International Journal of Pharmaceutics (v.452, #1-2)

Drug disposition: From cradle to cane by Rosalinde Masereeuw (1-2).

Profound changes in drug metabolizing enzyme expression occurs during development that impacts drug efficacy and the risk of adverse events in the neonate and young child. A review of our current knowledge suggests individual hepatic drug metabolizing enzymes can be categorized into one of three classes based on developmental trajectories. The time frame for the perinatal changes observed for both Class 1 and Class 3 enzymes varies considerably between different enzymes. However, for a given enzyme, significant interindividual variation is observed in the timing of the perinatal changes, creating windows of hypervariability. Genetic variation clearly impacts drug disposition in children. However, developmental factors can dominate pharmacogenetic factors. Thus, a major challenge in applying pharmacogenomics to improve pediatric drug safety is determining at what age functional genetic variants identified in adults become a major determinant of expression in children. Developmental and genetic data on drug metabolizing enzyme ontogeny, as well as age-dependent changes in other physiological factors impacting drug disposition, can be integrated into physiologically-based pharmacokinetic models. Such models have proven useful in predicting the range of expected metabolic capacities at a given age.
Keywords: Human hepatic drug metabolizing enzymes; Early-life stages; Ontogeny; Cytochrome P450; Flavin-containing monooxygenase; Glutathione S-transferase;

Drug dosing in infants should be based on their physiological characteristics and the pharmacokinetic and -dynamic profile of the compound. Since maturational physiological changes are most prominent in infancy, variability is the key feature of clinical pharmacology in infancy: developmental physiology drives developmental pharmacology. This is illustrated by the link between renal physiology and renal drug clearance and between hepatic physiology and hepatic drug elimination for some specific compounds. However, the maturational profiles of the individual elimination processes differ substantially at the enzyme and transporter level. This implies that it is important to integrate all ontogeny-related knowledge of the different elimination routes to predict compound specific, phenotypic in vivo observations of infancy.In addition to the introduction of already available in vivo observations to validate mechanistic (estimated to in vivo observations) or physiology based pharmacokinetic (PBPK, developmental physiology related estimated to in vivo observations) models, a simultaneous use of both approaches (mechanistic and PBPK) to search for discrepancies between both approaches may also unveil ‘missing’ links in maturational physiology or clinical pharmacology (e.g. ontogeny renal or hepatic drug transporters): developmental pharmacology drives developmental pharmacology.
Keywords: Developmental pharmacology; Maturation; Variability; Covariates; Newborn; Infant;

From gut to kidney: Transporting and metabolizing calcineurin-inhibitors in solid organ transplantation by Noël Knops; Elena Levtchenko; Bert van den Heuvel; Dirk Kuypers (14-35).
Since their introduction circa 35 years ago, calcineurin-inhibitors (CNI) have become the cornerstone of immunosuppressive therapy in solid organ transplantation. However, CNI's possess a narrow therapeutic index with potential severe consequences of drug under- or overexposure. This demands a meticulous policy of Therapeutic Drug Monitoring (TDM) to optimize outcome. In clinical practice optimal dosing is difficult to achieve due to important inter- and intraindividual variation in CNI pharmacokinetics. A complex and often interdependent set of factors appears relevant in determining drug exposure. These include recipient characteristics such as age, race, body composition, organ function, and food intake, but also graft-related characteristics such as: size, donor-age, and time after transplantation can be important. Fundamental (in vitro) and clinical studies have pointed out the intrinsic relation between the aforementioned variables and the functional capacity of enzymes and transporters involved in CNI metabolism, primarily located in intestine, liver and kidney. Commonly occurring polymorphisms in genes responsible for CNI metabolism (CYP3A4, CYP3A5, CYP3A7, PXR, POR, ABCB1 (P-gp) and possibly UGT) are able to explain an important part of interindividual variability. In particular, a highly prevalent SNP in CYP3A5 has proven to be an important determinant of CNI dose requirements and drug-dose-interactions. In addition, a discrepancy in genotype between graft and receptor has to be taken into account. Furthermore, common phenomena in solid organ transplantation such as inflammation, ischemia- reperfusion injury, graft function, co-medication, altered food intake and intestinal motility can have a differential effect on the expression enzymes and transporters involved in CNI metabolism. Notwithstanding the built-up knowledge, predicting individual CNI pharmacokinetics and dose requirements on the basis of current clinical and experimental data remains a challenge.
Keywords: Calcineurin-inhibitors; Tacrolimus; Cyclosporine; CYP3A5; CYP3A4; P glycoprotein;

Regulation of multidrug resistance protein 2 (MRP2, ABCC2) expression by statins: Involvement of SREBP-mediated gene regulation by Masaki Kobayashi; Keisuke Gouda; Ikumi Chisaki; Koji Asada; Jiro Ogura; Natsuko Takahashi; Toru Konishi; Yusuke Koshida; Shotaro Sasaki; Hiroaki Yamaguchi; Ken Iseki (36-41).
Multidrug resistance protein 2 (MRP2, ABCC2) is localized to the apical membrane of hepatocytes and played an important role in the biliary excretion of a broad range of endogenous and xenobiotic compounds and drugs, such as pravastatin. However, the effects of statins on MRP2 in the liver and the precise mechanisms of their actions have been obscure. The goal of this study was to determine the regulatory molecular mechanism for statin-induced MRP2 expression in hepatocytes. In vitro and in vivo studies suggested that pitavastatin increased MRP2 expression. Pitavastatin promoted liver X receptor (LXR) α/β translocation from the cytosol to nuclei, resulting in LXR activation. Deletion and mutational analysis suggested that the potential sterol regulatory element (SRE) played a major role in the observed modulation of MRP2 expression by pitavastatin. Furthermore pitavastatin increased the protein–DNA complex, and when SRE was mutated, stimulation of the protein–DNA complex by pitavastatin was decreased. It was demonstrated that pitavastatin upregulated MRP2 expression by an SREBP regulatory pathway in hepatocytes and that the actions of statins may lead to improve the biliary excretion of MRP2 substrates.
Keywords: Statin; Hepatocytes; Multidrug resistance protein 2; Sterol regulatory element-binding protein;

Display OmittedThe 3D hydrodynamics and shear rates distributions within the United States Pharmacopeia Apparatus 2 have been investigated in this paper. With the help of a CFD package, several geometric modifications to the device were evaluated in this study. Specially, we examine the influence of impeller clearance, blade diameter, shape of the vessel base and shape of the lower part of blade. Increasing the impeller clearance was observed to exacerbate the heterogeneity in shear and would likely result in greater variability in dissolution measurements. Use of moderate blade diameter and dished bottom were observed to reduce shear heterogeneity in the regions where tablets are most likely to visit during testing. The comparative analysis shows better reproducibility and accelerated dissolution rates with the modified vessel shape, the dished bottom can enhance mixing near the vessel base when compared with the flat bottom. Increasing length of the lower edge of the paddle was observed to generate high radial pumping and to enlarge the dead zone located at the center of the vessel base.
Keywords: USP II; Hydrodynamics; Shear rates; Computational fluid dynamics (CFD); Dissolution; Modeling;

Screening for stability and compatibility conditions of recombinant human epidermal growth factor for parenteral formulation: Effect of pH, buffers, and excipients by Héctor Santana; Yaima González; Patricia Targon Campana; Jesús Noda; Odalys Amarantes; Rosangela Itri; Alejandro Beldarraín; Rolando Páez (52-62).
A successful parenteral formulation can be developed by studying stability and compatibility of biopharmaceuticals as a function of solution composition. Here, we evaluate the influence of pH, buffers, ionic strength, protein concentration and presence of excipients on recombinant human epidermal growth factor (rhEGF) stability. The stability was accessed by reversed-phase high performance liquid chromatography (RP-HPLC), size exclusion chromatography (SEC-HPLC), enzyme-linked immunosorbent assay (ELISA), Far-UV circular dichroism (CD) and light scattering. The overall maximal stability was obtained in pH near to 7.0 in phosphate, Tris and histidine buffers as the results of the different methods revealed. The CD results revealed that this protein is stable in an extensive pH range. Aggregation of rhEGF was minimized at pH values ranged from 6.0 to 8.0 as indicated the SEC-HPLC and light scattering results. Nor the ionic strength neither the rhEGF concentration had significant effect on the reaction rate constants. Most rhEGF–excipient instability occurs among this protein and reducing sugars. Polymers like poly(ethylene glycol) (PEG) and polysorbates increased methionine oxidation. The rhEGF oxidation and deamidation were the most common degradation pathways. This research identified critical solution factors to be considered for the development of a successful rhEGF parenteral formulation.
Keywords: Epidermal growth factor; Solution stability; Excipients compatibility; Formulation;

Application of in-line near infrared spectroscopy and multivariate batch modeling for process monitoring in fluid bed granulation by Ravikanth Kona; Haibin Qu; Robert Mattes; Bela Jancsik; Raafat M. Fahmy; Stephen W. Hoag (63-72).
Fluid bed is an important unit operation in pharmaceutical industry for granulation and drying. To improve our understanding of fluid bed granulation, in-line near infrared spectroscopy (NIRS) and novel environmental temperature and RH data logger called a PyroButton® were used in conjunction with partial least square (PLS) and principal component analysis (PCA) to develop multivariate statistical process control charts (MSPC). These control charts were constructed using real-time moisture, temperature and humidity data obtained from batch experiments. To demonstrate their application, statistical control charts such as Scores, Distance to model (DModX), and Hotelling's T2 were used to monitor the batch evolution process during the granulation and subsequent drying phase; moisture levels were predicted using a validated PLS model. Two data loggers were placed one near the bottom of the granulator bowl plenum where air enters the granulator and another inside the granulator in contact with the product in the fluid bed helped to monitor the humidity and temperature levels during the granulation and drying phase. The control charts were used for real time fault analysis, and were tested on normal batches and on three batches which deviated from normal processing conditions. This study demonstrated the use of NIRS and the use of humidity and temperature data loggers in conjunction with multivariate batch modeling as an effective tool in process understanding and fault determining method to effective process control in fluid bed granulation.
Keywords: Fluid-bed; Near-infrared spectroscopy; Process control chart; PyroButton®; Scores; Hotelling's T2;

Development of amorphous solid dispersion formulations of a poorly water-soluble drug, MK-0364 by S. Sotthivirat; C. McKelvey; J. Moser; B. Rege; W. Xu; D. Zhang (73-81).
The goal of this study was to demonstrate that MK-0364 solid dispersions can be developed as a means to increase the solubility and bioavailability of a poorly water-soluble drug, MK-0364. The potential solid dispersions would enable an oral solid dosage form as a monotherapy or combination product of MK-0364. Preliminary screening included sample preparation via a solvent casting method, physical characterization, and in vitro dissolution testing. Lead formulations were subsequently manufactured using hot melt extrusion (HME) and spray-drying (SD). All HME (without polyvinyl pyrrolidone) and SD formulations exhibit characteristics of a single phase glass including an amorphous halo when analyzed with X-ray powder diffraction (XRPD), a single glass transition temperature (T g) measured with differential scanning calorimetry (DSC), and supersaturation when dissolved in dissolution media. The oral absorption of MK-0364 from selected HME and SD formulations in monkeys results in marginally greater exposure with a consistently longer T max relative to a liquid filled capsule reference. Based on the processability, physical characterization, in vitro dissolution, and animal pharmacokinetic results, copovidone- and hydroxypropyl methylcellulose acetate succinate (HPMCAS)-based solid dispersion formulations are viable product concepts. The physical stability of both the solid dispersion formulations was also evaluated for 54 weeks under different conditions. The copovidone-based solid dispersion requires protection from moisture.
Keywords: MK-0364; Copovidone; HPMCAS; Solid dispersion; Hot melt extrusion; Spray drying;

The focus of the study was to explore the physical–mechanical properties of curing films and use these properties as a reference for models that relate NIR spectra to the extent of curing. Cast films were cured at 40, 50, and 60 °C from 1 to 48 h before being scanned on an NIR Spectrometer and analyzed for physical–mechanical properties using an Instron® material testing system. The studies show clear dependence of the physical–mechanical properties on the time and temperature used for curing. Principal component analysis (PCA) and parallel factor analysis (PARAFAC) were performed to investigate the effect of curing on the films. Both PCA and PARAFAC analysis of the NIR spectra showed that spectral features could be directly related to the changes associated with the triethyl citrate (TEC), which directly affected the physical–mechanical properties during curing. Partial least squares (PLS) models were developed that related NIR spectra to the physical–mechanical properties. The use of the Young's Modulus as a reference for NIRS model development resulted in good prediction and was determined to be the best reference for model development. This study demonstrated the NIRS could predict the Young's Modulus which may be used to indicate the extent of curing.
Keywords: Polymer film; Curing; Near infrared (NIR) spectroscopy; Mechanical properties; Elastic modulus;

Analysis of interplay between collagen porous membrane and poly(N-isopropyl acrylamide-co-diethylene glycol diacrylate) was made in order to use the interpenetrated semi-synthetic network as matrix for bioactive compounds. SEM and NIR-CI morphological view and characterization evidenced the homogeneity of the Norfloxacin distribution into the polymeric template.The study is devoted to the characterization of the semi-interpenetrating polymeric network (semi-IPN) structures, prepared as dual sensitive networks, based on poly(N-isopropyl acrylamide-co-diethylene glycol diacrylate) inserted into a collagen porous membrane with potential biomedical-applications. The pharmaceutical applications are related to the possibility of using the semi-synthetic networks for inclusion, retention, transportation and release of drug molecules. The insertion and the homogeneity distribution of the drug into the polymeric network were evaluated by near infrared-chemical imaging (NIR-CI) technique. The drug release was investigated from the kinetically viewpoint in simulated biological environment by using UV–vis spectrophotometric technique. The zeta potential measurement results showed meaningful change of the electric potential of the network surface at the interfacial double layer with the environment in the interdependence with the network composition and environment characteristics. The biodegradable character of the semi-synthetic network, also presented, undergoes with tissue engineering request for achievement of tissue substituents. Texture analysis of the semi-IPN was realized in order to evidence the potential applications of the prepared compounds in tissue engineering. The adhesion properties reveal the possibility to control the surface adhesion by: network composition, the ratio between the polymer types, and the crosslinking degree of polymeric networks. The evaluation of the semi-IPN characteristics in medical terms, concerned the surface electrical charge, the loading, retention and release properties of an active compound, the adhesion properties and the effect of collagenase enzyme over the collagen fibres as component in semi-IPN, and from the pharmaceutical terms emphasizes the potential applications of the new polymeric semi-IPN networks.
Keywords: Biodegradability; Zeta potential; Drug release; NIR-CI characterization; Texture analysis; Medical and pharmaceutical applications;

Interaction of bioactive glass with clodronate by Kirsi Rosenqvist; Sari Airaksinen; Sara J. Fraser; Keith C. Gordon; Anne Mari Juppo (102-107).
Bone tissue engineering is a rapidly growing area of research involving the use of bioactive glass (BG) alone and in combination with different materials. The objective of this study was to investigate the interaction of BG with clodronate. Characterisation of the interaction between BG and clodronate was undertaken using; scanning electron microscopy (SEM), X-ray powder diffraction (XRPD), Fourier transform Raman spectroscopy and Fourier transform infrared spectroscopy (FTIR). The interaction was examined in vitro with respect to the ion exchange and surface modification on the surface of the bioactive glass in the combination product. The results showed clear ion exchange enhancement by clodronate. Additionally, this ion exchange was more extensive and long lasting in the combination product than in BG alone. Clodronate promotes the activity of the BG and a calcium clodronate precipitation is formed. It can be assumed that this solid combination could be used in clinical applications. Therefore, it can be concluded that clodronate makes a beneficial environment for BG and could enhance also the apatite formation of BG.
Keywords: Bioactive glass; Clodronate; Calcium clodronate; Physicochemical characterisation; Bioactivity;

A bioactive drug can participate in its self-formulation and the present work demonstrates that this kind of drug-participating catanionic vesicles can be used as a safe and an efficient drug delivery system for sustained drug release.In the present study, the self-assembly properties, aggregation behavior and potential application of mixed samples formed by an active drug (diclofenac sodium, DS) and conventional surfactant (didodecyldimethyl ammonium bromide, DDAB) are investigated with surface tension, transmission electron microscope (TEM), dynamic light scattering (DLS), zeta potential, conductivity, in vitro drug release and hemolytic toxicity measurements. The physicochemical parameters such as critical micelle concentration (CMC), the surface tension at CMC (γ CMC), the maximum surface excess concentration (Γ max) and the minimum area per molecule headgroup at the air/water interface (A min) and degree of counterion binding (β) are obtained from the surface tension and electrical conductivity measurements. The results show that diclofenac sodium can decrease the surface tension of water and aggregate in the aqueous solution when its concentration is large enough. The CMC and γ CMC of the DS/DDAB mixed systems are found to have values between that of individual DS and DDAB solutions. TEM and DLS results demonstrate the formation of spherical vesicles in a wide range of the molar ratio of the two components. The amount of charge on the vesicles and their stability can be tuned by controlling the amount of drug and surfactant. To evaluate the potential use of the as-prepared DS/DDAB catanionic vesicles in drug delivery systems, the in vitro drug release and hemolytic toxicity are carried out. The results indicate that both the drug release behavior and the hemolytic toxicity are dependent on the composition of the samples, X 1 (X 1  =  n DS/n (DS+DDAB), decreasing with the decrease of X 1. The results of this work suggested that the drug-participating catanionic vesicles can be used as a safe and an efficient vehicle for sustained drug release
Keywords: Diclofenac sodium; Didodecyldimethyl ammonium bromide; Drug-participating catanionic vesicles; Sustained drug release; Hemolytic toxicity;

Involvement of cholesterol depletion from lipid rafts in apoptosis induced by methyl-β-cyclodextrin by Risako Onodera; Keiichi Motoyama; Ayaka Okamatsu; Taishi Higashi; Ryusho Kariya; Seiji Okada; Hidetoshi Arima (116-123).
Methyl-β-cyclodextrin (M-β-CyD), which is widely used as a lipid rafts disrupting agent, is known to induce cytotoxicity at high concentration. In the present study, we investigated the potential of M-β-CyD as an antitumor drug. M-β-CyD markedly caused apoptotic cell-death in KB cells, a human oral squamous carcinoma cell line, Ihara cells, a highly pigmented human melanoma cell line, and M213 cells, a human cholangiocarcinoma cell line, through cholesterol depletion in cell membranes. The DNA content and mitochondrial transmembrane potential in KB cells were significantly decreased after treatment with M-β-CyD. Additionally, M-β-CyD elevated the caspase-3/7 activity in KB cells. Meanwhile, M-β-CyD did not induce the formation of autophagic vacuoles in KB cells. M-β-CyD drastically inhibited the tumor growth after intratumoral injection to Colon-26 cells-bearing mice. These results strongly suggest that M-β-CyD induced apoptosis in tumor cells and had the potential a novel antitumor agent and/or its lead compound.
Keywords: Apoptosis; Methyl-β-cyclodextrin; Cholesterol; Antitumor agent;

The effect of ultraviolet radiation on the anti-inflammatory effect of filters by C. Couteau; O. Couteau; C. Chauvet; E. Paparis; L.J.M. Coiffard (124-127).
A certain number of filters have notable anti-inflammatory properties with percentage inhibition of PMA-induced edema in mice at over 70%. The question arose as to whether this effect was likely to continue after UV irradiation. It can be noted that 7 filters retain an equivalent anti-inflammatory effect before and after 2 h of irradiation in a Suntest device (650 W/m2). For 9 filters, the anti-inflammatory effect decreases and for 5 filters, the anti-inflammatory effect increases. Various behaviors should be noted. 3 groups of substances can be distinguished: such as phenylbenzimidazole sulfonic acid which loses its anti-inflammatory character after irradiation (the percentage inhibition falls from 80 to 44%), oxybenzone which retains a constant anti-inflammatory character (89% inhibition before and after irradiation and also octyl methoxycinnamate which becomes very anti-inflammatory (with a percentage inhibition of 93%). The same phenomenon is observed in the case of commercial products. This should be made known as it can have a considerable impact on the results which are displayed on the packaging of sun products.
Keywords: Sun products; SPF; Anti-inflammatory effect; UV filters; Suntest;

Controlled delivery of naltrexone by an intraoral device: In vivo study on human subjects by Carlo Paderni; Giuseppina Campisi; Axel Schumacher; Thorsten Göttsche; Libero Italo Giannola; Viviana De Caro; Andy Wolff (128-134).
Naltrexone is widely used in the treatment of opiate addiction but its current peroral administration is characterized by low bioavailability with various side effects. The development of a long-acting transbuccal delivery device (IntelliDrug) for NLX may be useful to improve patient compliance and the therapy effectiveness.The aims of the study are (a) to test basic safety and effectiveness of controlled transbuccal drug delivery on human subjects; (b) to compare NLX bioavailability following transbuccal delivery vs per os conventional delivery; and (c) to test the hypothesis that transbuccal delivery is more efficient than the conventional route. In this randomized cross-over pilot study, 12 healthy subjects received in a different order 2 types of NLX administration, per os or transbuccal delivery, based on which group they were randomized to. For per os administration 50 mg NLX tablets were used, while for transbuccal administration, a NLX-loaded prototype of the IntelliDrug device was fixed on patients’ dental arch. Serial blood samples were drawn and analysed for the NLX concentration. The IntelliDrug prototype functioned properly and it did not exert any adverse side-effect. The transbuccal route resulted in administration efficiency 4–17 times higher than conventional per os route. Transbuccal delivery of NLX appears to be a more efficient drug administration route compared to peroral one. It allows to reach a given therapeutic blood level using a small drug dose.
Keywords: Addiction; Smoking cessation; Naltrexone hydrochloride; Transbuccal drug delivery; Transmucosal delivery;

Poor mechanical properties of crystalline drug particles require wet granulation technique for tablet production which is uneconomical, laborious, and tedious. The present investigation was aimed to improve flow and mechanical properties of racecadotril (RCD), a poorly water soluble antidiarrheal agent, by a crystallo-co-agglomeration (CCA) technique. The influence of various excipients and processing conditions on formation of directly compressible agglomerates of RCD was evaluated. Principal component analysis and Box–Behnken experimental design was implemented to optimize the agglomerates with good micromeritics and mechanical properties.The overall yield of the process was 88–98% with size of agglomerates between 351 and 1214 μm. Further, higher rotational speed reduced the size of agglomerates and disturbed sphericity. The optimized batch of agglomerates exhibited excellent flowability and crushing strength.The optimized batch of RCD agglomerates was characterized by fourier transform infrared spectroscopy, differential scanning calorimetry, powder X-ray diffractometry and gas chromatography which illustrated absence of drug–excipient interaction with minimal entrapment of residual solvent. Hence, it may be concluded that both excipients and processing conditions played a vital role to prepare spherical crystal agglomerates of RCD by CCA and it can be adopted as an excellent alternative to wet granulation.
Keywords: Principal component analysis; Box–Behnken design; Agglomerative hierarchy cluster analysis; Crystallo-co-agglomeration; Racecadotril;

Topical corticosteroid delivery into human skin using hydrofluoroalkane metered dose aerosol sprays by Monica L. Reid; Faiza Benaouda; Rajeshree Khengar; Stuart A. Jones; Marc B. Brown (157-165).
Drug loaded hydrofluoroalkane (HFA) sprays can generate effective pharmaceutical formulations, but a deeper understanding of the manner in which these dynamic systems drive the process of in situ semi-solid dosage form assembly is required. The aim of this study was to investigate the effect of the matrix assembly and composition on drug localisation in human skin. Comparing the characteristics of sprays constituting HFA 134a, ethanol (EtOH), poly(vinyl pyrrolidone) K90, isopropyl myristate (IPM), and poly(ethylene glycol) (PEG) demonstrated that the addition of non-volatile solvents acted to delay EtOH evaporation, control the degree of drug saturation (DS) and enhance the corticosteroid delivery from HFA spray formulations. In a dose matched skin penetration study the HFA sprays containing only EtOH as a co-solvent delivered 2.1 μg BMV (DS 13.5) into the tissue, adding IPM to the EtOH HFA delivered 4.03 μg BMV (DS 11.2), whist adding PEG to the EtOH HFA delivered 6.1 μg BMV (DS 0.3). Compared to commercial cream (delivering 0.91 μg BMV) the EtOH/PEG HFA spray deposited over 6 times (p  < 0.05) more drug into the skin. Post spray deposition characterisation of the semi-solid suggested that the superior performance of the EtOH/PEG HFA spray was a consequence of retarding EtOH evaporation and presenting the drug in an EtOH rich PEG residual phase, which promoted BMV passage through the SC and into epidermis.
Keywords: Supersaturation; Volatile evaporation; HFA spray; Skin delivery; Corticosteroids;

Porous orthopedic steel implant as an antibiotic eluting device: Prevention of post-surgical infection on an ovine model by Marina Gimeno; Pedro Pinczowski; Francisco J. Vázquez; Marta Pérez; Jesús Santamaría; Manuel Arruebo; Lluís Luján (166-172).
Traumatology and orthopedic surgery can benefit from the use of efficient local antibiotic-eluting systems to avoid bacterial contamination of implanted materials. In this work a new percutaneous porous-wall hollow implant was successfully used as a local antibiotic-eluting device both in vitro and in vivo. The implant is a macroporous 316L stainless steel filter tube with a nominal filtration cut-off size of 200 nm with one open end which was used to load the synthetic antibiotic linezolid and an opposite blind end. The antibiotic release kinetics from the device on a simulated biological fluid under in vitro conditions demonstrated an increased concentration during the first five days that subsequently was sustained for at least seven days, showing a kinetic close to a zero order release. Antibiotic-loaded implants were placed in the tibia of four sheep which were trans-surgically experimentally infected with a biofilm forming strain of Staphylococcus aureus. After 7 and 9 days post infection, sheep did not show any evidence of infection as demonstrated by clinical, pathological and microbiological findings. These results demonstrate the capability of such an antibiotic-loaded implant to prevent infection in orthopedic devices in vivo. Further research is needed to assess its possible use in traumatology and orthopedic surgery.
Keywords: Medical device; Steel implant; Orthopedics; Antibiotic; Ovine model;

Solubility enhancement of paclitaxel using a linear-dendritic block copolymer by Zhengyuan Zhou; Antony D’Emanuele; David Attwood (173-179).
The solubilising capacities of micelles of a linear-dendritic copolymer (BE-PAMAM), formed by conjugating the poly(butylene oxide) (B)–poly(ethylene oxide) (E) block copolymer B16E42 (BE) with a G2 PAMAM dendrimer, have been compared with those of the diblock copolymer B16E42 for the anti-cancer drug paclitaxel. The BE-PAMAM copolymer showed a greater solubility enhancement than BE under equivalent conditions. Drug-loading efficiency was improved using a solvent-loading method compared with the conventional solution-loading method. The solubility of paclitaxel was increased 3700-fold by micellar encapsulation in a 2% (w/v) BE–PAMAM copolymer solution at 37 °C using this solubilisation technique. Dynamic light scattering and transmission electron microscopy studies indicated a transition of spherical to worm-like micelles of the BE copolymer induced by the encapsulation of drug molecules. A sustained release of encapsulated drug was observed, with approximately 80% and 60% paclitaxel being released from 2% (w/v) solutions of BE and BE-PAMAM respectively after 24 h of dialysis at 37 °C.
Keywords: Paclitaxel; Dendrimer; Micellisation; Drug solubilisation; Block copolymer; Worm-like micelles;

Determination for dry layer resistance of sucrose under various primary drying conditions using a novel simulation program for designing pharmaceutical lyophilization cycle by Tatsuhiro Kodama; Hiroyuki Sawada; Hiroshi Hosomi; Masahito Takeuchi; Naoki Wakiyama; Etsuo Yonemochi; Katsuhide Terada (180-187).
Dry layer resistance, which is the resistance of dried cake against water vapor flow generated from sublimation, is one of the important parameters to predict maximum product temperature and drying time during primary drying in lyophilization. The purpose of this study was to develop the predictive model of dry layer resistance under various primary drying conditions using the dry layer resistance obtained from a preliminary lyophilization run. When the maximum dry layer resistance was modified under the assumption that the chamber pressure is zero, the modified dry layer resistance, which is defined as specific dry layer resistance, correlated well with the sublimation rate. From this correlation, the novel predictive model including the empirical formula of sublimation rate and specific dry layer resistance is proposed. In this model, the dry layer resistance under various conditions of shelf temperature and chamber pressure was successfully predicted based on the relationship of the sublimation rate and specific dry layer resistance of the edge and center vials obtained from the product temperature in one preliminary cycle run. It is expected that this predictive model could be a practical and useful tool to predict product temperature during primary drying.
Keywords: Dry layer mass transfer resistance; Simulation; Predictive model; Heat and mass transfer model;

Antibacterial properties of cyclodextrin–antiseptics-complexes determined by microplate laser nephelometry and ATP bioluminescence assay by Susanne Finger; Cornelia Wiegand; Hans-Jürgen Buschmann; Uta-Christina Hipler (188-193).
Cyclodextrins (CDs) are able to form inclusion complexes with other molecules, thereby, protecting these guest molecules from degradation, enhancing their biocompatibility or influencing their physiological distribution while retaining their activity. Here, antibacterial effects of CD-complexes with the antiseptics chlorhexidine diacetate (CHX), iodine (IOD) and polihexanide (PHMB) were determined using two different in vitro methods, microplate laser nephelometry and an ATP bioluminescence assay. Laser nephelometry is a direct method for monitoring and evaluating growth of micro-organisms by measurement of the turbidity of the solution. In contrast, the ATP bioluminescence assay determines specifically the amount of metabolic active bacterial cells. The antibacterial effects of CD–antiseptics-complexes were examined for Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis and the results of both methods were compared in respect of calculated means of half maximal inhibitory concentrations (IC50) and statistical evaluated Pearson's correlation coefficients (r). It could be demonstrated that both methods showed a high comparability although they differ in the parameters tested. This study revealed that CD-complexes with CHX and PHMB were most effective against E. coli and the tested staphylococci. While CD–IOD-complexes obtained high activity against K. pneumoniae, P. aeruginosa was distinctly more resistant compared to the other bacteria.
Keywords: Antibacterial; Antiseptic; Bioluminescence; Cyclodextrin-complex; Disinfectant; Laser nephelometry;

Novel alternatives to reduce powder retention in the dry powder inhaler during aerosolization by Desmond Heng; Sie Huey Lee; Wai Kiong Ng; Hak-Kim Chan; Jin Wang Kwek; Reginald B.H. Tan (194-200).
Dry powder inhalers (DPIs) are used predominantly for the treatment of pulmonary diseases by delivering drugs directly into the lungs. The drug delivery efficiency is typically low and there is significant drug retention inside the DPI. An innovative ‘green’ initiative aimed at minimizing drug wastage via channeling the residual drug into the useful inhaled therapeutic fraction was pioneered. Drug retention could be minimized via coating the drug capsule and delivery device with pharmaceutically acceptable force-control agents. This coating reduces the adhesion between the drug particles and the internal surfaces of the DPI, which in turn increases the fine particle dose by as much as 300%.
Keywords: Dry powder inhaler; Capsule; Device; Surface coating; Powder retention; Biomimicry; Force control agents;

The aims of the present study were to characterize the microstructure and study the skin permeation enhancement of formulations containing the alkaloidal extract from Tabernaemontana divaricata. The extract was loaded in the formulations composed of Zingiber cassumunar oil, Triton X-114, ethanol and water with the oil:surfactant ratios of 1:5 and 2:5. The formulations were characterized by photon correlation spectroscopy, polarizing light microscopy, differential scanning calorimetry, and viscosity measurement. A reverse micellar phase, w/o microemulsions, liquid crystalline systems, liquid crystal in microemulsion systems and coarse emulsions were formed along the aqueous dilution line of both oil:surfactant ratios. Formulations with the ratio of 1:5 containing 0.1 μg/ml extract showed a significantly higher acetylcholinesterase inhibition than those with the ratio of 2:5. The skin of stillborn piglet was used in the permeation study. The liquid crystalline and microemulsion systems significantly increased the transdermal delivery of the extract within 24 h. It was concluded that the alkaloidal extract from T. divaricata stem loaded in liquid crystalline or microemulsion systems comprising Z. cassumunar oil/Triton X-114/ethanol/water may act as an alternative percutanous formulations for enhancing the acetylcholine level in Alzheimer's patients.
Keywords: Liquid crystalline; Microemulsion; Alkaloid; Tabernaemontana divaricata; Anticholinesterase activity;

Research of novel biocompatible radiopaque microcapsules for arterial embolization by Xiao-Jing Lu; Yuan Zhang; Dai-Chao Cui; Wen-Jing Meng; Ling-Ran Du; Hai-Tao Guan; Zhuo-Zhao Zheng; Nai-Qi Fu; Tian-Shi Lv; Li Song; Ying-Hua Zou; Wan-Liang Lu; Tian-Yuan Fan (211-219).
After subcutaneous injection to mice and embolization to a rabbit kidney, radiopaque microcapsules were detected clearly by in vivo imaging system and computed tomography (CT), respectively.Embolic agents, such as microparticles, microspheres or beads used in current embolotherapy are mostly radiolucent, which means the agents are invisible under X-ray imaging during and after the process of embolization, and the fate of these particles cannot be precisely assessed. In this research, a radiopaque embolic agent was developed by encapsulating lipiodol in polyvinyl alcohol. The lipiodol-containing polyvinyl alcohol microcapsules (LPMs) were characterized and evaluated for their morphology, size distribution, lipiodol content, lipiodol release, elasticity, and deliverability through catheter. The radiopacity of LPMs in vials and in living mice was both detected by an X-ray imaging system. The biocompatibility of LPMs was investigated with L929 cells and in mice after subcutaneous injection. Embolization of LPMs to a rabbit kidney was performed under digital subtraction angiography (DSA) and the radiopacity of LPMs was verified by computed tomography (CT).
Keywords: Microcapsules; Embolization; X-ray imaging; Lipiodol; Polyvinyl alcohol;

Development of novel solid dispersion of tranilast using amphiphilic block copolymer for improved oral bioavailability by Satomi Onoue; Yoshiki Kojo; Hiroki Suzuki; Kayo Yuminoki; Keitatsu Kou; Yohei Kawabata; Yukinori Yamauchi; Naofumi Hashimoto; Shizuo Yamada (220-226).
The present study aimed to develop novel solid dispersion (SD) of tranilast (TL) using amphiphilic block copolymer, poly[MPC-co-BMA] (pMB), to improve the dissolution and pharmacokinetic behavior of TL. pMB-based SD of TL (pMB-SD/TL) with drug loading of 50% (w/w) was prepared by wet-mill technology, and the physicochemical properties were characterized in terms of morphology, crystallinity, dissolution, and hygroscopicity. Powder X-ray diffraction and polarized light microscopic experiments demonstrated high crystallinity of TL in pMB-SD/TL. The pMB-SD/TL exhibited immediate micellization when introduced to aqueous media, forming fine droplets with a mean diameter of ca. 122 nm. There was marked improvement in the dissolution behavior for the pMB-SD/TL even under acidic conditions, although the supersaturated TL concentration gradually decreased. NMR analyses demonstrated interaction between TL and pMB, as evidenced by the chemical shift drifting and line broadening. Pharmacokinetic behaviors of orally dosed TL formulations were evaluated in rats using UPLC/ESI-MS. After oral administration of pMB-SD/TL (10 mg TL/kg) in rats, enhanced TL exposure was observed with increases of C max and AUC by 125- and 52-fold, respectively, compared with those of crystalline TL. From these findings, pMB-based SD formulation approach might be an efficacious approach for enhancing the therapeutic potential of TL.
Keywords: Tranilast; Solid dispersion; Dissolution; Bioavailability; Crystallinity;

Solvent evaporation/extraction method is widely used for core–shell microspheres fabrication. However, the solvent evaporation rate, as an essential factor for polymers phase separation, is difficult to control which results in failure of complete phase separation between polymers. At the present study, the selective dissolution technique was used to improve the phase separation, and successfully fabricate core–shell microspheres for controlled delivery of drug with reduced initial burst release. The core–shell microspheres were prepared with poly(l-lactic-co-glycolic acid) (PLGA) and poly(l-lactic acid) (PLLA), and aspirin was used as model drug. Ethyl acetate (EtAc) was applied to ameliorate the phase separation during the preparation process. The ratio of dichloromethane (DCM)/EtAc seriously affected the distribution of polymer molecules and the formation of the core–shell structure. The internal morphology of the microspheres varied depending on the amount of EtAc. Core–shell structure with a dense core of PLLA and a shell of PLGA was well formed when 2 ml EtAc was used. The differential scanning calorimeters (DSC) results showed two distinct melting points which confirmed a completely polymer phased separation occurred. These microspheres showed sustained release of aspirin for at least 456 h with a little burst release (3.49%).
Keywords: Core–shell microspheres; Solvent evaporation/extraction method; Burst release; Phase separation;

Controlled release of cyclosporine A self-nanoemulsifying systems from osmotic pump tablets: Near zero-order release and pharmacokinetics in dogs by Xi Zhang; Yueneng Yi; Jianping Qi; Yi Lu; Zhiqiang Tian; Yunchang Xie; Hailong Yuan; Wei Wu (233-240).
It is very important to enhance the absorption simultaneously while designing controlled release delivery systems for poorly water-soluble and poorly permeable drugs (BCS IV). In this study, controlled release of cyclosporine (CyA) was achieved by the osmotic release strategy taking advantage of the absorption-enhancing capacity of self-nanoemulsifying drug delivery systems (SNEDDSs). The liquid SNEDDS consisting of Labrafil M 1944CS, Transcutol P and Cremophor EL was absorbed by the osmotic tablet core excipients (sucrose, lactose monohydrate, polyethylene oxide, and partly pregelatinized starch) and then transformed into osmotic tablets. Near zero-order release could be achieved for CyA-loaded nanoemulsions reconstituted from the SNEDDS. In general, the influencing factor study indicated that the release rate increased with increase of inner osmotic pressure, ratio of osmotic agent to suspending agent, content of pore-forming agent, and size of release orifice, whereas the thickness of the membrane impeded the release of CyA nanoemulsion. Pharmacokinetic study showed steady blood CyA profiles with prolonged T max and MRT, and significantly reduced C max for self-nanoemulsifying osmotic pump tablet (SNEOPT) in comparison with highly fluctuating profiles of the core tablet and Sandimmune Neoral®. However, similar oral bioavailability was observed for either controlled release or non-controlled release formulations. It was concluded that simultaneous controlling on CyA release and absorption-enhancing had been achieved by a combination of osmotic tablet and SNEDDS.
Keywords: Controlled release; Self-nanoemulsifying; Drug delivery; Osmotic tablet; Cyclosporine A;

Gastro-floating tablets of cephalexin: Preparation and in vitro/in vivo evaluation by Lifang Yin; Chao Qin; Kaisheng Chen; Chunli Zhu; Hui Cao; Jianping Zhou; Wei He; Qiang Zhang (241-248).
Gastro-floating tablets of cephalexin were developed to prolong the residence time in major absorption sites. Gastro-floating tablets were prepared and optimized using hydroxypropyl methylcellulose (HPMC K100M) as matrix and sodium bicarbonate as a gas-forming agent. The properties of the tablets in terms of floating lag time, floating time and in vitro release were evaluated. Furthermore, in vivo pharmacokinetic study in fed and fasted beagle dogs was performed. The gastro-floating tablets had short floating lag time and exhibited a satisfactory sustained-release profile in vitro. Compared with conventional capsules, the gastro-floating tablets presented a sustained-release behavior with a relative bioavailability of 99.4%, while the reference sustained-release tablets gave a relative bioavailability of only 39.3%. Meanwhile, the food had significant effect on the pharmacokinetics of sustained-release tablets. It was concluded that the gastro-floating tablets had a sustained-release effect in vitro and in vivo, as well as desired pharmacokinetic properties in both fed and fasted conditions.
Keywords: Cephalexin; Gastro-floating; Sustained-release; Absorption study; Bioavailability;

Quantitative analysis of the layer separation risk in bilayer tablets using terahertz pulsed imaging by Masahiro Niwa; Yasuhiro Hiraishi; Norio Iwasaki; Katsuhide Terada (249-256).
Layer separation is a critical defect in many bilayer tablets. Despite its importance for product quality, few studies have investigated its root cause. We evaluated bilayer tablets with varying layer separation tendencies using terahertz pulsed imaging (TPI) in comparison with other analytical methods such as tensile strength measurements, friability testing, scanning electron microscopy (SEM), and X-ray computed tomography (XRCT). The layer separation risk was determined by friability testing and shown to be correlated with the final compression pressure used for bilayer tablet fabrication. TPI could nondestructively detect cracks between the component layers that lead to layer separation. The adhesion integrity of the interface was quantified by the interface index, a unique value derived from the time-domain terahertz waveform. The interface index showed good correlation to the layer separation tendency and could distinguish interface quality among seven batches of bilayer tablets. In contrast, SEM and XRCT detected structural defects but could not distinguish batches with high or low layer separation risk. TPI revealed the relationship between compression pressure and interface quality. Thus, TPI can aid in quality control by providing a precise estimate of the layer separation risk and robust quality of bilayer tablet development with better understanding of layer separation.
Keywords: Bilayer tablet; Layer-separation; Terahertz pulsed imaging; X-ray CT;

Stabilization of monomeric insulin is a primary requirement for preserving the efficacy of the final formulation. Degraded and/or aggregated protein as well as the presence of any of the conventional excipients can result in immunogenic or anaphylactic reactions, and reduced bioavailability of the protein drug. The aim of this work was to select novel RNA-based stabilizers of bovine insulin which would recognize and bind to the monomeric protein and help retain its bioactivity. RNA aptamers were selected by an in vitro selection method. They were screened for their ability to inhibit insulin fibrillation using agitation as a stress condition. The in vitro activity of insulin was determined by phosphorylation of downstream proteins in the cell. In vivo bioactivity was determined in a diabetic rat model. RNA aptamers, which bound to insulin with very low dissociation constants and high specificity, were selected. These sequences were aligned and consensus regions were found. The RNA sequences had no effect on the signalling cascade initiated by insulin. The bioactivity of insulin, as measured by its ability to lower plasma glucose level in a diabetic rat model, also remained unchanged. RNA aptamers are a novel class of protein stabilizers which have the ability to disrupt protein–protein interactions and hence inhibit protein aggregation. Their non-toxic and non-immunogenic nature makes such formulations safe for use.
Keywords: Aptamer; Diabetes; Fibrillation; Insulin; Protein stabilization;

Controlled release of morphine from a poloxamer 407 gel by Mark M.P.M. Jansen; Jacques M. Verzijl; David M. Burger; Yechiel A. Hekster (266-269).
Display OmittedTreatment of painful ulcers is discouraging. Topical morphine has been described as a useful therapeutic adjunct in some patients. In the development of a new analgesic product, we studied the in vitro release characteristics of a new topical formulation containing 0.5% (w/w) morphine–HCl in a poloxamer 407 (P407) based gel.A diffusion cell was used for measurement of in vitro release characteristics. The donor compartment (DC) and the receptor compartment (RC) were separated by a 5000 Da cellulose acetate membrane.The morphine–HCl release from this developed P407 based gel followed zero-order kinetics with a constant release of 150 μg cm−2  h−1. Our results support the use of this P407 gel as a sustained release topical formulation in the pharmacological treatment of painful ulcers. Future research welcomes a formulation with release characteristics leading to less frequent application.
Keywords: Morphine; Topical; Controlled release; Poloxamer 407;

We demonstrate a rapidly formed cubic liquid crystalline phase, i.e. typically 1 g cubic phase in less than 1 min confirmed by X-ray diffraction, consisting of an ether lipid, 1-glyceryl monooleyl ether (GME), an aprotic solvent (propylene glycol or pentane-1,5-diol) and water. The efficacy of the cubic formulation was tested in vivo by administrating formulations containing 3% (w/w) of the HCl salts of δ-aminolevulinic acid (ALA) or methylaminolevulinate (MAL) to hairless mice. The endogenous formation of protoporphyrin IX (PpIX) was monitored spectrophotometrically as a marker for cellular uptake of active compound. As reference, a commercial product containing 16% (w/w) MAL in an oil-in-water emulsion (Metvix®), and a cubic phase based on an ester lipid (glyceryl monooleate, GMO), previously shown to facilitate topical delivery of both ALA and MAL, were applied. It was found that in general the cubic phases gave rise to higher fluorescence levels than the mice exposed to the commercial product. The instantly formed cubic formulations based on GME demonstrated the same efficiency as the GMO based formulations. The results imply that instantly formed cubic formulations opens up new opportunities, particularly for transdermal drug delivery of substances subject to stability problems in, e.g. aqueous environments.
Keywords: Instantly formed cubic phase; Topical delivery; Glyceryl monooleyl ether; Glyceryl monooleate; Aminolevulinic acid; Methylaminolevulinate;

Mucus can change the permeation rank order of drug candidates by Ellen Hagesaether; Elisabeth Christiansen; Maria Elisabeth Due-Hansen; Trond Ulven (276-282).
The aim of the study was to test the effect of mucus on the permeability of newly developed structurally related free fatty acid receptor 1-agonists TUG-488, TUG-499 and TUG-424, which were compared to the more hydrophilic ketoprofen and the more hydrophobic testosterone as reference drugs. The model membrane was cell monolayers consisting of methotrexate treated HT29 cells grown for approximately one, two or three weeks, and thereby differing in the amount of goblet cells and hence mucus.The results show that the permeation of all compounds was high and that mucus in most cases only had a minor influence. However, for one of the drug candidates, TUG-499, mucus had a clear impact, and this could not be explicitly related to the hydrophobicity of this compound. Secreted mucus thus changed the obtained rank order of permeation. This was especially apparent when the experiments were carried out at a lower agitation. These results indicate that an experimental system without mucus can give a faulty rank order of permeation compared to mucous membranes when structurally related drug candidates are tested.
Keywords: Permeability; Mucus; HT29-MTX cells;

Engineering poly(ethylene oxide) buccal films with cyclodextrin: A novel role for an old excipient? by Agnese Miro; Ivana d’Angelo; Antonella Nappi; Pietro La Manna; Marco Biondi; Laura Mayol; Pellegrino Musto; Roberto Russo; Maria Immacolata La Rotonda; Francesca Ungaro; Fabiana Quaglia (283-291).
Inspired by the multiple roles cyclodextrins can play in polymeric systems, here we engineered poly(ethylene oxide) (PEO) films with (2-hydroxypropyl)-β-cyclodextrin (CD) as multipurpose ingredient. To shed light on the potential of CD in formulating PEO buccal films for the delivery of poorly water-soluble drugs, we preliminarily assessed thermal and mechanical properties as well as wettability of films prepared at different PEO/CD ratios. PEO/CD platform containing 54% by weight of CD was chosen as the optimized composition since it matched acceptable mechanical properties, in terms of tensile strength and elasticity, with a good wettability. The platform was tested as buccal delivery system for triamcinolone acetonide (TrA), a lipophilic synthetic corticosteroid sparely water soluble. Confocal Raman imaging clearly showed that CD was homogeneously (i.e. molecularly) dispersed in PEO. Nevertheless, homogenous drug distribution in the film without TrA crystallization occurred only in the presence of CD. Finally, CD-containing PEO film placed in simulated buccal fluids provided a useful speed-up of TrA release rate while showing slower dissolution as compared to PEO film. These results, as well as compliance with quality specifications of pharmaceutical manufacturing products, strongly support the soundness of the strategy and prompt toward further applications of PEO/CD films in buccal drug delivery.
Keywords: Buccal films; Cyclodextrins; Poly(ethylene oxide); Polymer properties; Raman spectroscopy;

The shape of nanoparticles in drug targeting applications has been recently suggested as being an interesting parameter for modulating their fate in the body after delivery. In view of investigating the incidence of this parameter on in vivo distribution, poly(γ-benzyl-l-glutamate) (PBLG) has been selected for preparing non-spherical nanoparticles. PBLG is a degradable polymer forming α-helices rods, which can easily self-assemble by nanoprecipitation into oblate-shaped nanoparticles. Varying the molecular weight of the polymer from 28 to 85 kg mol−1 resulted in elongation ratios between 1.5 and 3.5, respectively, while the nanoparticles exhibited a fairly constant particle volume. Their non spherical shape was attributed to an ordered packing of the PBLG rods resulting in a liquid crystal-like self organization of PBLG rods with varying lengths. This set of morphological characteristics makes these nanoparticles a valuable tool for gaining a better understanding of their fate in biological fluids, mechanisms of interactions with cells and in vivo distribution studies
Keywords: Nanoparticles; Shape; Morphology; PBLG; Polypeptides; Self-assembly;

(1) Olanzapine (OZ)-loaded micellar nanocarriers (F11), (2) histopathological assessment of the local toxicity on the sheep nasal mucosa, (3) minor changes in the olfactory epithelium of the sheep nasal mucosa, (4) estimation of 99mTc-OZ biodistribution and pharmacokinetics in the brain and the blood of male Wister albino rats, and (5) Oz concentration in male Wister albino rat brain at different time intervals following administration of intranasal 99mTc-OZ solution, intranasal 99mTc-F11 micellar nanocarriers and intravenous 99mTc-F11 micellar nanocarriers, mean ± S.D., n  = 3.Olanzapine (OZ) is atypical antipsychotic drug that suffers from low brain permeability due to efflux by P-glycoproteins and hepatic first-pass metabolism. The current work aimed to develop OZ-loaded micellar nanocarriers and investigate their nose-to-brain targeting potential. OZ-loaded (5 mg/ml) micelles (F1–F12) were prepared, using a Pluronic® mixture of L121 and P123, adopting thin-film hydration method. The micelles were evaluated for turbidity, particle size, morphology, drug-entrapment efficiency (EE%), drug-loading characteristics, in vitro drug release and ex vivo nasal toxicity in sheep. The in vivo biodistribution and pharmacokinetic studies in the brain/blood following intravenous (i.v.) and intranasal (i.n.) administrations of technetium-labeled OZ-loaded micelles and OZ-solution were evaluated in rats. Spherical micelles ranging in size from 18.97 to 380.70 nm were successfully developed. 1H NMR studies confirmed OZ incorporation into micelle core. At a drug:Pluronic® L121:Pluronic® P123 ratio of 1:8:32 (F11), the micelles achieved a conciliation between kinetic and thermodynamic stability, high drug-EE%, controlled drug-release characteristics and evoked minor histopathological changes in sheep nasal mucosa. The significantly (P  < 0.05) higher values for F11 micelles (i.n.); brain/blood ratio (0.92), drug targeting index (5.20), drug targeting efficiency (520.26%) and direct transport percentage (80.76%) confirm the development of a promising non-invasive OZ-loaded nose-to-brain delivery system.
Keywords: Olanzapine; Polymeric mixed micelles; Radiolabeling; Nose-to-brain delivery; In vivo biodistribution;

A retinyl palmitate-loaded solid lipid nanoparticle system: Effect of surface modification with dicetyl phosphate on skin permeation in vitro and anti-wrinkle effect in vivo by Ho Seong Jeon; Jo Eun Seo; Min Soo Kim; Mean Hyung Kang; Dong Ho Oh; Sang Ok Jeon; Seong Hoon Jeong; Young Wook Choi; Sangkil Lee (311-320).
Surface-modified solid lipid nanoparticles (SLNs) containing retinyl palmitate (Rpal) were prepared by the hot-melt method using Gelucire 50/13® and Precirol ATO5®. Dicetyl phosphate (DCP) was added to negatively charge the surfaces of the SLNs and thereby enhance the skin distribution properties of Rpal. In vitro skin permeation and in vivo anti-aging studies were performed using SLNs dispersed in a hydrogel. The SLNs were under 100 nm in size with an even polydispersity index (PDI), and the high absolute zeta-potential value was sufficient to maintain the colloidal stability of the SLNs. DCP-modified negative SLNs (DCPmod-SLNs) enhanced the skin distribution of Rpal 4.8-fold and delivered Rpal to a greater depth than did neutral SLNs. The in vivo anti-wrinkle effect of the DCPmod-SLN formulation was Rpal dose-dependent. However, the anti-wrinkle effects of the DCPmod-SLN formulations were significantly different from that of the negative control and effectively prevented the reduction of elastin and superoxide dismutase by UV irradiation. In conclusion, the DCPmod-SLN system presented is a good candidate for topical Rpal delivery.
Keywords: Retinyl palmitate; Solid lipid nanoparticles; Surface modification; Dicetyl phosphate; Anti-wrinkle effect;

Artemether has been used for a long time in the treatment of malaria as safe and non expensive drug. It possesses potent anticancer effects in cancer cell lines. Our aim was to develop transferrin-modified-artemether lipid nanospheres as targeted anticancer drug delivery system. In this study, artemether intravenous delivery system was prepared by emulsifying method as lipid nanospheres containing mixture of soya oil and crodamol as the core and soya lecithin and Tween 80 as coating layer. According to the physicochemical characterization, the process and formulation variables were optimized by orthogonal design and ANOVA analysis. Based on the electrostatic interaction, transferrin (TR) was physically adsorbed onto the coating layer; the effect of medium pH and the charge of the nanocarriers on the adsorption were investigated. The in vitro characterizations were carried out including, the zeta potential, AFM, TEM, FTIR, 1H NMR and gel filtration. ART-LNSs with high entrapment efficiency, small size of about 50 nm and monodispersity were formulated. Optimized and stable TR-LNSs, a lipoprotein like structure and size, were produced. We showed a method by which TR can be bound to lipid nanospheres without the need for chemical modification as a base for the development of safe, effective and non expensive anticancer drug delivery system.
Keywords: Artemether; Lipid nanospheres; Transferrin; Physical adsorption;

Electrospun chitosan-based nanofiber mats loaded with Garcinia mangostana extracts by Natthan Charernsriwilaiwat; Theerasak Rojanarata; Tanasait Ngawhirunpat; Monrudee Sukma; Praneet Opanasopit (333-343).
The aim of this study was to prepare electrospun chitosan-based nanofiber mats and to incorporate the fruit hull of Garcinia mangostana (GM) extracts into the mats. Chitosan-ethylenediaminetetraacetic acid/polyvinyl alcohol (CS-EDTA/PVA) was selected as the polymers. The GM extracts with 1, 2 and 3 wt% α-mangostin were incorporated into the CS-EDTA/PVA solution and electrospun to obtain nanofibers. The morphology and diameters of the mats were analyzed using scanning electron microscopy (SEM). The mechanical and swelling properties were investigated. The amount of GM extracts was determined using high-performance liquid chromatography (HPLC). The antioxidative activity, antibacterial activity, extract release and stability of the mats were evaluated. In vivo wound healing tests were also performed in Wistar rats. The results indicated that the diameters of the fibers were on the nanoscale and that no crystals of the extract were observed in the mats at any concentration. The mats provided suitable tensile strength and swelling properties. All of the mats exhibited antioxidant and antibacterial activity. During the wound healing test, the mats accelerated the rate of healing when compared to the control (gauze-covered). The mats maintained 90% of their content of α-mangostin for 3 months. In conclusion, the chitosan-based nanofiber mats loaded with GM extracts were successfully prepared using the electrospinning method. These nanofiber mats loaded with GM extracts may provide a good alternative for accelerating wound healing.
Keywords: Electrospinning; Chitosan; Garcinia mangostana; Wound healing;

Enhanced brain distribution and pharmacodynamics of rivastigmine by liposomes following intranasal administration by Zhen-Zhen Yang; Yan-Qing Zhang; Zhan-Zhang Wang; Kai Wu; Jin-Ning Lou; Xian-Rong Qi (344-354).
Alzheimer’s disease (AD) is a common progressive neurodegenerative disorder associated with cholinergic neurons degeneration. The blood–brain barrier (BBB) not only provides protection for the brain but also hinders the treatment and diagnosis of this neurological disease, because the drugs must cross BBB to reach the lesions. The present work was aimed at formulating rivastigmine liposomes (Lp) and cell-penetrating peptide (CPP) modified liposomes (CPP-Lp) to improve rivastigmine distribution in brain and proceed to enhance pharmacodynamics by intranasal (IN) administration and minimize side effects. The results revealed that Lp especially the CPP-Lp can enhance the permeability across the BBB by murine brain microvascular endothelial cells model in vitro. IN administration of rivastigmine solution and rivastigmine liposomes demonstrated the capacity to improve rivastigmine distribution and adequate retention in CNS regions especially in hippocampus and cortex, which were the regions most affected by AD, than that of IV administration. Importantly, the lagging but intense inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities were relative to the extended release, absorption and retention. In addition, there was very mild nasal toxicity of liposomal formulations. The data suggest that rivastigmine liposomes especially CPP-Lp improve the brain delivery and enhance pharmacodynamics which respect to BBB penetration and nasal olfactory pathway into brain after IN administration, and simultaneously decrease the hepatic first pass metabolism and gastrointestinal adverse effects.
Keywords: Intranasal administration; Liposomes; Cell penetrating peptide (CPP); Biodistribution; Pharmacodynamics; Rivastigmine;

Trans-resveratrol loaded chitosan nanoparticles modified with biotin and avidin to target hepatic carcinoma by Le Bu; Liang-Chun Gan; Xiao-Qiang Guo; Feng-Zheng Chen; Qin Song; Qi-Zhao; Xiao-Jun Gou; Shi-Xiang Hou; Qian Yao (355-362).
Conventional liver targeted system focuses on delivering drugs to liver, bringing toxicity on hepatic normal tissues. The purpose of this study is to construct a new system capable of specially targeting to hepatic carcinoma instead of the whole liver. Based on the fact that nanoparticles (NPs) bound with either biotin or avidin tend to accumulate in tumors and avidin-attached reagents were quickly eliminated from blood circulation and assembled in liver, trans-resveratrol loaded chitosan nanoparticles (CS-NPs), CS-NPs with the surface modified either by biotin (B-CS-NPs) or by both biotin and avidin (A-B-CS-NPs) were prepared and their physiochemical properties were investigated. The in vitro release profiles of the three NPs all conformed to bioexponential equation. Pharmacokinetic experiment indicated that A-B-CS-NPs rapidly assembled in liver after injection, with the highest liver targeting index of 2.70, while the modification of biotin attenuated the liver targeting ability of NPs. Inhibitory study on HepG2 cells declared that compared to trans-resveratrol solution and CS-NPs, both B-CS-NPs and A-B-CS-NPs significantly improved the anticancer activity. When incubated with HepG2 cells at high concentration for longer time, A-B-CS-NPs exhibited superior cytotoxicity than B-CS-NPs. This study exclaims that A-B-CS-NPs may be a potent drug delivery vector specially targeting to hepatic carcinoma.
Keywords: Chitosan nanoparticles; Biotin; Avidin; Target; Hepatic carcinoma; Pharmacokinetics;

Synthesis and characterization of multi-functional linear-dendritic block copolymer for intracellular delivery of antitumor drugs by Hongzhi Qiao; Juan Li; Yang Wang; Qineng Ping; Guangji Wang; Xiaochen Gu (363-373).
A novel amphiphilic linear-dendritic block copolymer, semi-polyamidoamine-b-poly(d,l-lactic acid) (PALA) was synthesized and evaluated for its potential as a drug delivery system in this study. PALA was self-assembled in aqueous solution to form nanomicelles with low critical micelle concentration; antitumor drug docetaxel (DTX) was successfully encapsulated into micelles. The prepared micelles demonstrated pH-induced charge conversion and dimension changes. In vitro drug release suggested susceptibility of DTX-loaded micelles to acidic microenvironment. Hemolysis and cytotoxicity testing also indicated in vitro biocompatibility of PALA. Pharmacokinetic study in rats proved that DTX-loaded PALA micelles enhanced the AUC of DTX and prolonged drug clearance in comparison to conventional DTX injection (Taxotere®). It was concluded that self-assembled micelles from linear-dendritic block copolymer PALA demonstrated potential for intracellular delivery of anticancer molecules, and that its safety and efficacy in chemotherapy should be further studied.
Keywords: Linear-dendritic block copolymers; pH-sensitive; Specific drug targeting; Cellular uptake; Pharmacokinetics;

Bile salts enhance the intestinal absorption of lipophilic drug loaded lipid nanocarriers: Mechanism and effect in rats by Zhiwen Zhang; Fang Gao; Shijun Jiang; Lingli Chen; Zeying Liu; Haijun Yu; Yaping Li (374-381).
Bile salts improve the cellular uptake of lipophilic drug loaded lipid nanocarriers (CLN) in Caco-2 cell monolayers via the active processes and enhance the intestinal absorption through the lymphatic pathway.The purpose of this study was to elucidate the effect and possible mechanism of bile salts on the intestinal absorption of lipophilic drug loaded lipid nanocarriers in rats. Effects of sodium cholate (SC) on the characteristics, intestinal absorption, cellular uptake in Caco-2 cell monolayers and intestinal lymphatic transport of candesartan cilexetil loaded lipid nanocarriers (CLN) were investigated to clarify the possible mechanism. The intestinal absorption of candesartan from CLN was evidently improved over 16-fold compared with free drug suspension, and further significantly enhanced 1.79-fold after the addition of SC. The cellular uptake of CLN in Caco-2 cell monolayers at 37 ̊C and its colocalization with endoplasmic reticulum were obviously increased in the presence of SC. Moreover, the intestinal lymphatic transport of CLN was obviously enhanced by SC. These results implicated that bile salts could improve the cellular uptake of CLN in Caco-2 cell monolayers via the active processes and promote the intestinal absorption of CLN through the intestinal lymphatic pathway. Therefore, bile salts could be an important physiological factor affecting the intestinal absorption of lipophilic drugs loaded lipid nanocarriers.
Keywords: Bile salts; Lipid nanocarriers; Lipophilic drugs; Intestinal absorption; Caco-2 cells; Lymphatic transport;

Mesoporous carbon as a novel drug carrier of fenofibrate for enhancement of the dissolution and oral bioavailability by Xia Niu; Long Wan; Zhong Hou; Tianyi Wang; Changshan Sun; Jin Sun; Peng Zhao; Tongying Jiang; Siling Wang (382-389).
The purpose of this study was to develop mesoporous carbon loaded with a poorly watersoluble drug to enhance the drug dissolution and improve the oral bioavailability. Mesoporous carbon was synthesized using Pluronic 127 triblock polymer (F127), TEOS and phenolic resins. Fenofibrate (FFB) was chosen as a model drug and loaded onto mesoporous carbon using three different loading methods involving incipient wetness impregnation, and the solvent and melting methods. The effect of the physical state and the specific surface area were investigated using nitrogen adsorption, transmission electron microscopy (TEM), powder X-ray diffraction (XRD) and differential scanning calorimetry (DSC). It was found that the physicochemical properties of the drug as well as the drug loading methods had critical effects on the drug release rate. In vitro drug release studies showed that incorporation of FFB in mesoporous carbon greatly enhanced the dissolution rate in comparison with that of the pure crystalline drug. Moreover, the oral bioavailability of the drug from mesoporous carbon was higher than that of FFB commercial capsules. Furthermore, mesoporous carbon produced no irritation of the mucosa of the gastrointestinal tract as shown by gastric mucosa irritation test.
Keywords: Mesoporous carbon; Poorly soluble drug; Fenofibrate; In vitro dissolution; Oral bioavailability;

Sub-unit vaccine against S. aureus-mediated infections: Set-up of nano-sized polymeric adjuvant by C. Colonna; R. Dorati; B. Conti; P. Caliceti; I. Genta (390-401).
The aim of this work was the design of a novel adjuvanted system for vaccination against S. aureus-mediated infections: in particular, poly-lactide-co-glycolide (PLGA) nanoparticles were developed in order to efficiently load and boost a sub-unit model vaccine, namely a purified recombinant collagen binding bacterial adhesin fragment (CNA19).At first, the assessment of the actual immunogenicity of free CNA19 via subcutaneous administration was evaluated, in order to consider it as subunit antigen model. Secondly, for the development of CNA19 loaded PLGA nanoparticles, a preliminary study was focused on the production of well-formed nanoparticles by w/o/w double emulsion method exploiting ultrasonication cycles under mild conditions, then the optimization of the freeze-drying conditions and different CNA19 loading methods were considered (encapsulation, adsorption of on blank or CNA19 encapsulated nanoparticles). The set-up preparation method (process yield of about 83%) permitted to obtain CNA19 loaded nanoparticles with spherical shape, narrow size distribution (187.41 ± 51.2 nm), a slightly negative zeta-potential (−2.91 ± 0.64 mV) and to elicit satisfactory protein encapsulation efficiency (75.91 ± 4.22%) and loading capacity (8.59 ± 0.33 μg CNA19/nanoparticles mg). Then, CNA19 loaded PLGA nanoparticles were characterized by (i) an in vitro release test performed at different temperatures, namely 4 ̊C, 25 ̊C and 37 ̊C, testing the antigen integrity (SDS-PAGE) and activity (ELISA); (ii) an in vitro stability study in terms of dimension and surface charge performed in a 21 days period of time. At 37 ̊C there was evidence of a sustained release of the antigen, in active form, for almost 240 h with a burst release of about 20% in the first 2 h. At 4 ̊C stability tests and activity assays allowed to identify storage conditions useful to maintain CNA19 activity and easily NP re-suspendability with intact physical characteristics. Furthermore the evaluation of CNA19 loaded nanoparticles cytotoxicity (up to 10.652 mg PLGA/ml) by MTT assay and the study of cellular up-take assessed on human fibroblasts confirmed the feasibility to formulate a dosage form useful for vaccination against S. aureus-mediated infections.
Keywords: S. aureus-mediated infections; Sub-unit vaccine; Polymeric nanoparticles; Nano-sized adjuvant; In vitro characterization;

Nanodrugs play important roles in enhancing the sustainability of pharmaceutical manufacturing via their ability to enhance the bioavailability of poorly soluble drugs, resulting in less drug wastage and less mass/energy consumed in their manufacturing. Despite their sustainability enhancement capability, solid dosage form manufacturing of nanodrugs remains lacking from the sustainability perspective. One example is the granulation of nanodrugs prior to tablet preparation, where existing methods (e.g. wet granulation, spray granulation, spray drying) require high energy and time expenses, or are highly intricate often leading to product inconsistencies. Herein we present an alternative nanodrug granulation method via electrostatic adsorption of the nanodrugs onto chitosan microparticles acting as granulation substrates. The method is sustainable involving only mixing of aqueous suspensions of the nanodrugs and substrates under ambient conditions, followed by washing and drying. We investigate the effects of substrate’s physical characteristics and nanodrug to substrate ratio on the nanodrug loading in the granules, content uniformity, nanodrug recovery, and granule flowability. Ciprofloxacin and curcumin nanoplexes prepared by drug–polyelectrolyte complexation are used as the model nanodrugs with neutrally, positively, and negatively charged chitosan microparticles as the substrates. Granules having 25% (w/w) nanodrug loading at 50% (w/w) recovery with good flowability have been successfully prepared.
Keywords: Drug nanoparticles; Sustainable pharmaceutical manufacturing; Wet granulation; Solid dosage form; Nanomedicines;

Development of docetaxel-loaded solid self-nanoemulsifying drug delivery system (SNEDDS) for enhanced chemotherapeutic effect by Youn Gee Seo; Dae Hwan Kim; Thiruganesh Ramasamy; Jeong Hwan Kim; Nirmal Marasini; Yu-Kyoung Oh; Dong-Wuk Kim; Jin Ki Kim; Chul Soon Yong; Jong Oh Kim; Han-Gon Choi (412-420).
The main purpose of this study was to investigate the potential of self-nano-emulsifying drug delivery system (SNEDDS) in improving the bioavailability of docetaxel (DCT) and its chemotherapeutic effect. The DCT-loaded SNEDDS was prepared by employing rational blends of capryol 90, labrasol, and transcutol HP using ternary phase diagram. The liquid nano-emulsion was spray-dried into solid SNEDDS (D-SNEDDS) using an inert porous carrier, colloidal silica. The optimized formulation was characterized in terms of physico-chemical and pharmacokinetic parameters. Furthermore, anti-tumor efficacy of D-SNEDDS was compared with commercial marketed product, Taxotere®. The various compositions of SNEDDS were screened and found optimal at a volume ratio of 10/75/15 for capryol 90, labrasol, and transcutol HP, respectively. We observed a high oral bioavailability of 17% DCT for D-SNEDDS than compared to only 2.6% for pure DCT solution. Notably, D-SNEDDS exhibited an augmented anti-tumor efficacy with a reduced toxicity profile when compared with intravenously administered Taxotere®, the commercialized formulation of DCT. Taken together, D-SNEDDS could be a potential candidate for an oral dosage form of DCT with enhanced antitumor activity and reduced toxicity.
Keywords: Anti-tumor efficacy; Bioavailability; Docetaxel; Self-nanoemulsifying drug delivery systems; Toxicity;

Enhanced antitumor efficacy by methotrexate conjugated Pluronic mixed micelles against KBv multidrug resistant cancer by Yanzuo Chen; Wei Zhang; Jijin Gu; Qiuyue Ren; Zhuoyang Fan; Weitong Zhong; Xiaoling Fang; Xianyi Sha (421-433).
A methotrexate (MTX) conjugated polymeric mixed micelles for MDR cancer therapy was developed in this study. To the best of our knowledge, MTX was firstly reported to be conjugated with Pluronic P105 (P105-MTX). The Pluronic F127 and P105-MTX polymeric mixed micelles (F127/P105-MTX) were fabricated by thin-film hydration technique, and performed superiority over physically entrapped MTX mixed micelles in drug loading capacity. The drug loading of MTX in F127/P105-MTX was found to be 3.42-fold higher than that of physically entrapped MTX mixed micelles. By conjugated to Pluronic, the amount of MTX in mixed micelles was increased 3.42-fold. In vitro cytotoxicity, cell apoptosis and cell cycle arrest studies also demonstrated that F127/P105-MTX had better antitumor efficacy in KBv MDR cells compared to that of physically entrapped mixed micelles. In comparison with MTX injection, F127/P105-MTX can significantly enhance blood circulation time of MTX in rats. Moreover, a much stronger antitumor efficacy in KBv xenografts mice was observed in F127/P105-MTX group than that of MTX. Therefore, MTX-conjugated mixed micelles might be an effective platform for delivering chemotherapeutic agents to MDR tumors.
Keywords: Methotrexate; Mixed micelles; Multidrug resistant; Drug delivery; Pluronic block copolymer;

Process development for spray drying of sticky pharmaceuticals; case study of bioadhesive nicotine microparticles for compressed medicated chewing gum by Camilla Sander; Henrik Stillhof Nielsen; Susanne Roslev Søgaard; Celina Støving; Mingshi Yang; Jette Jacobsen; Jukka Rantanen (434-437).
Spray drying of pharmaceutical compounds with sticky properties is a challenging task and may require substantial time and resources. By including small-scale studies of single droplet drying kinetics a relatively high number of experiments with less material is allowed. This means one can construct a more robust design space according to Quality by Design (QbD) formulation development principles. In the current study we present a case study on the development of spray dried microparticles comprising nicotine bitartrate and hypromellose or alginate polymer, for incorporation into medicated chewing gum. By illustration of initial studies on single droplet drying kinetics, subsequent characterization of microparticles, and final characterization of compressed chewing gum this paper summarizes the entire development process.
Keywords: Spray drying; Nicotine; Single droplet drying kinetics; Chewing gum; Bioadhesion;