International Journal of Pharmaceutics (v.398, #1-2)

Oral pulsatile delivery: Rationale and chronopharmaceutical formulations by Alessandra Maroni; Lucia Zema; Maria Dorly Del Curto; Giulia Loreti; Andrea Gazzaniga (1-8).
Oral pulsatile/delayed delivery systems are designed to elicit programmable lag phases preceding a prompt and quantitative, repeated or prolonged release of drugs. Accordingly, they draw increasing interest because of the inherent suitability for accomplishing chronotherapeutic goals, which have recently been highlighted in connection with a number of widespread chronic diseases with typical night or early-morning recurrence of symptoms (e.g. bronchial asthma, cardiovascular disease, rheumatoid arthritis, early-morning awakening). In addition, time-based colonic release can be attained when pulsatile delivery systems are properly adapted to overcome unpredictable gastric emptying and provide delay phases that would approximately match the small intestinal transit time. Oral pulsatile delivery is pursued by means of a variety of release platforms, namely reservoir, capsular and osmotic devices. The aim of the present review is to outline the rationale and main formulation strategies behind delayed-release dosage forms intended for the pharmacological treatment of chronopathologies.
Keywords: Oral drug delivery; Pulsatile release; Delayed release; Chronotherapy; Lag time;

Advancement in the fields of material science, analytical methodologies, instrumentation, automation, continuous monitoring, feed forward/feed back control and comprehensive data collection have led to continual improvement of pharmaceutical tablet manufacturing technology, notably the multi-layer tablets. This review highlights the material attributes, formulation design, process parameters that impact the performance, and manufacturability of the multi-layer tablets. It also highlights on critical-to-quality elements that needs to be addressed in the regulatory submission.
Keywords: Bi-layer tablets; Multi-layer tablets; Compaction; Material attributes; Process parameter; Tensile strength; Tablet breaking force;

Assessment of sodium hyaluronate gel as vehicle for intracameral delivery of cefuroxime in endophthalmitis prophylaxis by M. Uhart; F. Pirot; A. Boillon; E. Senaux; L. Tall; E. Diouf; C. Burillon; K. Padois; F. Falson; G. Leboucher; C. Pivot (14-20).
Sodium cefuroxime is a second-generation cephalosporin widely used at 10 mg/mL for endophthalmitis prophylaxis after cataract surgery. Sodium cefuroxime solution is usually conditioned in pre-filled syringes then frozen for storage. In the present study, 0.2% sodium hyaluronate gel, natural extracellular polymer used in wound healing, was compared to conventional saline solution (0.9% sodium chloride) as drug delivery systems for cefuroxime loading in pre-filled syringes. Therefore, the temperature (4 and 25 °C) and time of storage (up to 21 days) varied in order to appreciate both cefuroxime and vehicle stability. Furthermore, the kinetics of drug release from both hyaluronate gel and saline solution were compared since in vitro sets of dialysis experiments.Results indicated that cefuroxime loaded in either saline solution or hyaluronate hydrogel was found stable in pre-filled syringes stored at 4 °C for 21 days, whereas cefuroxime degradations products appeared from the 2nd day of storage at 25 °C. Both drug delivery systems were found bioequivalent, although statistically slower cefuroxime dialysis was evidenced by using sodium hyaluronate vehicle. Noteworthy, cefuroxime concentration in drug delivery systems during dialysis experiment remained greater than the minimum inhibitory concentrations reported for resistant strains.In conclusion, the present stability and release study confirmed that sodium hyaluronate hydrogel is a promising vehicle for cefuroxime intracameral delivery in endophthalmitis prophylaxis.
Keywords: Stability; Cefuroxime; Hyaluronate; Ophthalmic delivery; Endophthalmitis;

Effect of electrokinetic stabilizers on the physicochemical properties of propofol emulsions by Yun-Seok Rhee; Chun-Woong Park; Tack-oon Oh; Ju-Young Kim; Jung-Myung Ha; Beom-Jin Lee; Kyu-Hyun Lee; Sang-Cheol Chi; Eun-Seok Park (21-27).
The aims of the present study were to elucidate the potential mechanism of propofol emulsion destabilization following the addition of lidocaine, and to evaluate the effects of various electrokinetic stabilizers on the physicochemical properties of lidocaine–propofol emulsions. The assessments included pH observations and determination of the maximum globule diameter (MGD) and zeta potential (ZP). The MGD of propofol emulsions increased up to several tens μm following the addition of 50 mg of lidocaine to 200 mg of propofol, and the proposed destabilization mechanism involves localization of protonated lidocaine molecules between lecithin molecules in propofol emulsions, which consequently leads to increased ZP. The ZP of propofol emulsions containing acidic amino acid or neutral amino acid increased following the addition of lidocaine, and a charge reversal occurred. Therefore, the MGD of emulsions increased to several tens (m. However, the MGD of emulsions that contained basic amino acids or basic compounds remained below 5 (m, despite the addition of large amounts lidocaine (50 mg), and the ZP did not pass through the point of zero charge. In conclusion, our results provide not only further insight into the physical stability of propofol emulsions containing lidocaine, but also a better understanding of the administration of propofol in existing applications.
Keywords: Injectables; Physical stability; Propofol; Lidocaine; Emulsion; Zeta potential;

Modelling the effect of mixture components on permeation through skin by T. Ghafourian; E.G. Samaras; J.D. Brooks; J.E. Riviere (28-32).
A vehicle influences the concentration of penetrant within the membrane, affecting its diffusivity in the skin and rate of transport. Despite the huge amount of effort made for the understanding and modelling of the skin absorption of chemicals, a reliable estimation of the skin penetration potential from formulations remains a challenging objective. In this investigation, quantitative structure–activity relationship (QSAR) was employed to relate the skin permeation of compounds to the chemical properties of the mixture ingredients and the molecular structures of the penetrants. The skin permeability dataset consisted of permeability coefficients of 12 different penetrants each blended in 24 different solvent mixtures measured from finite-dose diffusion cell studies using porcine skin. Stepwise regression analysis resulted in a QSAR employing two penetrant descriptors and one solvent property. The penetrant descriptors were octanol/water partition coefficient, log  P and the ninth order path molecular connectivity index, and the solvent property was the difference between boiling and melting points. The negative relationship between skin permeability coefficient and log  P was attributed to the fact that most of the drugs in this particular dataset are extremely lipophilic in comparison with the compounds in the common skin permeability datasets used in QSAR. The findings show that compounds formulated in vehicles with small boiling and melting point gaps will be expected to have higher permeation through skin. The QSAR was validated internally, using a leave-many-out procedure, giving a mean absolute error of 0.396. The chemical space of the dataset was compared with that of the known skin permeability datasets and gaps were identified for future skin permeability measurements.
Keywords: Skin; QSAR; Formulation; Mixture; Permeation; Penetration;

A pattern-fitting procedure using an X-ray diffraction pattern was applied to the quantitative analysis of binary system of crystalline pharmaceuticals in tablets. Orthorhombic crystals of isoniazid (INH) and mannitol (MAN) were used for the analysis. Tablets were prepared under various compression pressures using a direct compression method with various compositions of INH and MAN. Assuming that X-ray diffraction pattern of INH–MAN system consists of diffraction intensities from respective crystals, observed diffraction intensities were fitted to analytic expression based on X-ray diffraction theory and separated into two intensities from INH and MAN crystals by a nonlinear least-squares procedure. After separation, the contents of INH were determined by using the optimized normalization constants for INH and MAN. The correction parameter including all the factors that are beyond experimental control was required for quantitative analysis without calibration curve. The pattern-fitting procedure made it possible to determine crystalline phases in the range of 10–90% (w/w) of the INH contents. Further, certain characteristics of the crystals in the tablets, such as the preferred orientation, size of crystallite, and lattice disorder were determined simultaneously. This method can be adopted to analyze compounds whose crystal structures are known. It is a potentially powerful tool for the quantitative phase analysis and characterization of crystals in tablets and powders using X-ray diffraction patterns.
Keywords: X-ray diffraction; Quantitative analysis; Crystals; Pattern-fitting; Preferred orientation;

Comparative study of non-destructive methods to quantify thickness of tablet coatings by C. Cahyadi; A.D. Karande; L.W. Chan; P.W.S. Heng (39-49).
The Supercell coater is a newly introduced coater which utilizes air fluidization for tablet coating. The aim of this study was to define a suitable, fast and non-destructive method for the quantification of coat thickness for Supercell-coated tablets. Various coat thickness characterization methods were carried out on tablets coated at different process conditions. These include the use of optical microscopy, micrometer, X-ray fluorescence (XRF), Raman and near-infrared (NIR) spectroscopy. Coat thicknesses obtained from direct measurements were used to calibrate the spectral data from spectroscopic methods for model generation. The models were subsequently validated to evaluate their prediction capabilities, especially the ability to differentiate tablets coated at different conditions. XRF spectroscopy was viewed to be more suitable for the assessment of process yield and efficiency but both Raman and NIR spectroscopy were shown to be more appropriate methods for the rapid prediction and evaluation of coat thickness. However, only Raman spectroscopy was able to differentiate tablets coated under different conditions accurately. In conclusion, direct thickness measurements were more time-consuming but provided assured coat thickness data. On the other hand, XRF, Raman and NIR spectroscopy methods were viable alternatives to provide complementary information for the study of tablet coatings.
Keywords: Supercell coater; Tablet coating; Coat thickness; Raman spectroscopy; Near-infrared spectroscopy; X-ray fluorescence spectroscopy;

Crystal structures, X-ray powder patterns, infrared spectra and DSC data of three solvates of erythro-(±)-mefloquinium chloride are reported.The crystal structure of a monohydrate of (±)-mefloquinium chloride, (±)-[MEFHCl·(H 2 O)], has been obtained from data collected at 120 K. Further details of the crystal structure of a hemihydrate, (±)-[MEFHCl·½(H 2 O)], previously determined at room temperature, are also reported from data collected at 120 K. The structural data, along with X-ray powder patterns, infrared spectra (both mid and near IR ranges) and DSC data, provide definitive evidence for the two hydrates. Further attempts to completely resolve the crystal structure of a tetragonal solvated phase failed due to the considerable disorder, involving the solvates, either water or methanol, even at 120 K, however X-ray powder pattern and infrared spectral and DSC data for this phase are also reported. Comparisons of the data obtained for the hydrates in this study, with X-ray powder patterns and mid-infrared spectra of previously reported forms of (±)-mefloquinium chloride have highlighted erroneous assignments and allowed structural identification of previously and differently designated forms.
Keywords: Mefloquine; Solvates; Hydrates; Crystal structures; Near IR; Powder diffraction patterns; DSC;

Influence of relative humidity on the interaction between different aryl propionic acid derivatives and poly(vinylpyrrolydone) K30: Evaluation of the effect on drug bioavailability by Ledjan Malaj; Roberta Censi; Matteo Mozzicafreddo; Luca Pellegrino; Mauro Angeletti; Roberto Gobetto; Piera Di Martino (61-72).
The present work assessed the physical interactions between several aryl propionic acid derivatives and polyvinyl(pyrrolidone) K30 (PVP), stored together at 298 ± 0.5 K at different relative humidities (RH 55, 75 and 86%). Results were compared to those obtained at low RH (22%), published in a previous paper. The water uptake percentage of binary mixtures were intermediate between that of pure PVP and pure drugs. By X-ray powder diffraction, for all the drugs, it was possible to note a marked decrease in crystallinity degree, in particular at highest RH%. The loss in crystallinity degree may be considered an evidence of the physicochemical interaction between the polymer and the drug, supporting the formation of a solid dispersion. By high-resolution 1H solid-state NMR spectrometry, it was possible to observe an increase of drug–polymer interaction with aging, with the only exception of ibuprofen. Molecular docking proved the establishment of Van der Waals and electrostatic interactions for all the mixtures, and for mixtures with fenbufen and naproxen, also hydrogen bonds. The application of Gordon–Taylor rule to the thermal analysis revealed that the requirement of volume additivity of this rule was not fulfilled for any mixture, and a negative deviation from theoretical behaviour was always observed. The hydration of drug–PVP mixtures had important repercussion on drug solubility and intrinsic dissolution rate (IDR). In general, an increase in water solubility and consequently an increase in IDR were observed, with few exceptions, at highest RH%.
Keywords: Interaction; Solid dispersion; Water in solids; Physical characterization; Preformulation;

The purpose of the present study was to investigate the oral absorption simulation of free base drugs. In the case of a low solubility free base drug, a portion of drug particles remains incompletely dissolved during the stomach transit and can reach the small intestine. As the pH is neutralized in the small intestine, the solubility of the drug decreases and the concentration gradient around the particles becomes a negative value. The drug particles would then grow because of this negative concentration gradient resulting in a reduction of the dissolved drug concentration. The modified Nernst Brunner equation was used to simulate both particle dissolution and growth (particle growth is the opposite phenomena of particle dissolution). Albendazole, aprepitant, dipyridamole, gefitinib and ketoconazole were used as model drugs (all free solid form (not salts)). The effect of stomach pH on oral absorption was appropriately simulated. Based on the simulation results, it was suggested that the dissolution patterns in the gastrointestinal tract were significantly different depending on the dose–solubility ratio in the stomach.
Keywords: Free base; Solubility; Dissolution rate; Computational simulation; Oral absorption;

Effect of different enhancers on the transdermal permeation of insulin analog by K.M. Yerramsetty; V.K. Rachakonda; B.J. Neely; S.V. Madihally; K.A.M. Gasem (83-92).
Using chemical penetration enhancers (CPEs), transdermal drug delivery (TDD) offers an alternative route for insulin administration, wherein the CPEs reversibly reduce the barrier resistance of the skin. However, there is a lack of sufficient information concerning the effect of CPE chemical structure on insulin permeation. To address this limitation, we examined the effect of CPE functional groups on the permeation of insulin. A virtual design algorithm that incorporates quantitative structure–property relationship (QSPR) models for predicting the CPE properties was used to identify 43 potential CPEs. This set of CPEs was pre-screened using a resistance technique, and the 22 best CPEs were selected. Next, standard permeation experiments in Franz cells were performed to quantify insulin permeation.Our results indicate that specific functional groups are not directly responsible for enhanced insulin permeation. Rather, permeation enhancement is produced by molecules that exhibit positive log  K ow values and possess at least one hydrogen donor or acceptor. Toluene was the only exception among the 22 potential CPEs considered. In addition, toxicity analyses of the 22 CPEs were performed. A total of eight CPEs were both highly enhancing (permeability coefficient at least four times the control value) and non-toxic, five of which are new discoveries.
Keywords: Transdermal; Drug delivery; Permeation enhancer; Insulin;

In vitro dissolution of oral modified-release tablets and capsules in ethanolic media by Anjanette P. Smith; Terry W. Moore; Benjamin J. Westenberger; William H. Doub (93-96).
In 2005, Palladone, an extended-release capsule, was withdrawn from the market after clinical testing showed subjects who took the product with alcohol had increased levels of drug in their blood. To better understand this phenomenon, we studied the in vitro drug release of 27 oral modified-release products in alcohol-containing media. In 40% alcoholic medium, 9 of 10 capsules and 2 of 17 tablets show accelerated drug release. When a high percentage of the total dose is released in a short period of time, the extended-release product is then performing like an immediate release formulation. Products were also tested in 5% and 20% alcoholic media and in simulated gastric fluid (without enzyme) containing 20% alcohol. No tested capsules or tablets exhibited a significant increase in drug release in media containing only 5% alcohol. The current study indicates that in vitro dissolution may provide evidence regarding the ruggedness of formulations to ingested alcohol.
Keywords: Dose dumping; Ethanol; Dissolution; Modified release; Tablets and capsules;

The application of multivariate wavelet texture analysis (MWTA) is presented and discussed as it is applied to three different types of pharmaceutical materials: (a) tablet cores, (b) wet granules and (c) controlled release tablets. The application of MWTA is initially proposed as a quantitative replacement to the human visual judgment of the textural appearance of the different materials. In all cases, the metrics obtained with MWTA agree with visual assessment on the progression of textural features such as erosion and surface roughness. This work further demonstrates that MWTA also represents a useful tool to increase the understanding of the manufacturing process, as it provides diagnostics to relate process parameters with textural features of the material that are difficult or costly to measure otherwise (such as granule size for wet material or surface appearance for a controlled release product). MWTA is also presented as a potential tool for real-time release for those cases where the textural features can be proven to provide accurate enough predictions of the final product performance; as shown here with the obtained prediction of dissolution from the controlled release tablet using the texture of the product as an input.
Keywords: Multivariate wavelet texture analysis; Drug product; PLS; PCA; Quality by Design; Design space;

Multiple water-in-oil-in-water (W/O/W) emulsions are of major interest as potential skin delivery systems for water-soluble drugs like oligonucleotides due to their distinct encapsulation properties. However, multiple emulsions are highly sensitive in terms of variations of the individual components. The presence of osmotic active ingredients in the inner water phase is crucial for the generation of stable multiple emulsions. In order to stabilize the emulsions the influence of NaCl, MgSO4, glucose and glycine and two cellulose derivatives was investigated. Briefly, multiple W/O/W emulsions using Span 80 as a lipophilic emulsifier and different hydrophilic emulsifiers (PEG-40/50 stearate, steareth-20 and polysorbate 80) were prepared. Stability of the emulsions was analyzed over a period of time using rheological measurements, droplet size observations and conductivity analysis.In this study we show that additives strongly influence the properties stability of multiple emulsions. By increasing the concentration of the osmotic active ingredients, smaller multiple droplets are formed and the viscosity is significantly increased. The thickening agents resulted in a slightly improved stability.The most promising emulsions were chosen and further evaluated for their suitability and compatibility to incorporate a DNAzyme oligonucleotide as active pharmaceutical ingredient.
Keywords: W/O/W multiple emulsion; Osmolyte; Hydrophilic surfactant; Electrolyte; DNAzyme; Oligonucleotides;

Determination of partition and binding properties of solutes to stratum corneum by Liming Wang; Longjian Chen; Guoping Lian; Lujia Han (114-122).
The binding property of a number of relatively hydrophilic solutes to native and delipidized stratum corneum (SC) and their partition coefficients to extracted lipid have been measured by equilibration experiments to expand the current database which consisted of mostly hydrophobic solutes. Using the extended database, quantitative structure property relationships (QSPR) have been proposed for predicting the partition and binding coefficients of both hydrophobic and hydrophilic solutes to the SC protein, and lipid. Solute partition to the SC lipid is best fitted by PC lip / w = K ow 0.69 and solute binding to the SC protein is best described by PC pro / w = 4.2 K ow 0.31 . The two QSPR models of solute partition to the SC lipid and binding to the SC protein have been further combined into a two-phase model to predict the overall partition coefficient of solutes to the stratum corneum (K sc/w). Our study not only extends the database of solute partition and binding properties of the SC to include hydrophilic solutes, but also demonstrates that the thermodynamic equilibrium properties of the SC partition and binding can be fitted with good accuracy by combining QSPR models with the multiphase and heterogeneous structures of the SC.
Keywords: Stratum corneum; Skin lipid; Skin protein; Solute partition; Solute binding;

To avoid the clinical inconvenience of repeated injection of the immune modulator thymopentin (TP5), biodegradable implants comprising a mixed polymer matrix of poly(lactide acid) (PLA) and poly(lactide-co-glycolide acid) (PLGA) were produced using a simple extrusion method. Drug release from these TP5-loaded implants was characterized both in vitro and in vivo. Pharmacodynamic studies were carried out in immunosuppressed rats using the ratio of CD4+/CD8+ cells, determined by flow cytometry, as an index of immunity. The results indicated that the entrapment efficiency of the implants was greater than 98%, but the release rate of TP5 depended on the drug loading. Implants containing less than 10% TP5 showed consistent release over 30 days, with low burst-release both in vitro and in vivo. Improved immunity and survival rates were observed in rats treated by TP5 injection and in rats given middle-to-high dose implants. When the release of TP5 exceeded 0.1 mg/kg body weight/day the CD4+/CD8+ ratios increased in the 3 weeks after implantation, reaching a maximum (91.6% of the normal level) by the end of the third week. The TP5-loaded implants presented here provide a promising alternative to injections and the results support the further development of controlled-release TP5 formulations.
Keywords: Thymopentin (TP5); Biodegradable implants; Release; Flow cytometry; Immunity;

Preparation of pingyangmycin PLGA microspheres and related in vitro/in vivo studies by Bing Han; Hao-Tian Wang; Huai-Yu Liu; Hua Hong; Wei Lv; Zu-Hui Shang (130-136).
Using a multiple emulsion solvent evaporation method, pingyangmycin was entrapped in poly(lactic-co-glycolic acid) (PLGA) to prepare a long-acting pingyangmycin PLGA microsphere formulation that can be sustainably released with high entrapment efficiency. Meanwhile, the effects of stirring speed during the multiple emulsion solvent evaporation process were also taken into consideration. Investigation of the in vitro release properties showed that the microsphere formulations could sustainably release the drug over nearly 28 d, and, moreover, it could stably control pingyangmycin release over nearly 24 d when intramuscularly injected into dogs. No serious toxic effect was observed in an acute toxicity test in mice. A subcutaneous xenotransplant model of hepatoma H22 in mice was established for pharmacodynamic studies and the results showed that the process of preparing pingyangmycin PLGA microsphere formulations was feasible and that intramuscular injection of this microsphere formulation resulted in anti-tumor activity in vivo.
Keywords: Pingyangmycin; Poly(lactic-co-glycolic acid); Microsphere; In vitro release; In vivo release; Toxicology; Pharmacodynamics;

Alternative oral exemestane formulation: Improved dissolution and permeation by Burçin Yavuz; Erem Bilensoy; İmran Vural; Murat Şumnu (137-145).
Exemestane (EXE) is an irreversible aromatase inactivator used for the treatment of advanced postmenopausal breast cancer. EXE is orally active but its bioavailability is about 5% due to its low solubility in water and the extensive first pass effect. It is known that cyclodextrin (CD) complexation enhances solubility and oral bioavailability of poorly soluble drugs. Thus, it was aimed to design and develop cyclodextrin complexes in powder and tablet forms containing EXE to improve aqueous solubility and in vitro permeability. In this study, inclusion complexes of EXE were prepared with three different CD derivatives (methyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin and hydroxypropyl-γ-cyclodextrin) and by two different preparation methods (kneading and colyophilization) and the complexes were characterized with 1H NMR, FT-IR, SEM, X-ray and DSC analyses. Both inclusion complexes and tablet formulations prepared using EXE:CD inclusion complexes showed significant improvement in the dissolution profile of this oral antiestrogen drug. Furthermore, Caco-2 cell permeation studies revealed that apparent permeability constant for EXE was increased by 3-fold via cyclodextrin complexation. In conclusion, complexation of EXE with cyclodextrin derivatives, randomly methylated-β-cyclodextrin in particular, results in a more efficient tablet formulation with improved dissolution and better permeation suggesting an enhancement in oral bioavailability of the drug.
Keywords: Exemestane; Cyclodextrin; Anticancer drugs; Oral drug delivery; In vitro dissolution; In vitro permeation;

Sustained prolonged topical delivery of bioactive human insulin for potential treatment of cutaneous wounds by Michael Hrynyk; Manuela Martins-Green; Annelise E. Barron; Ronald J. Neufeld (146-154).
Skin damaged by heat, radiation, or chemical exposure is difficult to treat and slow to heal. Indeed full restoration of the tissue is difficult to obtain. Sub-dermal insulin injection was recently shown to stimulate wound healing of the skin by accelerating wound closure, stimulating angiogenesis and inducing a regenerative process of healing. We have developed a topical delivery vehicle that is capable of releasing therapeutic levels of bioactive insulin for several weeks with the potential to stimulate and sustain healing. By encapsulating the crystalline form of insulin within poly(d,l-lactide-co-glycolide) microspheres, we succeeded in stabilizing and then releasing bioactive insulin for up to 25 days. To measure bioactivity we used Rat L6 myofibroblasts, stimulated them with this slow release insulin and determined activation of the receptors on the cell surface by quantifying AKT phosphorylation. There was only a minor and gradual decrease in AKT phosphorylation over time. To determine whether the slow release insulin could stimulate keratinocyte migration, wounding was simulated by scratching confluent cultures of human keratinocytes (HaCaT). Coverage of the scratch “wounds” was significantly faster in the presence of insulin released from microspheres than in the insulin-free control. Extended and sustained topical delivery of active insulin from a stable protein crystal-based reservoir shows promise in promoting tissue healing.
Keywords: Wound healing; Insulin; Microspheres; L6 myoblasts; Controlled release; HaCaT; PLGA;

Among the different experimental methods that can be used to quantify the evolution of drug crystallinity in polymer-containing amorphous solid dispersions, powder X-ray diffractometry (PXRD) is commonly considered as a frontline method. In order to achieve accurate quantification of the percent drug crystallinity in the system, calibration curves have to be constructed using appropriate calibration samples and calculation methods. This can be non-trivial in the case of partially crystalline solid dispersions where the calibration samples must capture the multiphase nature of the systems and the mathematical model must be robust enough to accommodate subtle and not so subtle changes in the diffractograms. The purpose of this study was to compare two different calculation and model-building methods to quantify the proportion of crystalline drug in amorphous solid dispersions containing different ratios of drug and amorphous polymer. The first method involves predicting the % drug crystallinity from the ratio of the area underneath the Bragg peaks to total area of the diffractogram. The second method is multivariate analysis using a Partial Least-Squares (PLS) multivariate regression method. It was found that PLS analysis provided far better accuracy and prediction of % drug crystallinity in the sample. Through the application of PLS, root-mean-squared error of estimation (RMSEE) values of 2.2%, 1.9%, and 4.7% drug crystallinity was achieved for samples containing 25%, 50%, and 75% polymer, respectively, compared to values of 11.2%, 17.0%, and 23.6% for the area model. In addition, construction of a PLS model enables further analysis of the data, including identification of outliers and non-linearity in the data, as well as insight into which factors are most important to correlate PXRD diffractograms with % crystallinity of the drug through analysis of the loadings.
Keywords: Partial Least-Squares model; Crystallinity; Amorphous solid dispersion; Powder X-ray diffractometry;

Liver-specific expression of an exogenous gene controlled by human apolipoprotein A-I promoter by Yurong Hu; Xueling Ren; Hui Wang; Yue Ma; Lei Wang; Yingying Shen; Kazuhiro Oka; Zhenzhong Zhang; Yun Zhang (161-164).
Liver-specific gene therapy is advantageous to minimize the possible adverse effects caused by non-target gene expression. The CMV promoter of the enhanced green fluorescent protein (EGFP) expressing plasmid pCMV-EGFP was replaced with the liver-specific promoter apolipoprotein A-I (ApoAI) generating pApoAI-EGFP plasmid. In vitro expression experiments performed in various cell lines including HepG2, SMMC-7721, MCF7, ACC-2 and Lo2 indicated that pCMV-EGFP treatment caused gene expression in all the cell lines, whereas pApoAI-EGFP treatment only induced EGFP expression in cells of liver origin including the liver cancer cells HepG2 and SMMC-7721 and the normal liver cells Lo2. Either pCMV-EGFP or pApoAI-EGFP was formulated as pegylated immuno-lipopolyplexes (PILP), a novel and efficient gene delivery system. Following intravenous administration of the PILP in H22 tumor-bearing mice, there was significant EGFP expression in liver, tumor, spleen, brain and lung in the pCMV-EGFP treated mice, whereas in the pApoAI-EGFP treated mice there was only gene expression in liver and tumor and the non-liver organ gene expression was eliminated. This study suggests that the use of the PILP technology and liver-specific promoter enables efficient and liver-specific expression of an exogenous gene.
Keywords: Liver-specific expression; Hepatocellular carcinoma; Pegylated immuno-lipopolyplexes; Gene therapy; Promoter; Apolipoprotein A-I;

Atopic dermatitis (AD) is chronically relapsing eczematous skin disorder having significant impact worldwide. Tacrolimus is the drug-of-choice which inhibits T-cell activation resulting in suppression of inflammation. However, despite being effective, most common adverse events of tacrolimus are low-and-variable bioavailability, burning sensation and pruritus at application site, which prompt for development of novel carrier that could effectively target tacrolimus to site-of-action without producing undesirable side-effects. Tacrolimus-loaded lipid-nanoparticles (T-LN) were prepared and optimized. DSC and FT-IR have been employed to study drug-excipient incompatibility and encapsulation of drug in lipid which was further confirmed by 1H NMR. In vitro studies revealed much higher drug release, skin penetration and enhanced skin accumulation as compared to reference Protopic®. In vitro and in vivo occlusion studies demonstrated similar occlusiveness for T-LN and reference however; T-LN showed significantly higher drug levels penetrating into deeper skin layers where dendritic cells responsible for immunopathogenesis of AD mainly reside. In-vivo skin retention demonstrated 3.36, 30.81 and 28.68-times higher stratum corneum, epidermal and dermal levels respectively compared to reference. Visualization of cutaneous uptake in-vivo using CLSM confirmed targeting to deeper skin layers and Draize test showed no skin irritation with PII 0.00. Thus T-LN displayed superior performance, effective skin targeting and improved safety as compared to reference.
Keywords: Tacrolimus; Drug targeting; Skin penetration enhancement; Lipid nanoparticles;

Quick and efficient extraction of uranium from a contaminated solution by a calixarene nanoemulsion by Aurélie Spagnul; Céline Bouvier-Capely; Marc Adam; Guillaume Phan; François Rebière; Elias Fattal (179-184).
This work aims to evaluate the efficiency of a calixarene emulsion for uranium extraction from a contaminated solution prior to apply such a delivery system to uranium skin decontamination. For this purpose, various experimental parameters that can influence the efficiency of the calixarene emulsion on uranium extraction were determined. The results show that the calixarene nanoemulsion effect can be observed after a very short time of contact with uranium-contaminated solution (5 min) and that it is still efficient in case of small volumes of contaminated solution. The pH of the contaminated solution was found to be the most important parameter affecting the calixarene nanoemulsion efficiency with a dramatic reduction of the uranium extraction rate in case of acidification of the contaminated medium. This lack of efficiency can be overcome by buffering the nanoemulsion continuous phase. The obtained results reveal that the calixarene nanoemulsion could represent a promising system for the emergency treatment of uranium cutaneous contamination.
Keywords: Uranium; Decontamination; Calixarene; Nanoemulsion; In vitro characterization;

PAMAM dendrimers as solubilizers and hosts for 8-methoxypsoralene enabling transdermal diffusion of the guest by Katarzyna Borowska; Barbara Laskowska; Agnieszka Magoń; Bogdan Mysliwiec; Marek Pyda; Stanisław Wołowiec (185-189).
PAMAM dendrimers form host–guest complexes with 8-methoxypsoralene (8-MOP) – the photosensitizer for PUVA therapy. The stoichiometry of complexes was studied by 1H NMR spectroscopy in solution and by differential scanning calorimetry in neat mixtures containing 8-MOP and dendrimers of generations G2.5, G3, G3.5, and G4. The dendrimers showed solubilization effect for 8-MOP resulting in increase of 8-MOP concentration in methanol up to 15 molecules of 8-MOP per G2.5 and G3 and 30 molecules of 8-MOP per G3.5, and G4. Isolation of oily host–guest complexes containing 3 or 7 molecules of 8-MOP per G3 and G4, respectively corroborate well with DSC results; glass transition temperature of neat host–guest complexes increases with number of host molecules in comparison with G3 or G4, until the capacity of host is exceeded. The oily host–guest complexes of stoichiometry 3:1 and 7:1 of 8-MOP to G3 and G4, respectively are well soluble in water. The 3:1 host–guest complexes diffused slowly through polyvinyldifluoride and pig ear skin membranes, when released from o/w emulsion. The host–guest complex 8-MOP-G3 was proposed as convenient formulation for psoralene skin administration in PUVA therapy.
Keywords: Psoralene; PAMAM dendrimers; Host–guest complexes; Transdermal permeation;

Transferrin mediated solid lipid nanoparticles containing curcumin: Enhanced in vitro anticancer activity by induction of apoptosis by Rohit S. Mulik; Jukka Mönkkönen; Risto O. Juvonen; Kakasaheb R. Mahadik; Anant R. Paradkar (190-203).
Photodegradation and low bioavailability are major hurdles for the therapeutic use of curcumin. Aim of the present study was to formulate transferrin-mediated solid lipid nanoparticles (Tf-C-SLN) to increase photostability, and enhance its anticancer activity against MCF-7 breast cancer cells. Tf-C-SLN were prepared by homogenization method and characterized by size, zeta potential, entrapment efficiency and stability, transmission electron microscopy (TEM), X-ray diffraction (XRD) and in vitro release study. Microplate analysis and flow cytometry techniques were used for cytotoxicity and apoptosis study. The physical characterization showed the suitability of method of preparation. TEM and XRD study revealed the spherical nature and entrapment of curcumin in amorphous form, respectively. The cytotoxicity, ROS and cell uptake was found to be increased considerably with Tf-C-SLN compared to curcumin solubilized surfactant solution (CSSS) and curcumin-loaded SLN (C-SLN) suggesting the targeting effect. AnnexinV-FITC/PI double staining, DNA analysis and reduced mitochondrial potential confirmed the apoptosis. The flow cytometric studies revealed that the anticancer activity of curcumin is enhanced with Tf-C-SLN compared to CSSS and C-SLN, and apoptosis is the mechanism underlying the cytotoxicity. The present study indicated the potential of Tf-C-SLN in enhancing the anticancer effect of curcumin in breast cancer cells in vitro.
Keywords: Apoptosis; Curcumin; Cytotoxicity; Breast cancer; Flow cytometry;

Reverse micelle-loaded lipid nano-emulsions: New technology for nano-encapsulation of hydrophilic materials by Nicolas Anton; Halina Mojzisova; Emilien Porcher; Jean-Pierre Benoit; Patrick Saulnier (204-209).
This study presents novel, recently patented technology for encapsulating hydrophilic species in lipid nano-emulsions. The method is based on the phase-inversion temperature method (the so-called PIT method), which follows a low-energy and solvent-free process. The nano-emulsions formed are stable for months, and exhibit droplet sizes ranging from 10 to 200 nm. Hydrophilic model molecules of fluorescein sodium salt are encapsulated in the oily core of these nano-emulsion droplets through their solubilisation in the reverse micellar system. As a result, original, multi-scaled nano-objects are generated with a ‘hydrophilic molecule in a reverse-micelles-in-oil-in-water’ structure. Once fluorescein has been encapsulated it remains stable, for thermodynamic reasons, and the encapsulation yields can reach 90%. The reason why such complex objects can be formed is due to the soft method used (PIT method) which allows the conservation of the structure of the reverse micelles throughout the formulation process, up to their entrapment in the nano-emulsion droplets. In this study, we focus the investigation on the process itself, revealing its potential and limits. Since the formulation of nanocarriers for the encapsulation of hydrophilic substances still remains a challenge, this study may constitute a significant advance in this field.
Keywords: Nano-emulsion; Reverse micelle; Nano-emulsification; Phase inversion temperature; PIT method; Low-energy method; Hydrophilic material;

Bioadhesive nanoparticles of fungal chitosan for oral DNA delivery by Laurence Plapied; Gaëlle Vandermeulen; Benoît Vroman; Véronique Préat; Anne des Rieux (210-218).
Chitosan is an ideal candidate for oral DNA delivery due to its mucoadhesive properties. Chitosan (CS) produced under GMP conditions from fungal source was used to encapsulate a plasmid DNA coding for a reporter gene. Nanoparticles made by complex coacervation of CS and DNA had a size around 200 nm, a positive zeta potential, a high association of DNA and protected the plasmid against nuclease degradation. Their transfection ability was assessed in differentiated intestinal Caco-2 cells. An N/P ratio of 4 and a DNA concentration of 8 μg/ml were the optimal conditions leading to a transfection efficiency similar to the one reached with polyethyleneimine (PEI)–DNA complexes without cytotoxicity. M cells in monolayers influenced DNA uptake up to 8 μg of DNA/ml when complexed with CS. Fungal trimethylchitosan was also tested but the complexes interactions were too strong to induce transfection in vitro. Confocal microscopy studies showed that CS/DNA and PEI/DNA nanoparticles were found at the apical surface of cell monolayers and DNA was co-localized within the nucleus. Quantification seemed to show that more DNA was associated with the cells when incubated with CS nanoparticles and that the presence of M cells slightly influenced DNA uptake when complexed with CS. In conclusion, we developed a new nanocarrier made of fungal CS promising for oral gene delivery and oral DNA vaccination.
Keywords: Gene; Caco-2 cells; M cells; Chitosan; Trimethylchitosan; Nanoparticles;

Disulfide crosslinked stearoyl carrier peptides containing arginine and histidine enhance siRNA uptake and gene silencing by Ko Tanaka; Takanori Kanazawa; Takaya Ogawa; Yuuki Takashima; Tsunehiko Fukuda; Hiroaki Okada (219-224).
The siRNA has been expected to apply for several diseases such as cancer since siRNA specifically silences the disease-associated genes. However, effective gene carriers should be developed to overcome the low siRNA stability in vivo, form stable complexes and facilitate intracellular uptake of siRNA. In this study, to develop a safe and efficient siRNA carrier, stearoyl (STR) peptides with Cys (C), Arg (R), and His (H) residues that can form disulfide cross linkages via Cys (C) were synthesized, and their suitability as siRNA carriers was evaluated. The particle size of STR-CH2R4H2C/siRNA complexes was about 100 nm. The cellular uptake ability after transfection with FAM-siRNA with STR-CH2R4H2C, CH2R4H2C, or STR-GH2R4H2G was significantly higher than that with FAM-siRNA only. STR-CH2R4H2C showed the highest cellular uptake ability when compared with CH2R4H2C and STR-GH2R4H2G. STR-CH2R4H2C did not induce substantial cytotoxicity. The intratumor injection of STR-CH2R4H2C/vascular endothelial growth factor (VEGF) siRNA (siVEGF) complexes achieved a high anti-tumor effect in tumor bearing mice. These results suggest STR-CH2R4H2C has potential of effective siRNA carrier possible to exercise silencing effect in vitro and in vivo.
Keywords: Artificial cell penetrating peptide; Disulfide linkage; Anti-tumor effects; siRNA delivery;

The ratio of unsaturated fatty acids in biosurfactants affects the efficiency of gene transfection by Yoshikazu Inoh; Tadahide Furuno; Naohide Hirashima; Dai Kitamoto; Mamoru Nakanishi (225-230).
An unsaturated hydrocarbon chain in phospholipid was reported to affect a phase transition and a fusogenic activity after mixing membranes, and consequently to achieve a high DNA transfection efficiency. We previously showed that a biosurfactant mannosylerythritol lipid-A (MEL-A) enhances the gene transfection efficiency of cationic liposomes. Here, we have studied the effects of unsaturated fatty acid ratio of MEL-A on the physicochemical properties and gene delivery into cells of cationic liposomes using MEL-A with three different unsaturated fatty acid ratios (9.1%, 21.5%, and 46.3%). The gene transfer efficiency of cationic liposomes containing MEL-A (21.5%) was much higher than that of those containing MEL-A (9.1%) and MEL-A (46.3%). MEL-A (21.5%)-containing cationic liposomes induced highly efficient membrane fusion after addition of anionic liposomes and led to subsequent DNA release. Imaging analysis revealed that MEL-A (21.5%)-containing liposomes fused with the plasma membrane and delivered DNA into the nucleus of NIH-3T3 cells, MEL-A (46.3%)-containing liposomes fused with the plasma membrane did not deliver DNA into the nucleus, and MEL-A (9.1%)-containing liposomes neither fused with the plasma membrane nor delivered DNA into the nucleus. Thus, it is understandable that the unsaturated fatty acid ratio of MEL-A strongly influences the gene transfection efficiency of cationic liposomes.
Keywords: Biosurfactants; Cationic liposome; Gene transfection; Unsaturation; Fatty acid;

Evaluation of ISCOM matrices clearance from rabbit nasal cavity by gamma scintigraphy by Ravi S. Pandey; Anil K. Babbar; Ankur Kaul; Anil K. Mishra; Vinod K. Dixit (231-236).
Immune stimulating complexes and/or ISCOM matrices (adjuvant nanoparticles without antigen as a structural component) found potential applications as nasal vaccine adjuvant/delivery system owing to virus like particulate structure and saponin as potent Th1 adjuvant. One of important limiting factor for nasal vaccine delivery is the limited time available for absorption within the nasal cavity due to mucociliary clearance. In this report the clearance rate of ISCOM matrices from nasal cavity of rabbit was determined by gamma scintigraphy. ISCOM matrices were radiolabelled with 99mTc by direct labelling method using stannous chloride as a reducing agent. 99mTc labelled ISCOM matrices were administered into the nostril of female New Zealand rabbits and 1 min static views were repeated each 15 min until 4 h. Clearance rate of ISCOM matrices from nasal cavity was calculated after applying the physical decay corrections. The mean labelling efficiency for ISCOM matrices were calculated as ∼58.4%. ISCOM matrices showed slower clearance rate compared to sodium pertechnetate control solution (p  < 0.005) from nasal cavity that may be due to particulate and hydrophobic characters of ISCOM particles even though it was also cleared within 4 h from nasal cavity. Mucoadhesive ISCOM formulations that retain in nasal cavity for longer duration of time may reduce the dose/frequency of vaccine for nasal immunization.
Keywords: Nasal vaccination; ISCOM matrices; Gamma scintigraphy; Mucocilliary clearance;

Poly(α-glutamic acid) combined with polycation as serum-resistant carriers for gene delivery by Cuifeng Wang; Min Feng; Jingjing Deng; Yuefang Zhao; Xin Zeng; Lina Han; Shirong Pan; Chuanbin Wu (237-245).
The transfection efficiency of cationic polymers decreases dramatically in the presence of serum, which hampers the in vivo application of these polymers for gene delivery. Due to its shielding effect of poly(α-glutamic acid) (PGA) from negatively charged serum proteins, it was introduced into DNA polyplexes to overcome the serum inhibitory effect. In the present studies, the transfection efficiency of DNA/PEI/PGA terplex system was compared to PEI 25 kDa and Lipofectamine 2000 in the presence of serum. The successful formation of DNA/PEI/PGA terplexes was confirmed by their near-neutral surface charge. Interaction between components in the terplex system demonstrated that PGA was competing with DNA to combine with PEI. PEI/PGA combined carriers were not cytotoxic at a C/N ratio higher than 0.3. The in vitro transfection efficiency of DNA/PEI/PGA terplexes was not significantly different from those of DNA/PEI25 kDa in serum-free medium. Importantly, in serum-containing medium, the DNA terplexes at their optimal C/N ratios maintained the same level of transfection efficiency as that of serum-free medium, even though the transfection efficiency of PEI 25 kDa and Lipofectamine 2000 was significantly decreased under serum-containing conditions. CLSM results confirmed that the cellular import of pDNA delivered by PEI/PGA combined carriers was more efficient than PEI 25 kDa alone under serum-containing conditions. Therefore, PGA could be used as a versatile serum-resistant reagent to overcome the serum inhibitory effect of polycations for gene delivery.
Keywords: Poly(α-glutamic acid); DNA terplexes; Serum inhibitory effect;

A critique and two corrigenda by Alexander T. Florence (246).

The original article to which this Corrigendum refers was published in International Journal of Pharmaceutics 367 (1–2) (2009) 1–15.
Keywords: Transdermal drug delivery; Pharmacokinetics; Interindividual variation; QSPR; Skin absorption; Transdermal patch;

Response to Farahmand and Maibach's Corrigenda by John C. Kissel; Annette L. Bunge (254-256).