European Journal of Pharmaceutics and Biopharmaceutics (v.77, #1)

APV Diary (I).

A colloidal carrier system with an oily colloidal phase (O/W microemulsion) is shown on the right hand side which is used to improve dermal and transdermal drug delivery of extremely lipophilic drugs. On the left hand side a colloidal carrier system with an aqueous colloidal phase (W/O microemulsion) is shown suitable for dermal and transdermal drug delivery of extremely hydrophilic drugs such as peptides and proteins.Nanocarriers (NCs) are colloidal systems having structures below a particle or droplet size of 500 nm. In the previous years, the focus for the application of NCs was primarily placed on the parenteral and oral application. However, NCs applied to the skin are in the center of attention and are expected to be increasingly applied as the skin offers a lot of advantages for the administration of such systems. For the use of NCs to the skin, one has to differentiate between the desired effects: the local effect within the skin (dermal drug delivery) or a systemic effect accompanied by the permeation through the skin (transdermal drug delivery).Both for dermal and transdermal drug delivery, the stratum corneum (SC), the main barrier of the skin, has to be overcome.SC is one of the tightest barriers of the human body. Therefore, it is the primary goal of new NC to overcome this protective and effective barrier. For that purpose, new NCs such as microemulsions, vesicular (liposomes) and nanoparticular NCs are developed and investigated. This article evaluates the potentials of these NCs for dermal and transdermal drug delivery.
Keywords: Nanocarriers; Microemulsions; Liposomes; Vesicles; Nanoparticles; Dermal and transdermal drug delivery;

Sustained release of proteins from a modified vaginal ring device by Ryan J. Morrow; A. David Woolfson; Louise Donnelly; Rhonda Curran; Gavin Andrews; Dietmar Katinger; R. Karl Malcolm (3-10).
Vaginal ring devices can accommodate solid, insert dosage forms for sustained protein release.A new vaginal ring technology, the insert vaginal ring (InVR), is presented. The InVR overcomes the current shortfall of conventional vaginal rings (VRs) that are generally ineffectual for the delivery of hydrophilic and/or macromolecular actives, including peptides, proteins and antibodies, due to their poor permeation characteristics in the hydrophobic polymeric elastomers from which VRs are usually fabricated. Release of the model protein BSA from a variety of insert matrices for the InVR is demonstrated, including modified silicone rods, directly compressed tablets and lyophilised gels, which collectively provided controlled release profiles from several hours to beyond 4 weeks. Furthermore, the InVR was shown to deliver over 1 mg of the monoclonal antibody 2F5 from a single device, offering a potential means of protecting women against the transmission of HIV.
Keywords: Vaginal ring; Protein drug delivery; Vaccine; Microbicide; 2F5; Lyophilisation;

Preparation and evaluation of alginate–chitosan microspheres for oral delivery of insulin by Yueling Zhang; Wei Wei; Piping Lv; Lianyan Wang; Guanghui Ma (11-19).
The insulin-loaded alginate-chitosan microspheres prepared by SPG membrane emulsification and two-step solidification could realize the pH-sensitive release of insulin.The alginate–chitosan microspheres with narrow size distribution were prepared by membrane emulsification technique in combination with ion (Ca2+) and polymer (chitosan) solidification. The preparation procedure was observed, and the physical properties (particle size distribution, surface morphology, chitosan distribution, zeta potential) of the microspheres were characterized. Subsequently, the microspheres were employed to load model peptide of insulin. The effect of loading ways on the loading efficiency and immunological activity of insulin were investigated. It was shown that the higher loading efficiency (56.7%) and remarkable activity maintenance (99.4%) were obtained when the insulin was loaded during the chitosan solidification process (Method B). Afterward, the release profile in vitro for the optimal insulin-loaded microspheres was investigated. Under the pH conditions of gastrointestinal environment, only 32% of insulin released during the simulated transit time of drug (2 h in the stomach and 4 h in the intestinal). While under the pH condition of blood environment, insulin release was stable and sustained for a long time (14 days). Furthermore, the chemical stability of insulin released from the microspheres was well preserved after they were treated with the simulated gastric fluid containing pepsin for 2 h. Finally, the blood glucose level of diabetic rats could be effectively reduced and stably kept for a long time (∼60 h) after oral administration of the insulin-loaded alginate–chitosan microspheres. Therefore, the alginate–chitosan microspheres were found to be promising vectors showing a good efficiency in oral administration of protein or peptide drugs.
Keywords: Narrow size distribution; Alginate–chitosan microspheres; Protein or peptide; Oral administration; Insulin; Sustained release;

Nanostructured porous silicon microparticles enable sustained peptide (Melanotan II) delivery by Miia Kilpeläinen; Juha Mönkäre; Maria A. Vlasova; Joakim Riikonen; Vesa-Pekka Lehto; Jarno Salonen; Kristiina Järvinen; Karl-Heinz Herzig (20-25).
Peptide administration with nanoporous silicon microparticles delays and prolongs in vivo effects, when compared with a peptide solution. This indicates sustained peptide release from porous silicon.Peptide molecules can improve the treatment of a number of pathological conditions, but due to their physicochemical properties, their delivery is very challenging. The study aim was to determine whether nanostructured porous silicon could sustain the release and prolong the duration of action of a model peptide Melanotan II (MTII). Thermally hydrocarbonized nanoporous silicon (THCPSi) microparticles (38–53 μm) were loaded with MTII. The pore diameter, volume, specific surface area and loading degree of the microparticles were analyzed, and the peptide release was evaluated in vitro. The effects of MTII on heart rate and water consumption were investigated in vivo after subcutaneous administration of the MTII loaded microparticles. A peptide loading degree of 15% w/w was obtained. In vitro studies (PBS, pH 7.4, 37 °C) indicated sustained release of MTII from the THCPSi microparticles. In vivo, MTII loaded THCPSi induced an increase in the heart rate 2 h later than MTII solution, and the effect lasted 1 h longer. In addition, MTII loaded THCPSi changed the water consumption after 150 min, when the immediate effect of MTII solution was already diminished. The present study demonstrates that MTII loading into nanosized PSi pore structure enables sustained delivery of an active peptide.
Keywords: In vivo; Peptide delivery; Porous silicon; Microparticles; Nanostructure; Sustained release;

High loading efficiency and sustained release of siRNA encapsulated in PLGA nanoparticles: Quality by design optimization and characterization by Dongmei Cun; Ditte Krohn Jensen; Morten Jonas Maltesen; Matthew Bunker; Paul Whiteside; David Scurr; Camilla Foged; Hanne Mørck Nielsen (26-35).
The loading of PLGA nanoparticles with siRNA was optimized using design of experiments and the particles provided an siRNA burst release followed by a triphasic sustained release.Poly(dl-lactide-co-glycolide acid) (PLGA) is an attractive polymer for delivery of biopharmaceuticals owing to its biocompatibility, biodegradability and outstanding controlled release characteristics. The purpose of this study was to understand and define optimal parameters for preparation of small interfering RNA (siRNA)-loaded PLGA nanoparticles by the double emulsion solvent evaporation method and characterize their properties. The experiments were performed according to a 25−1  fractional factorial design based on five independent variables: The volume ratio between the inner water phase and the oil phase, the PLGA concentration, the sonication time, the siRNA load and the amount of acetylated bovine serum albumin (Ac-BSA) in the inner water phase added to stabilize the primary emulsion. The effects on the siRNA encapsulation efficiency and the particle size were investigated. The most important factors for obtaining an encapsulation efficiency as high as 70% were the PLGA concentration and the volume ratio whereas the size was mainly affected by the PLGA concentration. The viscosity of the oil phase was increased at high PLGA concentration, which explains the improved encapsulation by stabilization of the primary emulsion and reduction of siRNA leakage to the outer water phase. Addition of Ac-BSA increased the encapsulation efficiency at low PLGA concentrations. The PLGA matrix protected siRNA against nuclease degradation, provided a burst release of surface-localized siRNA followed by a triphasic sustained release for two months. These results enable careful understanding and definition of optimal process parameters for preparation of PLGA nanoparticles encapsulating high amounts of siRNA with immediate and long-term sustained release properties.
Keywords: siRNA; Poly(dl-lactide-co-glycolide acid); Sustained release; Nanoparticles; Drug delivery; Nanomedicine;

Chitosan-graft-spermine as a gene carrier in vitro and in vivo by Hu-Lin Jiang; Hwang-Tae Lim; You-Kyoung Kim; Rohidas Arote; Ji-Young Shin; Jung-Taek Kwon; Ji-Eun Kim; Ji-Hye Kim; Duyeol Kim; Chanhee Chae; Jae-Woon Nah; Yun-Jaie Choi; Chong-Su Cho; Myung-Haing Cho (36-42).
Proposed reaction scheme for synthesis of CHI-g-SPE and GFP expression in the lungs of miceChitosan has been proposed as a non-viral gene carrier because of its biodegradable and biocompatible cationic polymeric properties. However, the transfection efficiency of chitosan-DNA complexes is still too low for clinical trials. To improve transfection efficiency, we prepared a chitosan-graft-spermine (CHI-g-SPE) copolymer by an imine reaction between periodate-oxidized chitosan and spermine. The CHI-g-SPE copolymer was complexed with plasmid DNA in various copolymer-DNA weight ratios, and the complexes were characterized. The CHI-g-SPE copolymer showed good DNA binding ability and high protection of DNA from nuclease attack. The CHI-g-SPE/DNA complexes had well-formed spherical shapes and a nanoscale size with homogenous size distribution. The CHI-g-SPE copolymer had low cytotoxicity and CHI-g-SPE/DNA complexes showed transfection efficiency that was enhanced over that of chitosan-DNA. Furthermore, aerosol delivery of CHI-g-SPE/GFP complexes showed higher GFP expression compared with chitosan/GFP complexes, without toxicity. Our results indicate that the CHI-g-SPE copolymer has potential as a gene carrier.
Keywords: Gene therapy; Non-viral gene delivery; Chitosan; Spermine; Cytotoxicity; Aerosol delivery;

Plasma concentration–time profile following administration of 5 mg vinpocetine from SE proniosomal patch and Vinporal® tablet (mean ± SD, n  = 12). Significant difference.Vinpocetine (Vin) existing oral formulations suffer poor bioavailability (∼7%) since Vin undergoes a marked first-pass effect (∼75%) and its absorption is dissolution rate-limited. In this study, a novel sustained release proniosomal system was designed using sugar esters (SEs) as non-ionic surfactants in which proniosomes were converted to niosomes upon skin water hydration following topical application under occlusive conditions. Different in vitro aspects (encapsulation efficiency, vesicle size and shape, effect of occlusion, in vitro release, skin permeation and stability) were studied leading to an optimized formula that was assessed clinically for transdermal pharmacokinetics and skin irritation.All formulae exhibited high entrapment efficiencies, regardless of the surfactant HLB. Vesicle size analysis showed that all vesicles were in the range from 0.63 μm to 2.52 μm which favored efficient transdermal delivery. The extent of drug permeation through the skin from the optimized formula – containing laurate SE with shorter fatty acid chain length and high HLB – was quite high (91%) after 48 h under occlusive conditions. The extent of absorption of Vin from proniosomes was larger when compared to the oral tablet with a relative bioavailability (F rel) of 206%. Histopathological evaluation revealed only moderate skin irritation when using SEs compared to skin inflammation when using Tween 80. Sugar esters proniosomes may be a promising carrier for vinpocetine, especially due to their simple scaling up and their ability to control drug release.
Keywords: Proniosomes; Niosomes; Sugar esters; Vinpocetine; Transdermal delivery; Skin irritation;

The self-assembly of MPEG-dihexPLA into polymeric micelles incorporates efficiently Cyclosporin A and was found to be biocompatible.The immunosuppressive agent Cyclosporin A (CsA) has very poor solubility in water and, in consequence, non-aqueous formulations have been developed for its intravenous administration to treat patients with transplant rejection. In this article, aqueous micelle solutions of novel amphiphilic copolymers based on methoxy-poly(ethylene glycol) (MPEG) and hexyl-substituted poly(lactides) (hexPLA) were studied for possible incorporation and formulation of CsA, and for their biocompatibility towards novel pharmaceutical applications. Above the critical micellar concentration (CMC), MPEG–hexPLA block-copolymers self-assemble into unimodal micelles with diameters of around 30 nm, either unloaded or drug-loaded. The best shelf-life stability of these formulations was observed when stored at 4 °C with a drug loss inferior to 7% after 1 year. The polymer and micelle toxicities were evaluated in vitro for three different cell lines and in vivo using the chick embryo chorioallantoic membrane (CAM) model. The hemolytic property was assessed using human blood samples. As the studies revealed, MPEG–hexPLAs are non-toxic and do not show hemolysis; the same was found for the comparable MPEG–PLAs, both as unimers below their CMC and as polymeric micelles up to copolymer concentrations of 20 mg/mL. At this concentration, CsA was efficiently incorporated into MPEG–hexPLA micelles up to 6 mg/mL, which corresponds to a 500-fold increase of its water solubility. The current recommended clinical concentration administered per infusion (0.5–2.5 mg/mL) can be easily achieved and requires four times less copolymer than with the often-used Cremophor®EL surfactant. In this regard, MPEG–hexPLA micelle formulations can be an applicable formulation in transplant rejection treatments as an injectable CsA carrier system.
Keywords: Biocompatibility; CAM model; Cyclosporin A; Drug delivery; Polymeric micelles; Substituted polylactides;

Mesoporous silica coating gold nanorods was synthesized. The role of hyperthermia effect in enhancing release rates was investigated by subjecting loaded MCM-41-GNRs to near infra red (NIR) radiation at 800 nm. This would be of significance in targeted drug release using hyperthermia effect. Unlike hydroxyl apatite, loading MCM-41 with gold nanorods does not affect the release kinetics. Only when these samples are irradiated with NIR photons, does the release occur with enhanced rates. This property could be valuable in selected targeting of drugs.The early 5 h of coumarin I release from MCM-41-GNRs (○) and MCM-41-GNRs irradiated at 807 nm using a diode laser source (●). There is an increase in release by about 47.6%.The synthesis of three types of mesoporous materials is reported: pure mesoporous silica (MCM-41), a nanocomposite of mesoporous silica with hydroxyapatite (MCM-41-HA) and mesoporous silica/gold nanorods nanocomposite (MCM-41-GNRs). The mesoporous materials were characterized by X-ray diffraction, N2 adsorption isotherms, FTIR spectroscopy, transmission electron microscopy, and scanning electron microscopy. The samples were loaded with coumarin thiourea derivatives (I–IV) having functional groups of varying sizes and the in vitro release assays were monitored, and the release behavior was investigated as a function of soaking time in simulated body fluid. Two release stages were obtained in MCM-41, MCM-41-HA and MCM-41-GNRs loaded samples with the early release stages accounting for about 30% of loaded derivatives. These early release stages are characterized by Higuchi rate constant values nearly twice the values associated with the second release stages. The influence of substituent size on the release rate constants was explained in terms of sorption sites and hydrogen bonding with silanol groups on silicates. The release of coumarin derivatives loaded on MCM-41, MCM-41-HA and MCM-41-GNRs occurs over remarkably long time of the order of about 260 h with faster release rates in loaded MCM-41 and MCM-41-GNRs samples compared with MCM-41-HA ones. The role of hyperthermia effect in enhancing release rates was investigated by subjecting loaded MCM-41-GNRs to near infrared (NIR) radiation at 800 nm. This would be of significance in targeted drug release using hyperthermia effect. Unlike hydroxyl apatite, loading MCM-41 with gold nanorods does not affect the release kinetics. Only when these samples are irradiated with NIR photons, does the release occur with enhanced rates. This property could be valuable in selected targeting of drugs.
Keywords: Mesoporous silica; Hydroxyapatite nanocomposites; Gold nanorods; Coumarin thiourea; Controlled release; Hyperthermia effect;

The potential of liposomes as dental drug delivery systems by Sanko Nguyen; Marianne Hiorth; Morten Rykke; Gro Smistad (75-83).
Saliva constituents may interact with liposomes. An appropriate liposomal drug delivery system intended for use in the oral cavity seems to be dependent on the liposomal formulation.The potential of liposomes as a drug delivery system for use in the oral cavity has been investigated. Specifically targeting for the teeth, the in vitro adsorption of charged liposomal formulations to hydroxyapatite (HA), a common model substance for the dental enamel, has been conducted. The experiments were performed in human parotid saliva to simulate oral-like conditions. It was observed, however, that precipitation occurred in tubes containing DPPC/DPTAP or DPPC/DPPG-liposomes in parotid saliva with no HA present, indicating that constituents of parotid saliva reacted with the liposomes.The aggregation reactions of liposome–parotid saliva mixtures were examined by turbidimetry and by atomic force microscopy. Negatively charged DPPC/DPPS and DPPC/PI-liposomes were additionally included in these experiments. The initial turbidity of positive DPPC/DPTAP-liposomes in parotid saliva was very high, but decreased markedly after 30 min. AFM images showed large aggregates of micelle-like globules known to be present in saliva. The turbidity of the various negatively charged liposome and parotid saliva mixtures stayed relatively constant throughout the measuring time; however, their initial turbidities were different; mixtures with DPPC/DPPG-liposomes were the most turbid and DPPC/DPPA-liposomes the least. Pyrophosphate (PP) was added to the various liposome–parotid saliva mixtures to examine the effect of Ca2+ on the interactions. The effect of PP treatment of the negatively charged liposome–parotid saliva mixtures was most pronounced with DPPC/DPPG-liposome mixtures where it caused a sudden drop in turbidity. For positive DPPC/DPTAP liposome and parotid saliva mixtures, the effect of PP was minimal.These experiments showed that saliva constituents may interact with liposomes. An appropriate liposomal drug delivery system intended for use in the oral cavity seems to be dependent on the liposomal formulation. Based on the present results, negatively charged DPPC/DPPA-liposomes seem to be most suitable for use in the oral cavity as they were found to be the least reactive with the components of parotid saliva.
Keywords: Liposomes; Parotid saliva; Oral cavity; Dental enamel; Atomic force microscopy; Dental drug delivery;

Brain targeting with surface-modified poly(d,l-lactic-co-glycolic acid) nanoparticles delivered via carotid artery administration by Kohei Tahara; Yuta Miyazaki; Yoshiaki Kawashima; Jörg Kreuter; Hiromitsu Yamamoto (84-88).
Polysorbate 80-PLGA nanoparticles were delivered to the brain tissue via carotid artery administration.In this study, we investigated surface-modified nanoparticles (NP) formulated using a biodegradable polymer, poly(d,l-lactide-co-glycolide) (PLGA), for targeting central nervous system (CNS) diseases. Polysorbate 80 (P80), poloxamer 188 (P188), and chitosan (CS) were used to modify the surfaces of PLGA NP to improve the brain delivery of NP. Surface-modified PLGA NP were formulated using an emulsion solvent diffusion method. 6-Coumarin was used as a fluorescent label for NP. The different formulations of 6-coumarin-loaded PLGA NP were injected into rats via carotid arteries. NP remaining in the brain were evaluated quantitatively, and brain slices were observed using confocal laser scanning microscopy (CLSM). Carotid artery administration was more effective for delivering NP into the brain compared to intravenous administration. After administration, NP concentrations in the brain were increased by NP surface modification, especially CS- and P80-PLGA NP. CLSM observations indicated that P80-PLGA NP could cross the blood–brain barrier and thus serve as a drug delivery system for the CNS. These results indicate that surface-modified PLGA NP have a high potential for use in CNS delivery systems.
Keywords: PLGA; Nanoparticle; Polysorbate 80; Poloxamer 188; Chitosan; Surface modification;

Incorporation of a synthetic mycobacterial monomycoloyl glycerol analogue stabilizes dimethyldioctadecylammonium liposomes and potentiates their adjuvant effect in vivo by Pernille Nordly; Karen Smith Korsholm; Esra Alici Pedersen; Tayba Sajid Khilji; Henrik Franzyk; Lene Jorgensen; Hanne Mørck Nielsen; Else Marie Agger; Camilla Foged (89-98).
MMG-1 is proposed to stabilize the DDA liposomes by increased hydration of the lipid bilayer due to hydrogen bond formation between the glycerol moiety of MMG-1 and the aqueous medium as well as by reducing the repulsion between adjacent cationic DDA molecules. This was supported by studying Langmuir-Blodgett monolayers of DDA and MMG-1.The combination of delivery systems such as cationic liposomes and immunopotentiating molecules is a promising approach for the rational design of vaccine adjuvants. In this study, a synthetic analogue of the mycobacterial lipid monomycoloyl glycerol (MMG), referred to as MMG-1, was synthesized and combined with the cationic surfactant dimethyldioctadecylammonium (DDA). The purpose of the study was to provide a thorough pharmaceutical characterization of the resulting DDA/MMG-1 binary system and to evaluate how incorporation of MMG-1 affected the adjuvant activity of DDA liposomes. Thermal analyses demonstrated that MMG-1 was incorporated into the DDA lipid bilayers, and cryo-transmission electron microscopy (TEM) confirmed that liposomes were formed. The particles had a polydisperse size distribution and an average diameter of approximately 400 nm. Evaluation of the colloidal stability indicated that at least 18 mol% MMG-1 was required to stabilize the DDA liposomes as the average particle size remained constant during storage for 6 months. The improved colloidal stability is most likely caused by increased hydration of the lipid bilayer. This was demonstrated by studying Langmuir–Blodgett monolayers of DDA and MMG-1 which revealed an increased surface pressure in the presence of high concentrations of MMG-1 when the DDA/MMG-1 monolayers were fully compressed, indicating an increased interaction with water due to enhanced hydration of the lipid head groups. Finally, immunization of mice with the tuberculosis fusion antigen Ag85B-ESAT-6 and DDA/MMG-1 liposomes induced a strong cell-mediated immune response characterized by a mixed Th1/Th17 profile and secretion of IgG1 and IgG2c antibodies. The Th1/Th17-biased immunostimulatory effect was increased in an MMG-1 concentration-dependent manner with maximal observed effect at 31 mol% MMG-1. Thus, incorporation of 31 mol% MMG-1 into DDA liposomes results in an adjuvant system with favorable physical as well as immunological properties.
Keywords: Liposome; Adjuvant; Vaccine; Dimethyldioctadecylammonium; Monomycoloyl glycerol; Nanomedicine;

Drug release from hydrophilic matrices composed of HPMC with different substituent heterogeneity. The more heterogeneously substituted batches interacted with the poorly soluble drug to greater extent, which significantly increased the polymer erosion from the matrix. The increased erosion contributed to that the poorly soluble drug was released with a faster rate than the more soluble model drug. In contrast, the drug release from matrixes composed of more homogeneously substituted HPMC batches followed the expected drug release behaviour, where the poorly soluble drug was released with lower rates and with a greater erosional contribution to the release mechanism. Consequently, both the substituent heterogeneity of HPMC as well as the polymer-drug interactions highly influences the drug release from matrix tablets.This study investigates the effect of the chemical heterogeneity of hydroxypropyl methylcellulose (HPMC) on the release of model drug substances from hydrophilic matrix tablets. The hypothesis was that the release of drug substances could be influenced by possible interactions with HPMC batches having different chemical heterogeneity. The cloud point of the most heterogeneous batch was more affected by the model drug substances, methylparaben and butylparaben, and most by butylparaben with the lowest solubility. The different clouding behaviour was explained by the heterogeneously substituted batches being more associative and the more lipophilic butylparaben being able to interact more efficiently with the hydrophobic HPMC transient crosslinks that formed. Interestingly, tablet compositions of the heterogeneously substituted HPMC batches released the more soluble methylparaben at lower rates than butylparaben. The explanation is that the hydrophobic HPMC interactions with butylparaben made the gel of the tablet less hydrated and more fragile and therefore more affected by erosional stresses. In contrast, drug release from compositions consisting of the more homogeneously substituted batches was affected to a minor extent by the drugs and was very robust within the experimental variations. The present study thus reveals that there can be variability in drug release depending on the lipophilicity of the drug and the substituent heterogeneity of the HPMC used.
Keywords: Hydrophilic matrix tablets; HPMC; Chemical heterogeneous; Solubility; Drug release; Polymer erosion;

Tapioca starch graft copolymers and Dome Matrix® modules II. Effect of modules assemblage on Riboflavin release kinetics by Marta Casas; Orazio Luca Strusi; M. Rosa Jiménez-Castellanos; Paolo Colombo (111-115).
The combination of the floatation capability of assembled modules and the prolonged drug release provided with the graft copolymers make these systems candidates as controlled release gastro-retentive dosage forms.This paper studies the Riboflavin release from systems made of assembled modules of Dome Matrix® technology using tapioca starch-ethylmethacrylate (TSEMA) and tapioca hydroxypropylstarch-ethylmethacrylate (THSEMA) graft copolymers produced by two different drying methods. Two different shape modules were manufactured for this study, i.e., female and male modules, in order to facilitate their assemblage in “void configuration”, a system with an internal void space. Drug release studies on void configurations based on THSEMA show faster releases than TSEMA; HPMC systems used as a comparative reference showed intermediate release. Moreover, using void configurations made with one module of TSEMA and the other of THSEMA is possible to average the drug release, without difference between the drying methods used for the polymers. With respect to the floatation characteristics, all the void configurations floated immediately and, due to the mass center of the system, the floatation position of the system was always axial with the female module up and the male down. The drug release studies performed with a sinker to force the immersion of the systems in the medium did not show differences with respect to the dissolution test without a sinker. The combination of floatation capability of the assembled modules and the prolonged drug release provided with the graft copolymers make these assembled modules candidates as controlled release gastro-retentive dosage forms.
Keywords: Dome Matrix®; Tapioca starch; Graft copolymers; Floatation; Assembled modules; Riboflavin;

Swelling behavior during drug release for a two-module assembled in void configuration with a third barrier module added on the top. During the swelling of void assembly, the swellable module (left) is integrated in the void assembly whereas the inert module (right) is pushed away.The aim of this work was to study the clindamycin release kinetics from floating delivery systems consisting of two modules assembled in void configuration, according to the modified release technology platform known as Dome Matrix®. Two modules differently shaped, i.e., female and male, formulated as swellable matrices and containing clindamycin, were assembled by friction interlocking. Then, by stacking additional female modules without drug on the assembled two-module floating system, modulation of clindamycin release rate and kinetics was attained. The additional modules stacked on the assembled system acted as a transient barrier to clindamycin release from the void configuration. Inertness, dissolution/erosion or swelling behavior characterized their performance as matrices in simulated gastric fluid.In particular, we found that stacking additional barrier modules on the bases of void configuration, the drug release rate and kinetics of the assembled system were modified in dependence on the composition of module added. In particular, the quickly soluble module exerted an influence on the release rate in the late time of delivery. The swellable module produced a significant reduction in release rate of void assembly, but the release mechanism remained the same. Finally, the inert module led to a substantial linearization of the release profile with a minimal reduction in release rate.
Keywords: Dome Matrix®; Clindamycin; Floating dosage form; Drug release modules;

Characterization of solid dispersions and optimization of flotation and release properties by genetic programming and ANNs.The present study investigates the use of nimodipine–polyethylene glycol solid dispersions for the development of effervescent controlled release floating tablet formulations. The physical state of the dispersed nimodipine in the polymer matrix was characterized by differential scanning calorimetry, powder X-ray diffraction, FT-IR spectroscopy and polarized light microscopy, and the mixture proportions of polyethylene glycol (PEG), polyvinyl-pyrrolidone (PVP), hydroxypropylmethylcellulose (HPMC), effervescent agents (EFF) and nimodipine were optimized in relation to drug release (% release at 60 min, and time at which the 90% of the drug was dissolved) and floating properties (tablet’s floating strength and duration), employing a 25-run D-optimal mixture design combined with artificial neural networks (ANNs) and genetic programming (GP). It was found that nimodipine exists as mod I microcrystals in the solid dispersions and is stable for at least a three-month period. The tablets showed good floating properties and controlled release profiles, with drug release proceeding via the concomitant operation of swelling and erosion of the polymer matrix. ANNs and GP both proved to be efficient tools in the optimization of the tablet formulation, and the global optimum formulation suggested by the GP equations consisted of PEG = 9%, PVP = 30%, HPMC = 36%, EFF = 11%, nimodipine = 14%.
Keywords: Solid dispersions; Nimodipine; Controlled release; Effervescent floating tablets; Artificial neural networks; Genetic programming;

Aerosol generation, deposition and transport in the PADDOCC system.Absorption studies with aerosol formulation delivered by metered dose inhalers across cell- and tissue-based in vitro models of the pulmonary epithelia are not trivial due to the complexity of the processes involved: (i) aerosol generation and deposition, (ii) drug release from the carrier, and (iii) absorption across the epithelial air–blood barrier. In contrast to the intestinal mucosa, pulmonary epithelia are only covered by a thin film of lining fluid. Submersed cell culture systems would not allow to studying the deposition of aerosol particles and their effects on this delicate epithelial tissue.We developed a new Pharmaceutical Aerosol Deposition Device on Cell Cultures (PADDOCC) to mimic the inhalation of a single metered aerosol dose and its subsequent deposition on filter-grown pulmonary epithelial cell monolayers exposed to an air–liquid interface. The reproducibility of deposition of these dry powder aerosols and subsequent drug transport across Calu-3 monolayers with commercially available dry powder inhalers containing salbutamol sulphate or budesonide could be demonstrated.In the context of developing new dry powder aerosol formulations, PADDOCC appears as a useful tool, allowing reducing animal testing and faster translation into clinical trials.
Keywords: Aerosol medicine; In vitro model; Air–liquid interface; Calu-3 cells; Sedimentation;

Influence of PEGylation with linear and branched PEG chains on the adsorption of glucagon to hydrophobic surfaces by Charlotte Pinholt; Jens Thostrup Bukrinsky; Susanne Hostrup; Sven Frokjaer; Willem Norde; Lene Jorgensen (139-147).
The adsorption of glucagon from aqueous solution to a hydrophobic surface is compared with the adsorption of glucagon PEGylated with a linear and a branched PEG chain, respectively.PEGylation has proven useful for prolonging the plasma half lives of proteins, and since approval of the first PEGylated protein drug product by the FDA in 1990, several PEGylated protein drug products have been marketed. However, the influence of PEGylation on the behavior of proteins at interfaces is only poorly understood. The aim of this work was to study the effect of PEGylation on the adsorption of glucagon from aqueous solution to a hydrophobic surface and to compare the effects of PEGylation with a linear and a branched PEG chain, respectively. The 3483 Da peptide glucagon was PEGylated with a 2.2 kDa linear and a branched PEG chain, respectively, and the adsorption behaviors of the three proteins were compared using isothermal titration calorimetry, fixed-angle optical reflectometry and total internal reflection fluorescence. PEGylation decreased the number of glucagon molecules adsorbing per unit surface area and increased the initial adsorption rate of glucagon. Furthermore, the results indicated that the orientation and/or structural changes of glucagon upon adsorption were affected by the PEGylation. Finally, from the isothermal titration calorimetry and the reflectometry data, it was observed that the architecture of the PEG chains had an influence on the observed heat flow upon adsorption as well as on the initial rate of adsorption, respectively.
Keywords: PEGylation; Protein adsorption; Glucagon; Isothermal titration calorimetry; Fixed-angle optical reflectometry; Total internal reflection fluorescence;

Correlation between in vitro and in vivo erosion behaviour of erodible tablets using gamma scintigraphy by Manish Ghimire; Lee Ann Hodges; Janet Band; Blythe Lindsay; Bridget O’Mahony; Fiona J. McInnes; Alexander B. Mullen; Howard N.E. Stevens (148-157).
Effect of manufacturing methods on in vitro and in vivo erosion profiles of wax-disintegrant based matrix tablets using gamma scintigraphy. In vitro and in vivo erosion behaviour of erodible tablets consisting of glyceryl behenate and low-substituted hydroxypropylcellulose manufactured using three different methods: direct compression (DC), melt granulation (MG) and direct solidification (DS) was investigated. In vitro erosion behaviour was studied using gravimetric and scintigraphic methods. For scintigraphic investigations, the radiolabel was adsorbed onto activated charcoal and incorporated into tablets at a concentration that did not affect the erosion profile. A clinical study was carried out in six healthy volunteers using gamma scintigraphy. Tablet erosion was affected by the preparation method and was found to decrease in the order of preparation method, DC > MG > DS tablets. The mean in vivo onset time for all tablets (DC: 6.7 ± 3.8 min, MG: 18.3 ± 8.1 min, DS: 67 ± 18.9 min) did not differ significantly from in vitro onset time (DC: 5.3 ± 1 min, MG: 16.8 ± 3.9 min, DS: 61.8 ± 4.7 min). The mean in vivo completion times were found to be 36.6 ± 9.7 (DC tablets), 70 ± 18.3 min (MG tablets) and 192.5 ± 39.9 min (DS tablets). Among the three different erodible tablets, MG tablets showed the highest correlation between in vitro and in vivo mean erosion profile and suggested a potential platform to deliver controlled release of water-insoluble compounds.
Keywords: Glyceryl behenate; Low-substituted hydroxypropylcellulose; Granulation method; Erosion; Gamma scintigraphy; In vitro/in vivo correlation (IVIVC);

Experimental setup.The aim of this study was to evaluate the suitability of Raman spectroscopy as a Process Analytical Technology (PAT) tool for the in-line determination of the active pharmaceutical ingredient (API) concentration and the polymer–drug solid state during a pharmaceutical hot-melt extrusion process.For in-line API quantification, different metoprolol tartrate (MPT) – Eudragit® RL PO mixtures, containing 10%, 20%, 30% and 40% MPT, respectively, were extruded and monitored in-line in the die using Raman spectroscopy. A PLS model, regressing the MPT concentrations versus the in-line collected Raman spectra, was developed and validated, allowing real-time API concentration determination. The correlation between the predicted and real MPT concentrations of the validation samples is acceptable (R 2  = 0.997). The predictive performance of the calibration model is rated by the root mean square error of prediction (RMSEP), which is 0.59%.Two different polymer–drug mixtures were prepared to evaluate the suitability of Raman spectroscopy for in-line polymer–drug solid state characterization. Mixture 1 contained 90% Eudragit® RS PO and 10% MPT and was extruded at 140 °C, hence producing a solid solution. Mixture 2 contained 60% Eudragit® RS PO and 40% MPT and was extruded at 105 °C, producing a solid dispersion. The Raman spectra collected during these extrusion processes provided two main observations. First, the MPT Raman peaks in the solid solution broadened compared to the corresponding solid dispersion peaks, indicating the presence of amorphous MPT. Second, peak shifts appeared in the spectra of the solid dispersion and solid solution compared to the physical mixtures, suggesting interactions between Eudragit® RS PO and MPT, most likely hydrogen bonds. These shifts were larger in the spectra of the solid solution. DSC analysis confirmed these Raman solid state observations and the interactions seen in the spectra. Raman spectroscopy is a potential PAT-tool for in-line determination of the API concentration and the polymer–drug solid state during pharmaceutical hot-melt extrusion.
Keywords: Raman spectroscopy; Hot-melt extrusion (HME); Process Analytical Technology (PAT); In-line measurements; Solid dispersions; Solid solutions;

Increased compactibility of acetames after roll compaction by Theresia Kuntz; Martin A. Schubert; Peter Kleinebudde (164-169).
For levetiracetam as a representative of acetames, increase in crushing force of tablets was accompained by increasing surface areas of the different processed starting materials.A common technique for manufacturing granules in a continuous way is the combination of roll compaction and subsequent milling. Roll compaction can considerably impact tableting performance of a material. The purpose of this study was to investigate the influence of roll compaction/dry granulation on the compaction behavior of acetames, a class of active pharmaceutical substances, which are mainly used for the treatment of central nervous diseases. Some representatives of acetames were roll compacted and then compressed into tablets. Compactibility of granules was compared with the compaction behavior of the directly compressed drug powders. In contrast to many other materials, the roll compaction step induced an increase in compactibility for all investigated acetames. Specific surface areas of the untreated and the roll compacted drugs were determined by nitrogen adsorption. The raise in compactibility observed was accompanied by an increase in specific surface area during roll compaction.
Keywords: Dry granulation; Roll compaction; Crushing force; Compactibility; Specific surface area; Acetames;

Solid lipid extrusion with small die diameters – Electrostatic charging, taste masking and continuous production by Rieke Witzleb; Venkata-Rangarao Kanikanti; Hans-Jürgen Hamann; Peter Kleinebudde (170-177).
A continuous solid lipid extrusion process from powder mixture to post-processed lipid pellets was established. For the first time lipid extrudates with a diameter of 0.2 mm were produced. Furthermore, electrostatic phenomena during solid lipid extrusion were investigated and a taste masked formulation with the bitter tasting drug praziquantel was developed.The aim of this work was to develop a continuous solid lipid extrusion process that includes post-process milling of the extrudates. Die diameters smaller than 0.5 mm should be used for taste masking of the bitter tasting anthelmintic praziquantel. During lipid extrusion with small die diameters, electrostatic charging of the extrudates occured. This could be avoided by adding liquid polyethylene glycol (PEG) as antistatic agent. Further, extrusion with PEG as antistatic agent was possible with small diameter down to 0.2 mm and with up to 80% praziquantel load. Dissolution of praziquantel extrudates was shown to be faster with smaller extrudate diameter due to surface enlargement. Anyhow, different praziquantel extrudates with small diameter, drug load up to 70% and PEG content up to 20%, were proven to be sufficiently taste masked in a randomised palatability study with 40 cats. Within a scale-up experiment, lipid extrusion and milling of the extrudates in a centrifugal mill afterwards were conducted continuously. Extrudates from continuous and batchwise production revealed small differences in terms of size distribution and surface habit, but were similar in drug dissolution rate.
Keywords: Continuous process; Solid lipid extrusion; Small die diameter; Electrostatic charging; Taste masking; Milling;

Stable dry powder inhaler formulation of tranilast attenuated antigen-evoked airway inflammation in rats by Yohei Kawabata; Yosuke Aoki; Takuya Matsui; Kiyoshi Yamamoto; Hideyuki Sato; Satomi Onoue; Shizuo Yamada (178-181).
Inhaled nanocrystalline solid dispersion formulation of tranilast (CSD/TL-RP) suppresses antigen-induced pulmonary injury in experimental asthma/COPD/model, but the physical mixture (PM/TL-RP) did not.Tranilast (TL) has been clinically used for the treatment of airway inflammatory diseases, although the clinical use of TL is limited because of its poor solubility and systemic side effects. To overcome these drawbacks, a novel respirable powder of TL (CSD/TL-RP) for inhalation therapy was developed using nanocrystal solid dispersion of TL (CSD/TL). Stability study on CSD/TL-RP was carried out with a focus on inhalation performance. Even after 6 months of storage at room temperature, there were no significant morphological changes in micronized particles on the surface of carrier particles as compared with that before storage. Cascade impactor analyses on CSD/TL-RP demonstrated high inhalation performance with emitted dose and fine particle fraction (FPF) of ca. 98% and 60%, respectively. Long-term storage of CSD/TL-RP resulted in only a slight decrease in FPF value (ca. 54%). Inhaled CSD/TL-RP could attenuate antigen-induced inflammatory events in rats, as evidenced by marked reduction of granulocytes in bronchoalveolar lavage fluid and inflammatory biomarkers such as eosinophil peroxidase, myeloperoxidase, and lactate dehydrogenase. These findings were consistent with decreased expression levels of mRNAs for nuclear factor-kappa B and cyclooxygenase-2, typical inflammatory mediators. Given these findings, inhalable TL formulation might be an interesting alternative to oral therapy for the treatment of asthma and other airway inflammatory diseases with sufficient dispersing stability.
Keywords: Tranilast; Solid dispersion; Dry powder inhaler; Dispersibility; Stability;

The establishment of an up-scaled micro-mixer method allows the standardized and reproducible preparation of well-defined plasmid/LPEI polyplexes by Julia Christina Kasper; David Schaffert; Manfred Ogris; Ernst Wagner; Wolfgang Friess (182-185).
A micro-mixer method for the preparation of plasmid/LPEI polyplexesPolyplexes based on linear polyethylenimine (LPEI) and plasmid DNA are known as efficient non-viral gene delivery systems. However, the requirement for freshly prepared complexes prior to administration due to their instability in aqueous suspension poses the risk of batch-to-batch variations. Therefore, the aim of the study was the establishment of a reproducible and up-scalable method for the preparation of well-defined polyplexes.Polyplexes consisting of pCMVLuc plasmid and 22 kDa linear polyethylenimine (LPEI) were prepared by classical pipetting or with a micro-mixer method using different mixing speeds and plasmid DNA concentrations (20–400 μg/mL). The z-average diameter of the polyplexes was measured by dynamic light scattering. Metabolic activity and transfection efficiency was evaluated on murine neuroblastoma cells after transfection with polyplexes.When varying mixing speeds of the micro-mixer, polyplex size (59–197 nm) and polydispersity index (0.05–0.19) could be directly controlled. The z-average diameter (65–170 nm) and polydispersity index (0.05–0.22) of the polyplexes increased with increasing plasmid DNA concentration (20–400 μg/mL).The established up-scaled micro-mixer method allows the standardized and reproducible preparation of well-defined, transfection-competent plasmid/LPEI polyplexes with high reproducibility.
Keywords: Non-viral gene delivery; Well-defined polyplexes; Up-scaled preparation; Influence of mixing speed; Influence of concentration;

Corrigendum to “Controlling the functional performance of emulsion-based delivery systems using multi-component biopolymer coatings” by Yan Li; Min Hu; Hang Xiao; Yumin Du; Eric Andrew Decker; David Julian McClements (186).