effect. We also studied the bidirectional transepithelial transport of [14C]PEA at cell monolayers cultured on permeable filters. Net transepithelial flux of [14C]PEA from apical-to-basolateral side exceeded basolateral-to-apical flux 5-fold. We conclude that PEA is transported into Caco-2 cells by a highly active, saturable, H+-dependent (antiport) process. The transport characteristics do not correspond to those of the known carriers for organic cations of the SLC22, SLC44, SLC47 and other families.
Keywords: Biogenic amines; Caco-2 cells; Drug delivery; Intestinal epithelium; Membrane transport; Phenylethylamine (PEA);

The purpose of this study was to investigate the human nasal epithelial cell line RPMI 2650 regarding its usefulness as in vitro model for drug permeation studies. Particularly, the influence of the air–liquid interface in culture and coculture with human nasal fibroblasts (HNF) on the differentiation and permeation barrier properties of the cell layer was examined. In addition to a non-contact coculture, we developed a three-dimensional construct of the human nasal mucosa composed of a collagen matrix with embedded HNF, covered by a RPMI 2650 epithelial cell layer.Microscopic examination as well as measurement of the transepithelial electrical resistance and permeation experiments showed the importance of cultivation at the air–liquid interface. Permeation studies were performed using a paracellular marker (sodium fluorescein), a transcellular marker (propranolol-HCl) and a model substance with high molecular weight (FITC-dextran, MW 4000). The epithelial model showed an organotypic permeation barrier for paracellular, transcellular and high MW permeation. Three-dimensional reconstructed human nasal mucosa showed four- to fivefold higher permeation coefficients. Regardless of the limits of these models, both offer promise to evaluate passive drug permeation through the nasal mucosa.
Keywords: RPMI 2650; Transepithelial electrical resistance; Air–liquid interface; Human nasal fibroblasts; Coculture; Nasal mucosa; Drug permeation; Paracellular permeation; Transcellular permeation;

We aimed to develop a phase selection methodology for a weak basic active pharmaceutical ingredient (API) that would require less than 10 mg of the API and monitor the real-time kinetic solubility of the API in two bio-relevant media. Three sets of kinetic solubility measurements were conducted for free form I and the disulfate salt of an API (compound A) in order to determine the better API phase for further development of the compound. Tests consisted of solid API dissolution in both simulated gastric fluid (SGF) and fasted-state intestinal fluid (FaSSIF), and precipitation kinetics by injection of liquid state API into FaSSIF. All dissolution tests were conducted above the saturated concentrations in order to determine the compounds’ thermodynamic and kinetic solubility to trace the API’s phase transitions during dissolution. The pharmacokinetic profiles of compound A following oral administration of two API phases were evaluated in dogs. Results of the three sets of kinetic solubility measurements showed different kinetic solubility profiles for the two API phases under gastrointestinal conditions, indicating that the disulfate salt is preferred over free form I due to its superior kinetic solubility profile. This conclusion is consistent with the bioavailability results obtained in dogs.
Keywords: Phase selection; Dissolution; Bio-relevant media; Pharmacokinetics; Kinetic solubility;

Comparing different salt forms of rotigotine to improve transdermal iontophoretic delivery by O.W. Ackaert; J. Eikelenboom; H.M. Wolff; J.A. Bouwstra (304-310).
The transdermal delivery of a new salt form of the dopamine agonist rotigotine, rotigotine·H3PO4 is presented and compared to rotigotine·HCl. A comparison was made on the level of solubility, passive and iontophoretic delivery. Different aspects of the delivery were investigated: delivery efficiency, maximum flux, donor pH, electro-osmotic contribution and transport number. Changing the salt form from rotigotine·HCl to rotigotine·H3PO4 increases significantly the solubility and rules out the influence of NaCl on the solubility by the absence of the common-ion effect. At low donor concentration, no difference in transdermal delivery was observed between the salt forms. Due to an increase in the maximum solubility of rotigotine·H3PO4, a 170% increase in maximum flux, compared to rotigotine·HCl, was achieved. A balance between solubility and delivery efficiency can be obtained by choosing the correct donor pH between 5 and 6. A slight increase in electro-osmotic contribution and transport number was observed. Using the parameters, determined by modeling the in vitro transport, in vivo simulations revealed that with iontophoresis therapeutic levels can be achieved with a rapid onset time and be maintained in a controlled manner by adjusting the current density.
Keywords: Solubility; Transdermal; Iontophoresis; Rotigotine; Salt form; Modeling;

Oral pH-responsive systems for drug delivery to the ileo-colonic region of the gastrointestinal tract show poor site specificity. Here, we describe a novel double-coating concept, based on the acrylic polymer EUDRAGIT® S, which provides improved functionality for targeting performance. The coating system comprises an inner layer of partially neutralised EUDRAGIT® S and buffer agent and an outer coat of standard EUDRAGIT® S. Tablets containing prednisolone were coated with double-layer formulations with different inner coat compositions. A conventional single coating was also applied for comparison purposes. Dissolution of the coated tablets was assessed using USP II apparatus in 0.1 M HCl for 2 h followed by pH 7.4 physiological bicarbonate buffer (Krebs buffer), a medium which closely resembles the ionic composition and buffer capacity of the fluid in the distal small intestine. Following acid exposure, drug release from the EUDRAGIT® S single-layer-coated tablets in pH 7.4 Krebs buffer was delayed for 120 min. Release from the double-coated tablets was significantly faster compared to the single-coated tablets and was found to be affected by the pH and buffer capacity of the inner coat. The drug release lag time from the optimised double-coating formulation with an inner coat consisting of 10% KH2PO4 (neutralisation pH of 8.0) was 40 min. The accelerated coat dissolution and subsequent rapid drug release from the double-coating system can potentially overcome the limitations of conventional EUDRAGIT® S coatings for ileo-colonic delivery.
Keywords: Colonic delivery; Ileal delivery; Bicarbonate buffers; Biorelevant dissolution; Enteric coatings; pH-responsive polymers; Enteric polymers; Polymethacrylates; Methacrylic acid and methyl methacrylate copolymer; Inflammatory bowel disease;

Artificial neural networks in the optimization of a nimodipine controlled release tablet formulation by Panagiotis Barmpalexis; Feras Imad Kanaze; Kyriakos Kachrimanis; Emanouil Georgarakis (316-323).
Artificial neural networks (ANNs) were employed in the optimization of a nimodipine zero-order release matrix tablet formulation, and their efficiency was compared to that of multiple linear regression (MLR) on an external validation set. The amounts of PEG-4000, PVP K30, HPMC K100 and HPMC E50LV were used as independent variables following a statistical experimental design, and three dissolution parameters (time at which the 90% of the drug was dissolved, t 90%, percentage of nimodipine released in 2 and 8 h, Y 2h, and Y 8h, respectively) were chosen as response variables. It was found that a feed-forward back-propagation ANN with eight hidden units showed better fit for all responses (R 2 of 0.96, 0.90 and 0.98 for t 90%, Y 2h and Y 8h, respectively) compared to the MLR models (0.92, 0.87 and 0.92 for t 90%, Y 2h and Y 8h, respectively). The ANN was further simplified by pruning, which preserved only PEG-4000 and HPMC K100 as inputs. Optimal formulations based on ANN and MLR predictions were identified by minimizing the standardized Euclidian distance between measured and theoretical (zero order) release parameters. The estimation of the similarity factor, f 2, confirmed ANNs increased prediction efficiency (81.98 and 79.46 for the original and pruned ANN, respectively, and 76.25 for the MLR).
Keywords: Artificial neural networks; Pruning; Controlled release; Euclidian distance; Nimodipine;

Role of surface free energy and spreading coefficient in the formulation of active agent-layered pellets by Gabriella Baki; János Bajdik; Dejan Djuric; Klaus Knop; Peter Kleinebudde; Klára Pintye-Hódi (324-331).
Formulation of layered pellets can be a useful method for the preparation of multiparticulate systems. The aims of this work were to study the properties of hydrophilic active agent (pirenzepine dihydrochloride) layers formed on different pellet cores, the efficacy of layering and the connection between the core and the layers. The carrier pellets were prepared from mixtures of a hydrophilic (microcrystalline cellulose) and a hydrophobic (magnesium stearate) component in different ratios. These cores were coated in a fluid bed apparatus with an aqueous solution of active agent, with or without the addition of hydroxypropyl methyl cellulose (HPMC) as an adhesive component. The wettability of the pharmaceutical powders was assessed by means of Enslin number and contact angle measurements, and the surface energy was determined. Spreading coefficients of the components were also calculated and correlated with pellet properties such as the content of active agent, the friability and the morphological appearance of the layered product. An increased friability of the layer formed and the lower effectiveness of the process were experienced with a reduction in the wetting of the core. The efficiency of layering on a less polar core could be increased by the addition of HPMC, but the sensitivity of these pirenzepine layers to mechanical stress was higher. The type of the abrasion of these particles was dissimilar to that for samples prepared without HPMC. Peeling of the layers containing HPMC was observed for hydrophobic cores, but this phenomenon was not detected for the hydrophilic ones. These results can be explained by the spreading coefficients, which revealed an insufficient adhesion of layers for the samples that exhibited peeling.
Keywords: Friability; HPMC; Layering; Peeling; Spreading coefficient; Wetting;

Ciprofloxacin hydrochloride has a short elimination half-life, a narrow absorption window and is mainly absorbed in proximal areas of GIT. The purpose of this study was to develop a gastroretentive controlled-release drug delivery system with swelling, floating, and adhesive properties. Ten tablet formulations were designed using hydroxypropylmethylcellulose (HPMC K15M) and/or sodium alginate (Na alginate) as release-retarding polymer(s) and sodium bicarbonate (NaHCO3) or calcium carbonate (CaCO3) as a gas former. Swelling ability, floating behaviour, adhesion period and drug release studies were conducted in 0.1 N HCl (pH 1.2) at 37 ± 0.5 °C. The tablets showed acceptable physicochemical properties. Drug release profiles of all formulae followed non-Fickian diffusion. Statistical analyses of data revealed that tablets containing HPMC K15M (21.42%, w/w), Na alginate (7.14%, w/w) and NaHCO3 (20%, w/w) (formula F7) or CaCO3 (20%, w/w) (formula F10) were promising systems exhibiting excellent floating properties, extended adhesion periods and sustained drug release characteristics. Both formulae were stored at 40 °C/75% RH for 3 months according to ICH guidelines. Formula F10 showed better physical stability. Abdominal X-ray imaging of formula F10, loaded with barium sulfate, in six healthy volunteers revealed a mean gastric retention period of 5.50 ± 0.77 h.
Keywords: Ciprofloxacin hydrochloride; Floating gastroretentive tablets; Release-retarding polymers; Gas former; Abdominal X-ray imaging;

Fusion production of solid dispersions containing a heat-sensitive active ingredient by hot melt extrusion and Kinetisol® dispersing by James C. DiNunzio; Chris Brough; Justin R. Hughey; Dave A. Miller; Robert O. Williams; James W. McGinity (340-351).
Many techniques for the production of solid dispersions rely on elevated temperatures and prolonged material residence times, which can result in decomposition of temperature-sensitive components. In this study, hydrocortisone was used as a model temperature-sensitive active ingredient to study the effect of formulation and processing techniques as well as to characterize the benefits of KinetiSol® Dispersing for the production of solid dispersions. Preformulation studies were conducted using differential scanning calorimetry and hot stage microscopy to identify optimum carriers for the production of amorphous solid dispersions. After identification, solid dispersions were prepared by hot melt extrusion and KinetiSol® Dispersing, with material characterized by X-ray diffraction, dissolution and potency testing to evaluate physicochemical properties. Results from the preformulation studies showed that vinylacetate:vinylpyrrolidone (PVPVA) copolymer allowed for hydrocortisone dissolution within the carrier at temperatures as low as 160 °C, while hydroxypropyl methylcellulose required temperatures upward of 180 °C to facilitate solubilization. Low substituted hydroxypropyl cellulose, a high glass transition temperature control, showed that the material was unable to solubilize hydrocortisone. Manufacturing process control studies using hot melt extruded compositions of hydrocortisone and PVPVA showed that increased temperatures and residence times negatively impacted product potency due to decomposition. Using KinetiSol® Dispersing to reduce residence time and to facilitate lower temperature processing, it was possible to produce solid dispersions with improved product potency. This study clearly demonstrated the importance of carrier selection to facilitate lower temperature processing, as well as the effect of residence time on product potency. Furthermore, KinetiSol® Dispersing provided significant advantages over hot melt extrusion due to the reduced residence times and lower required processing temperatures. This allowed for the production of solid dispersions with enhanced product potency.
Keywords: Hydrocortisone; Thermal stability; Solid dispersion; Thermal processing; Hot melt extrusion; KinetiSol® Dispersing; Degradation;

Properties of melt extruded enteric matrix pellets by Sandra U. Schilling; Navnit H. Shah; A. Waseem Malick; James W. McGinity (352-361).
The objective of this study was to investigate the properties of enteric matrix pellets that were prepared by hot-melt extrusion in a one-step, continuous process.Five polymers (Eudragit® L100-55, L100 and S100, Aqoat® grades LF and HF) were investigated as possible matrix formers, and pellets prepared with Eudragit® S100 demonstrated superior gastric protection and acceptable processibility. Extruded pellets containing Eudragit® S100 and up to 40% theophylline released less than 10% drug over 2 h in acid, however, the processibility and yields were compromised by the high amounts of the non-melting drug material in the formulation. Efficient plasticization of Eudragit® S100 was necessary to reduce the polymer’s glass transition temperature and melt viscosity. Five compounds including triethyl citrate, methylparaben, polyethylene glycol 8000, citric acid monohydrate and acetyltributyl citrate were investigated in terms of plasticization efficiency and preservation of the delayed drug release properties. The aqueous solubility of the plasticizer and its plasticization efficiency impacted the drug release rate from the matrix pellets. The use of water-soluble plasticizers resulted in a loss of gastric protection, whereas low drug release rates in acid were found for pellets containing insoluble plasticizers or no plasticizer, independent of the extent of Eudragit® S100 plasticization. The release rate of theophylline in buffer pH 7.4 was faster for pellets that were prepared with efficient plasticizers. The microstructure and solid-state properties of plasticized pellets were further investigated by scanning electron microscopy and powder X-ray diffraction. Pellets prepared with efficient plasticizers (TEC, methylparaben, PEG 8000) exhibited matrices of low porosity, and the drug was homogeneously dispersed in its original polymorphic form. Pellets containing ATBC or citric acid monohydrate had to be extruded at elevated temperature and showed physical instabilities in the form of recrystallization at room temperature. Enteric matrix pellets with a diameter below 1 mm and containing 30% theophylline could be successfully prepared by hot-melt extrusion when Eudragit® S100 plasticized with either TEC or methylparaben was employed as the matrix material.
Keywords: Matrix pellets; Hot-melt extrusion; Delayed release; Eudragit; Aqoat; Plasticizers;

Curing of aqueous polymeric film coatings: Importance of the coating level and type of plasticizer by Q.W. Yang; M.P. Flament; F. Siepmann; V. Busignies; B. Leclerc; C. Herry; P. Tchoreloff; J. Siepmann (362-370).
The aim of this study was to better understand the effects of the curing conditions on the resulting drug release patterns from pellets coated with aqueous polymer dispersions. Diltiazem HCl was used as model drug, ethylcellulose as polymer, triethyl citrate (TEC), dibutyl sebacate (DBS), and distilled acetylated monoglycerides (Myvacet) as plasticizers. Interestingly, the effects of the curing conditions strongly depended on the coating level and the type of plasticizer: in the case of TEC, the drug release rate monotonically decreased with increasing harshness of the curing conditions (time, temperature, and relative humidity), irrespective of the coating level. In contrast, in the case of DBS and Myvacet, this type of relationship was only observed at low coating levels (5%). At intermediate coating levels (around 7.5%), the curing conditions had virtually no effect on drug release. At high coating levels (⩾10%), the release rate initially increased and then decreased with increasing harshness of the curing conditions. This more complex behavior might be attributable to the superposition of two competing phenomena: improved film formation and drug migration into the polymeric membrane. Furthermore, it could be shown that the type of plasticizer had a major effect on drug release in not fully coalesced and equilibrated film coatings, whereas the release profiles were similar for all plasticizers in the case of completely formed and equilibrated film coatings. Importantly, the latter systems were stable for long term even during storage under stress conditions.
Keywords: Coating; Pellets; Plasticizer; Aqueous ethylcellulose dispersion; Curing;

Scratch resistance of plasticized hydroxypropyl methylcellulose (HPMC) films intended for tablet coatings by Kalle Hanhijärvi; Terhi Majava; Ivan Kassamakov; Jyrki Heinämäki; Juha Aaltonen; Jonne Haapalainen; Edward Hæggström; Jouko Yliruusi (371-376).
Scratch resistance (SR) of externally plasticized hydroxypropyl methylcellulose (HPMC) films intended for tablet film coatings was studied. Special attention was paid to effects of short-term aging and ultraviolet (UV) treatment on the SR properties of these films. Controlled scratching of the films was performed with a Lloyd LRX materials tester featuring a spherical steel tip. Scratch surface profiles were measured by scanning white light interferometry (SWLI). The influence of using an external plasticizer on the SR was studied by comparing scratch dimensions in non-plasticized films to samples plasticized either with glycerol or polyethylene glycol (PEG) 400. The study demonstrates that both the amount and type of plasticizer influences the SR of aqueous HPMC films. It also shows that SWLI can quantitatively evaluate the effect of plasticizer content and aging on the SR of pharmaceutical films. This knowledge could be used to optimize pharmaceutical film coating formulations.
Keywords: Film; Hydroxypropyl methylcellulose; Plasticizer; Scratch resistance; Scanning white light interferometry; Aging;

Investigation into the intragranular structures of microcrystalline cellulose and pre-gelatinised starch by Peter Laity; Andrew Cassidy; Jeremy Skepper; Bill Jones; Ruth Cameron (377-387).
The internal structures of commercial spheronised microcrystalline cellulose (s-MCC) and pre-gelatinised starch (PGS) granules were investigated, using a range of methods. Results from scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM) revealed hierarchical structures, with dimensions ranging from nanometres to a few micrometres, for both materials. Residual fragments of plant cell walls, consisting largely of crystalline fibrillar bundles, were indicated within s-MCC granules, while PGS granules appeared to consist of densely packed spherical features. The lack of any obvious regular periodicity associated with the intragranular sub-structures was entirely consistent with the power-law behaviour of the small-angle X-ray scattering (SAXS) patterns from these materials. The presence of intragranular porosity was inferred from TEM, AFM and N2-adsorption measurements, while the ability to deform these structures was clearly indicated by the irregular force–displacement curves recorded by AFM on the granule surfaces. Hence, the intragranular sub-structures observed for s-MCC and PGS appeared to be consistent with the possibility of entire granules undergoing affine deformation during compaction. Since this mechanism was postulated to explain changes in SAXS patterns from these materials following compaction, as reported elsewhere, the work reported here provides a considerably stronger basis for using 2D-SAXS to investigate powder compaction behaviour.
Keywords: Small-angle X-ray scattering (SAXS); Wide-angle X-ray scattering (WAXS); Scanning electron microscopy (SEM); Transmission electron microscopy (TEM); Atomic force microscopy (AFM); Nitrogen adsorption; Microcrystalline cellulose (MCC); Starch 1500, pre-gelatinised starch (PGS); Granule structure;

The scope of the work is twofold, first to introduce a new avalanche testing instrument and secondly to characterise flowability of pharmaceutical blends comprising of coarse and fine particles. The results were compared with established powder characterisation instruments like the angular shear cell and a flow through orifice tester. These different methods were applied to a broad concentration range of binary mixtures comprising coarse, well-flowing lactose and micronised, poorly flowing albendazole. Some of the mixtures were further analysed with scanning electron microscopy. The results showed clear changes in the flow behaviour of the mixtures that were considered as critical flow concentrations (CFCs). At least three drug concentrations were observed for which the flow behaviour essentially changed. Accordingly, different flow regions were identified, which were explained on the basis of changed particle packing configurations. A theoretical model successfully provided a first estimation of the initial two CFCs. In conclusion, the novel avalanche testing instrument provided complementary information to conventional flowability methodologies, and a thorough assessment of pharmaceutical blends is needed to avoid CFCs in view of a robust formulation development and hence with respect to building quality into the design of the solid dosage forms.
Keywords: Flowability; Binary mixtures; Powder avalanche; Avalanching powder analyser; Shear cell;

Lyophilization monophase solution technique for improvement of the physicochemical properties of an anticancer drug, flutamide by Nazik Elgindy; Kadria Elkhodairy; Abdallah Molokhia; Ahmed Elzoghby (397-405).
Flutamide (FLT), an anticancer drug for prostatic carcinoma, has poor aqueous solubility and low oral bioavailability. This study describes the ability of β-cyclodextrin (βCD) and hydroxypropyl-β-cyclodextrin (HPβCD) to form complexes with flutamide with enhanced solubility and dissolution rate in vitro. FLT–CD lyophilized dispersions (LDs) were prepared via lyophilization monophase solution technique using tertiary butyl alcohol (TBA) as a cosolvent. FLT showed an AL-type phase solubility diagram consistent with a linear increase in drug solubility as a function of CD concentration. Gas chromatography indicated that the LDs contain 0.02–0.03% w/w residual TBA. Based on the data from differential scanning calorimetry (DSC) and X-ray diffractometry (XRD), FLT was fully amorphous in 1:5 FLT-HPβCD LD as indicated by complete disappearance of FLT endothermic and diffraction peaks. The Fourier transform infrared (FTIR) spectra indicated that a FLT–CD interaction took place in the lyophilized complex. The particle sizes of 1:1 FLT-βCD and FLT-HPβCD LDs were 0.92 and 0.82 μm, with a high surface area (484.55 and 705.68 m2/g) and porosity (769.46 and 1020.99 e−3ml/g), respectively. The dissolution rate of FLT from its CD complexes was enhanced significantly. After 30 min in 0.1 N HCl, about 73% and 86% of FLT were dissolved from 1:5 FLT-βCD and FLT-HPβCD LDs, respectively, compared to only 13.45% of pure drug. No endothermic peak corresponding to FLT melting was detected in 1:5 FLT-HPβCD LD after storage at 20 °C and 45% relative humidity for 90 days thus indicating the stability of this binary system. These data suggest that cyclodextrins might be useful adjuncts in preparation of immediate-release formulations of FLT.
Keywords: Lyophilization; Monophase solution; Flutamide; Cyclodextrins; Inclusion complexes;

Classification of lyophilised mixtures using multivariate analysis of NIR spectra by Holger Grohganz; Margot Fonteyne; Erik Skibsted; Thomas Falck; Bent Palmqvist; Jukka Rantanen (406-412).
Excipient selection is critically affecting the processing and the stability of a lyophilised product. Near infra-red (NIR) spectroscopy was applied to investigate freeze-dried samples containing varying ratios of the commonly used excipients mannitol and sucrose. Further variation in the formulation was achieved by adding NaCl, CaCl2 and histidine and by exposing the samples to different conditions. Untreated NIR spectra are strongly affected by the physical nature of samples and can thus be useful for detecting production outliers. Applying standard normal variate (SNV) transformation highlights chemical information. The obtained NIR spectra of the freeze-dried samples were clustered by principal component analysis (PCA) after applying SNV correction in the range from 4200 to 7400 cm−1 (1350–2380 nm). Relative humidity under storage and the mannitol/sucrose ratio were clearly represented in the first two principal components, while influence of other excipients was observed in the 3rd and 4th principal component. It was investigated whether this could be due to an influence of the excipients on the mannitol crystallisation behavior. Performing PCA with two principal components of SNV-corrected spectra in the range 4200–4500 cm−1 (2220–1380 nm) led to the following observation: while the 1st principal component closely resembled the spectra of β-mannitol, the 2nd principal component contained additional features that were not attributable to β-mannitol but correlated well to the main absorbance band of δ-mannitol and mannitol hemihydrate. Therefore, it seems feasible that NIR can analyse versatile freeze-dried samples and classify these according to composition, water content and solid-state properties.
Keywords: NIR; Freeze-drying; Multivariate analysis; Excipients; Solid state;

ATR-FTIR spectroscopy and spectroscopic imaging of solvent and permeant diffusion across model membranes by W.J. McAuley; M.D. Lad; K.T. Mader; P. Santos; J. Tetteh; S.G. Kazarian; J. Hadgraft; M.E. Lane (413-419).
The uptake and diffusion of solvents across polymer membranes is important in controlled drug delivery, effects on drug uptake into, for example, infusion bags and containers, as well as transport across protective clothing. Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy has been used to monitor the effects of different solvents on the diffusion of a model compound, 4-cyanophenol (CNP) across silicone membrane and on the equilibrium concentration of CNP obtained in the membrane following diffusion. ATR-FTIR spectroscopic imaging of membrane diffusion was used to gain an understanding of when the boundary conditions applied to Fick’s second law, used to model the diffusion of permeants across the silicone membrane do not hold. The imaging experiments indicated that when the solvent was not taken up appreciably into the membrane, the presence of discrete solvent pools between the ATR crystal and the silicone membrane can affect the diffusion profile of the permeant. This effect is more significant if the permeant has a high solubility in the solvent. In contrast, solvents that are taken up into the membrane to a greater extent, or those where the solubility of the permeant in the vehicle is relatively low, were found to show a good fit to the diffusion model. As such these systems allow the ATR-FTIR spectroscopic approach to give mechanistic insight into how the particular solvents enhance permeation. The solubility of CNP in the solvent and the uptake of the solvent into the membrane were found to be important influences on the equilibrium concentration of the permeant obtained in the membrane following diffusion. In general, solvents which were taken up to a significant extent into the membrane and which caused the membrane to swell increased the diffusion coefficient of the permeant in the membrane though other factors such as solvent viscosity may also be important.
Keywords: ATR-FTIR spectroscopy; Imaging; Drug transport; Membrane diffusion; Penetration enhancers;