Pharmaceutical Research (v.31, #7)

Crystal polymorphism of pharmaceuticals has well-known profound effects on the physical, chemical, and pharmaceutical properties of drugs, which can result in changes in the solubility, stability, dissolution, bioavailability, and efficacy of drugs. In this review article, famotidine (FAM), which has a well-known trade name of Pepcid®, was selected as a model drug. Although FAM has three polymorphs (forms A, B and C), forms A and B have been commonly discussed. The active pharmaceutical ingredient (API) in the commercial version of FAM is the metastable form B. FAM has been a concern of FDA because of the physical properties, solubilities, bioavailabilities, or bioequivalencies of the different polymorphic forms. In addition, a patent infringement suit of FAM polymorph had been made sound legal arguments in the pharmaceutical market. We review the solid-state characteristics, thermodynamics, polymorphic transformation, and quality control of FAM in drug products. In particular, pharmaceutical processes, such as grinding, compression, and heating temperature have a significant effect on the polymorphic transformation of FAM. Moreover, environmental humidity and residual water content should be well controlled to prevent polymorphic transformation of FAM during pharmaceutical processing. Several thermal and spectroscopic analytical techniques used for qualitative and quantitative determinations of polymorphic transformation of FAM after different treatments or quality control of FAM in the commercial tablets before and after the expiration dates have been discussed.
Keywords: characteristics; famotidine; polymorphic transformation; quality control; thermodynamics

Co-delivery of Dexamethasone and Green Tea Polyphenols Using Electrospun Ultrafine Fibers for Effective Treatment of Keloid by Jinrong Li; Rong Fu; Long Li; Guang Yang; Shan Ding; Zhendong Zhong; Shaobing Zhou (1632-1643).
The electrospun polymer ultrafine fiber meshes wereused to co-deliver dexamethasone (DEX) and green tea polyphenols (GTP) in order to acquire a suitable balance between effective treament of keloid and safety to the skin.This co-delivery system was prepared with a simple electrospinning technology. Keloid model was established on the back of athymic nude mice with the human keloid tissues and the formulated fiber meshes were applied onto keloids for an in vivo evaluation on their therapeutic effects.Unlike other therapeutic formulations, these fiber meshes as a new surgical dressing possess multiple useful functions, including the capabilities of maintaining a moist environment, resisting bacterial infection and controlling the drug release. Hydrophobic DEX molecules inside the fiber meshes can be released successfully from the channels formed by the early release of the hydrophilic GTP molecules and then transported across the skin. A distinctive result acquired from histological analysis shows that after 3-month treatment, the DEX/GTP-loaded fiber meshes significantly induce the degradation of collagen fibers in keloid on the back of nude mice compared to the traditional treatment.The dressing formulation based on nanofibers provides a promising platform for the treatment of keloid.
Keywords: Biodegradable; Drug delivery; Electrospinning; Polymer; Ultrafine

Interindividual Variability in the Cardiac Expression of Anthracycline Reductases in Donors With and Without Down Syndrome by Adolfo Quiñones-Lombraña; Daniel Ferguson; Rachael Hageman Blair; James L. Kalabus; Almedina Redzematovic; Javier G. Blanco (1644-1655).
The intracardiac synthesis of anthracycline alcohol metabolites (e.g., daunorubicinol) contributes to the pathogenesis of anthracycline-related cardiotoxicity. Cancer patients with Down syndrome (DS) are at increased risk for anthracycline-related cardiotoxicity. We profiled the expression of anthracycline metabolizing enzymes in hearts from donors with- and without- DS.Cardiac expression of CBR1, CBR3, AKR1A1, AKR1C3 and AKR7A2 was examined by quantitative real time PCR, quantitative immunoblotting, and enzyme activity assays using daunorubicin. The CBR1 polymorphism rs9024 was investigated by allelic discrimination with fluorescent probes. The contribution of CBRs/AKRs proteins to daunorubicin reductase activity was examined by multiple linear regression. CBR1 was the most abundant transcript (average relative expression; DS: 81%, non-DS: 58%), and AKR7A2 was the most abundant protein (average relative expression; DS: 38%, non-DS: 35%). Positive associations between cardiac CBR1 protein levels and daunorubicin reductase activity were found for samples from donors with- and without- DS. Regression analysis suggests that sex, CBR1, AKR1A1, and AKR7A2 protein levels were significant contributors to cardiac daunorubicin reductase activity. CBR1 rs9024 genotype status impacts on cardiac CBR1 expression in non-DS hearts.CBR1, AKR1A1, and AKR7A2 protein levels point to be important determinants for predicting the synthesis of cardiotoxic daunorubicinol in heart.
Keywords: Aldo-keto reductases; Anthracycline-related cardiotoxicity; Anthracyclines; Carbonyl reductases; Down syndrome

Effect of Crystallinity on Electrostatic Charging in Dry Powder Inhaler Formulations by Jennifer Wong; Philip Chi Lip Kwok; Tim Noakes; Ali Fathi; Fariba Dehghani; Hak-Kim Chan (1656-1664).
This study aimed to characterize inherent charge generated by micron-sized drug-only formulations of amorphous and crystalline salbutamol sulfate (SS).Amorphous SS was produced by spray-drying whilst crystalline SS was produced by conditioning spray-dried SS with supercritical CO2 and menthol. Electrostatic charge of the powders was characterized in two ways. Firstly, the charge profile of the aerosols dispersed from an Aerolizer® was measured using a modified Electrostatic Low Pressure Impactor (ELPI™). Secondly, the net charge of the bulk powders generated from tumbling in containers composed of different materials (polyethylene, polyvinyl chloride, Teflon, nylon and stainless steel) was measured by a Faraday pail.Following aerosolization, crystalline SS appeared to show more consistent charging and mass deposition than amorphous SS. In the tumbling experiment crystalline SS had a significant correlation between net charge and work function, which was absent in amorphous SS. This may be due to the long-range crystal packing which was reflected as more predictable charging. In addition, the polarity of charging was attributed to the arrangement of SS molecules in the crystal lattice.The effect of crystallinity on the electrostatic charge behavior of inhalable micron-sized spherical drug particles with well-defined particle size distribution was investigated for the first time. The knowledge gained may assist in the development of optimized inhaled pharmaceutical products.
Keywords: crystallinity; dry powder inhaler (DPI); electrostatic charge; salbutamol sulfate; supercritical fluid

A Novel Formulation Based on 2,3-Di(tetradecyloxy)propan-1-amine Cationic Lipid Combined with Polysorbate 80 for Efficient Gene Delivery to the Retina by Gustavo Puras Ochoa; Jon Zárate Sesma; Mireia Agirre Díez; Ariadna Díaz-Tahoces; Marcelino Avilés-Trigeros; Santiago Grijalvo; Ramón Eritja; Eduardo Fernández; Jose Luis Pedraz (1665-1675).
The aim of the present study was to evaluate the potential application of a novel formulation based on a synthesized cationic lipid 2,3-di(tetradecyloxy)propan-1-amine, combined with polysorbate 80 to deliver the pCMS-EGFP plasmid into the rat retina.We elaborated lipoplexes by mixing the formulation containing the cationic lipid and the polysorbate 80 with the plasmid at different cationic lipid/DNA ratios (w/w). Resulted lipoplexes were characterized in terms of size, charge, and capacity to condense, protect and release the DNA. In vitro transfection studies were performed in HEK-293 and ARPE-19 cells. Formulations were also tested in vivo by monitoring the expression of the EGFP after intravitreal and subretinal injections in rat eyes.At 2/1 cationic lipid/DNA mass ratio, the resulted lipoplexes had 200 nm of hydrodynamic diameter; were positive charged, spherical, protected DNA against enzymatic digestion and transfected efficiently HEK-293 and ARPE-19 cultured cells exhibiting lower cytotoxicity than LipofectamineTM 2000. Subretinal administrations transfected mainly photoreceptors and retinal pigment epithelial cells; whereas intravitreal injections produced a more uniform distribution of transfection through the inner part of the retina.These results hold great expectations for other gene delivery formulations based on this cationic lipid for retinal gene therapy purposes.
Keywords: cationic lipids; gene therapy; lipoplexes; non-viral vectors; retina

Commonly Used Excipients Modulate UDP-Glucuronosyltransferase 2B7 Activity to Improve Nalbuphine Oral Bioavailability in Humans by Hong-Jaan Wang; Cheng-Huei Hsiong; Shung-Tai Ho; Min-Jen Lin; Tung-Yuan Shih; Pei-Wei Huang; Oliver Yoa-Pu Hu (1676-1688).
Nalbuphine (NAL) is a potent opioid analgesic, but can only be administered by injection. The major aim of this study was to develop an oral NAL formulation employing known excipients as UDP-glucuronosyltransferase 2B7 (UGT2B7) inhibitors to improve its oral bioavailability.Twenty commonly used pharmaceutical excipients were screened in vitro by using liver microsomes to identify inhibitors of UGT2B7, the major NAL metabolic enzyme. Tween 20 and PEG 400 were potent UGT2B7 inhibitors and both were co-administered (Tween-PEG) with NAL to rats and humans for pharmacokinetic and/or pharmacodynamic analyses.In animal studies, oral Tween-PEG (4 mg/kg of each) significantly increased the area under the plasma NAL concentration-time curve (AUC) and the maximal plasma concentration (Cmax) by 4- and 5-fold, respectively. The results of the pharmacodynamic analysis were in agreement with those of the pharmacokinetic analysis, and showed that Tween-PEG significantly enhanced the analgesic effects of orally administered NAL. In humans, oral Tween-PEG (240 mg of each) also increased NAL Cmax 2.5-fold, and AUC by 1.6-fold.Tween-PEG successfully improved oral NAL bioavailability and could formulate a useful oral dosage form for patient’s convenience.
Keywords: bioavailability; excipient; nalbuphine; pharmacodynamic; pharmacokinetic

Our previous studies indicated that drug leaked from discoidal reconstituted high density lipoprotein (d-rHDL) during the remodeling behaviors induced by lecithin cholesterol acyl transferase (LCAT) abundant in circulation, thus decreasing the drug amount delivered into the target. In this study, arachidonic acid (AA)-modified d-rHDL loaded with lovastatin (LT) were engineered as AA-LT-d-rHDL to explore whether AA modification could reduce the drug leakage during the remodeling behaviors induced by LCAT and further deliver more drug into target cells to improve efficacy.After successful preparation of AA-LT-d-rHDL with different AA modification amount, a series of in vitro remodeling behaviors were investigated. Furthermore, inhibition on macrophage-derived foam cell formation was chosen to evaluate drug efficacy of AA-LT-d-rHDL. In vitro physicochemical characterizations studies showed that all LT-d-rHDL and AA-LT-d-rHDL preparations had nano-size, negative surface charge, high entrapment efficiency (EE) and comparable drug loading efficiency (DL). With increment of AA modification amount, AA-LT-d-rHDL manifested lower reactivity with LCAT, thus significantly reducing the undesired drug leakage during the remodeling behaviors induced by LCAT, eventually exerting stronger efficacy on inhibition of macrophage-derived foam cell formation.AA-LT-d-rHDL could decrease the drug leakage during the remodeling behaviors induced by LCAT and fulfill efficient drug delivery.
Keywords: arachidonic acid; d-rHDL; drug leakage; LCAT; remodeling behaviors

Assessment of Physical Stability of an Antibody Drug Conjugate by Higher Order Structure Analysis: Impact of Thiol- Maleimide Chemistry by Jianxin Guo; Sandeep Kumar; Amarnauth Prashad; Jason Starkey; Satish K. Singh (1710-1723).
To provide a systematic biophysical approach towards a better understanding of impact of conjugation chemistry on higher order structure and physical stability of an antibody drug conjugate (ADC).ADC was prepared using thiol-maleimide chemistry. Physical stabilities of ADC and its parent IgG1 mAb were compared using calorimetric, spectroscopic and molecular modeling techniques.ADC and mAb respond differently to thermal stress. Both the melting temperatures and heat capacities are substantially lower for the ADC. Spectroscopic experiments show that ADC and mAb have similar secondary and tertiary structures, but these are more easily destabilized by thermal stress on the ADC indicating reduced conformational stability. Molecular modeling calculations suggest a substantial decrease in the conformational energy of the mAb upon conjugation. The local surface around the conjugation sites also becomes more hydrophobic in the ADC, explaining the lower colloidal stability and greater tendency of the ADC to aggregate.Computational and biophysical analyses of an ADC and its parent mAb have provided insights into impact of conjugation on physical stability and pinpointed reasons behind lower structural stability and increased aggregation propensity of the ADC. This knowledge can be used to design appropriate formulations to stabilize the ADC.
Keywords: antibody drug conjugates; fluorescent dyes; molecular model; physical stability; thiol-maleimide chemistry

Direct Microneedle Array Fabrication Off a Photomask to Deliver Collagen Through Skin by Jaspreet Singh Kochhar; Parthiban Anbalagan; Sandeep Balu Shelar; Jun Kai Neo; Ciprian Iliescu; Lifeng Kang (1724-1734).
To fabricate microneedle arrays directly off a photomask using a simple photolithographical approach and evaluate their potential for delivering collagen.A simple photolithographical approach was developed by using photomask consisting of embedded micro-lenses that govern microneedle geometry in a mould free process. Microneedle length was controlled by use of simple glass scaffolds as well as addition of backing layer. The fabricated arrays were tested for their mechanical properties by using a force gauge as well as insertion into human skin with trypan blue staining. Microneedle arrays were then evaluated for the delivery of fluorescent collagen, which was evaluated using a confocal laser scanning microscope.Microneedles with sharp tips ranging between 41.5 ± 8.4 μm and 71.6 ± 13.7 μm as well as of two different lengths of 1336 ± 193 μm and 957 ± 171 μm were fabricated by using the photomasks. The microneedles were robust and resisted fracture forces up to 25 N. They were also shown to penetrate cadaver human skin samples with ease; especially microneedle arrays with shorter length of 957 μm penetrated up to 72% of needles. The needles were shown to enhance permeation of collagen through cadaver rat skin, as compared to passive diffusion of collagen.A simple and mould free approach of fabricating polymeric microneedle array is proposed. The fabricated microneedle arrays enhance collagen permeation through skin.
Keywords: collagen; microneedles; photolithography; transdermal drug delivery

To explore the application of solution calorimetry for measuring drug solubility in experimentally challenging situations while providing additional information on the physical properties of the solute material.A semi-adiabatic solution calorimeter was used to measure the heat of dissolution of prednisolone and chlorpropamide in aqueous solvents and of griseofulvin and ritonavir in viscous solutions containing polyvinylpyrrolidone and N-ethylpyrrolidone.Dissolution end point was clearly ascertained when heat generation stopped. The heat of solution was a linear function of dissolved mass for all drugs (<10% RSD, except for chlorpropamide). Heats of solution of 9.8 ± 0.8, 28.8 ± 0.6, 45.7 ± 1.6 and 159.8 ± 20.1 J/g were obtained for griseofulvin, ritonavir, prednisolone and chlorpropamide, respectively. Saturation was identifiable by a plateau in the heat signal and the crossing of the two linear segments corresponds to the solubility limit. The solubilities of prednisolone and chlopropamide in water by the calorimetric method were 0.23 and 0.158 mg/mL, respectively, in agreement with the shake-flask/HPLC-UV determined values of 0.212 ± 0.013 and 0.169 ± 0.015 mg/mL, respectively. For the higher solubility and high viscosity systems of griseofulvin and ritonavir in NEP/PVP mixtures, respectively, solubility values of 65 and 594 mg/g, respectively, were obtained.Solution calorimetry offers a reliable method for measuring drug solubility in organic and aqueous solvents. The approach is complementary to the traditional shake-flask method, providing information on the solid properties of the solute. For highly viscous solutions, the calorimetric approach is advantageous.
Keywords: crystal lattice; drug-polymer dispersions; heat of solution; mixed solvents; solubility; solution calorimetry

In this study we explore the use of nano-scale localized thermal analysis (LTA) and transition temperature microcopy (TTM) as a novel combined approach to studying phase separation in HME dispersions of cyclosporine A in Eudragit EPO.Modulated temperature differential scanning calorimetry (MTDSC), attenuated total reflectance FTIR spectroscopy, nano-LTA and TTM were performed on raw materials and dispersions prepared by hot melt extrusion (HME) and spin coating. For samples prepared by HME, two mixing temperatures (110°C and 150°C) and residence times (5 and 15 min) were investigated.Spin coated samples showed an intermediate T g for the mixed systems consistent with molecular dispersion formation. The HME samples prepared at 110°C showed evidence of inhomogeneity using MTDSC and FTIR, while those produced at 150°C h showed evidence for the formation of a single phase system using MTDSC. The nanothermal methods, however, indicated the presence of phase separated cyclosporine A at the higher preparation temperature while the TTM was able to map regions of differing penetration temperatures, indicating the presence of compositionally inhomogeneous regions in all but the high processing temperature/high residence time samples.TTM is a potentially important new method for studying phase separation and that such separation may remain undetected or poorly understood using conventional bulk analytical techniques.
Keywords: hot melt extrusion; localized thermal analysis; solid dispersions; transition temperature microscopy

Development of 2D and 3D Mucus Models and Their Interactions with Mucus-Penetrating Paclitaxel-Loaded Lipid Nanocapsules by Anne-Claire Groo; Kristina Mircheva; Jérôme Bejaud; Caroline Ailhas; Ivan Panaiotov; Patrick Saulnier; Tzvetanka Ivanova; Frederic Lagarce (1753-1765).
To study, diffusion through mucus (3D model) of different formulations of paclitaxel loaded lipid nanocapsules (Ptx-LNCs), to interpret the results in the light of LNC behavior at air-mucus interface (2D model).LNC surface properties were modified with chitosan or poly(ethylene glycol) (PEG) coatings of different size (PEG 2,000 to 5,000 Da) and surface charges. LNC diffusion through 446 μm pig intestinal mucus layer was studied using Transwell®. LNCs were spread at the air-water-mucus interface then interfacial pressure and area changes were monitored and the efficiency of triglyceride (TG) inclusion was determined.Ptx-LNCs of surface charges ranging from −35.7 to +25.3 mV were obtained with sizes between 56.2 and 75.1 nm. The diffusion of paclitaxel in mucus was improved after encapsulation in neutral or positively charged particles (p < 0.05 vs Taxol®). No significative difference was observed in the 2,000–5,000 PEG length for diffusion both on the 2D or 3D models. On 2D model positive or neutral LNCs interacted less with mucus. Highest efficiency of TG inclusion was observed for particles with smallest PEG length.The results obtained with 2D and 3D model allowed us to select the best candidates for in vivo studies (neutral or positive LNCs with smaller PEG length).
Keywords: diffusion; mucus; nanoparticles; oral delivery; paclitaxel

Thermodynamic and Kinetic Investigation on the Crucial Factors Affecting Adefovir Dipivoxil-Saccharin Cocrystallization by Kun Ma; Ying Zhang; Hongliang Kan; Linfeng Cheng; Ling Luo; Qing Su; Jing Gao; Yuan Gao; Jianjun Zhang (1766-1778).
The aim of this study was to perform a thermodynamic and kinetic investigation on the crucial factors affecting the cocrystallization between adefovir dipivoxil (AD) and saccharin (SAC).Phase solubility diagrams and ternary phase diagrams were constructed based on the solubility data of AD, SAC and their cocrystals in ethanol, isopropanol and ethyl acetate at different temperatures. The conductimetric method was used to determine the induction time. A quantitative and intuitive technique modified from dissolution testing was employed to investigate the cocrystallization kinetics.AD-SAC cocrystals exhibited different crystal habits but only one cocrystal polymorph was confirmed. The effects of several crucial factors, including the input amounts of two components, AD/SAC ratio, solvent and temperature, on the crystallization of single-component alone, cocrystal formation, cocrystal stability, supersaturation, nucleation, crystal growth and cocrystal yield were determined. Thermodynamic and kinetic parameters provided the rationale for this spontaneous cocrystallization system without the need of solvent evaporation and temperature change.This systemic investigation enriched the present understanding of thermodynamics and kinetics of cocrystals and built the groundwork for AD-SAC cocrystal scale-up.
Keywords: adefovir dipivoxil; cocrystal; kinetics; nucleation; thermodynamics

The purpose of this study was to present a modified Andersen cascade impactor (ACI) as a platform to evaluate the deposition and subsequent transport of aerosol micropaticles across airway epithelial cells.The impaction plate of an ACI was modified to accommodate up to eight Snapwells. Aerodynamic particle size distribution of the modified ACI was investigated with two commercially available formulations of Ventolin® (salbutamol sulphate) and QVAR® (beclomethasone dipropionate). Deposition and transport of these drug microparticles across sub-bronchial epithelial Calu-3 cells were also studied.The modified ACI demonstrated reproducible deposition patterns of the commercially available pressurised metered dose inhalers compared to the standard ACI. Furthermore, the Calu-3 cells could be placed in different stages of the modified ACI. No significant effect was observed among the transport rate of different particle sizes deposited on Calu-3 cells within the range of 3.3 to 0.4 μm.The use of the cell compatible ACI to assess the fate of microparticles after deposition on the respiratory epithelia may allow for better understanding of deposited microparticles in vivo.
Keywords: aerodynamic size; Andersen cascade impactor; Calu-3; epithelial transport; QVAR; Ventolin

Bioactive Equivalence of Combinatorial Components Identified in Screening of an Herbal Medicine by Peng Liu; Hua Yang; Fang Long; Hai-Ping Hao; Xiaojun Xu; Ying Liu; Xiao-Wei Shi; Dan-Dan Zhang; Hao-Chuan Zheng; Qian-Ying Wen; Wen-Wen Li; Hui Ji; Xi-Juan Jiang; Bo-Li Zhang; Lian-Wen Qi; Ping Li (1788-1800).
To identify bioactive equivalent combinatorial components (BECCs) in herbal medicines. The exact composition of effective components in herbal medicines is often elusive due to the lack of adequate screening methodology. Herein, we propose a hypothesis that BECCs accounting for the whole efficacy of original herbal medicines could be discovered from a complex mixture of constituents.We developed a bioactive equivalence oriented feedback screening method and applied it to discover the BECCs from an herbal preparation Cardiotonic Pill (CP). The operations include chemical profiling of CP, followed by an iterative loop of determining, collecting and evaluating candidate BECCs.A combination of 18 compounds was identified as BECCs from CP, which accounts for 15.0% (w/w) of original CP. We have demonstrated that the BECCs were as effective as CP in cell models and in a rat model of myocardial infarction.This work answers the key question of which are real bioactive components for CP that have been used in clinic for many years, and provides a promising approach for discovering BECCs from herbal medicines. More importantly, the BECCs could be extended to improve quality control of herbal products and inspire an herbal medicines based discovery of combinatorial therapeutics.
Keywords: bioactive equivalence; Cardiotonic Pill; combinatorial components screening; herbal medicines

Population Pharmacokinetic Analysis of Simvastatin and its Active Metabolite with the Characterization of Atypical Complex Absorption Kinetics by Seok-Joon Jin; Kyun-Seop Bae; Sang-Heon Cho; Jin-Ah Jung; Unjib Kim; Sangmin Choe; Jong-Lyul Ghim; Yook-Hwan Noh; Hyun-Jung Park; Hee-sun Kim; Hyeong-Seok Lim (1801-1812).
The pharmacokinetics of simvastatin is complex with multiple peaks in the absorption phase, which cannot be adequately described by a conventional first order absorption model. The biotransformation of simvastatin into simvastatin acid, an active metabolite, is reversible. This study evaluated the pharmacokinetics of simvastatin and simvastatin acid, focusing on the absorption kinetics.Data were collected from three bioequivalence studies, in which subjects were administered 60 mg simvastatin, and from one crossover study, in which subjects were administered two doses randomly selected from 10, 20, 30, 40 to 80 mg simvastatin with washout period. The pharmacokinetics of simvastatin was assessed in 133 healthy males. Plasma concentrations of simvastatin and simvastatin acid were measured in 2,182 and 2,130 samples, respectively, and the pharmacokinetic data were analyzed using NONMEM.The time course of changes in the plasma simvastatin concentration was best described by a two-compartment linear model with three parallel absorption processes, each of which consisted of mixed zero-and first order absorption. Additions of inter-occasional variability to the absorption parameters significantly improved the model’s fit. The disposition parameter estimates were significantly different when different absorption models were applied, indicating the importance of the appropriate absorption modeling. Pharmacokinetic modeling preferred the inter-conversion between simvastatin and simvastatin acid.A pharmacokinetic model describing the complex, multiple peak, absorption kinetics of simvastatin was formulated using three parallel, mixed zero and first-order absorptions. This type of absorption model may be applicable to other drugs that show irregular, multiple-peak concentrations during their absorption phase.
Keywords: absorption model; multiple peaks; NONMEM; simvastatin; simvastatin acid

Similar Maximum Systemic but not Local Cyclooxygenase-2 Inhibition by 50 mg Lumiracoxib and 90 mg Etoricoxib: A Randomized Controlled Trial in Healthy Subjects by Lisa Felden; Carmen Walter; Carlo Angioni; Yannick Schreiber; Nils von Hentig; Nerea Ferreiros; Gerd Geisslinger; Jörn Lötsch (1813-1822).
Once daily doses of 100–400 mg lumiracoxib have been proposed to inhibit local prostaglandin synthesis longer than systemic prostaglandin synthesis due to local accumulation in inflamed, acidic tissue. Lower, less toxic doses, however, might still achieve the clinical goal and merit further contemplation.In a randomized, double-blind, placebo-controlled, three-way cross-over study, 18 healthy men received, with an interval of 24 h, two oral doses of 50 mg lumiracoxib or for comparison, 90 mg etoricoxib, for which local tissue accumulation has not been claimed as therapeutic component. Systemic and local drug concentrations, assessed by means of subcutaneous in-vivo microdialysis, were related to COX-2 inhibiting effects, quantified as inhibition of prostaglandin ex-vivo production in whole blood as well as local tissue prostaglandin (PG) concentrations.Twenty-four hours after the first dose, only etoricoxib was detectable in plasma and inhibited PGE2 production. In contrast, after the second dose, systemic PGE2 concentrations were significantly reduced by both coxibs, indicating similar maximum systemic effects of the selected doses. The local COX-2 inhibition by etoricoxib was most pronounced for PGD2. To the contrary, no indication was given of local inhibition of PG production by lumiracoxib at the dose tested.Doses of 50 mg lumiracoxib and 90 mg etoricoxib produced similar maximum inhibition of systemic COX-2 function whereas 50 mg lumiracoxib was ineffective in producing local COX-2 inhibition. At a 50 mg dosage, lumiracoxib does not provide peripheral effects that outlast its systemic actions in therapies of rheumatic diseases such as osteoarthritis.
Keywords: COX-2; microdialysis; pharmacokinetcs; prostaglandins

One Mouse, One Pharmacokinetic Profile: Quantitative Whole Blood Serial Sampling for Biotherapeutics by Alison P. Joyce; Mengmeng Wang; Rosemary Lawrence-Henderson; Cynthia Filliettaz; Sheldon S. Leung; Xin Xu; Denise M. O’Hara (1823-1833).
The purpose of this study was to validate the approach of serial sampling from one mouse through ligand binding assay (LBA) quantification of dosed biotherapeutic in diluted whole blood to derive a pharmacokinetic (PK) profile.This investigation compared PK parameters obtained using serial and composite sampling methods following administration of human IgG monoclonal antibody. The serial sampling technique was established by collecting 10 μL of blood via tail vein at each time point following drug administration. Blood was immediately diluted into buffer followed by analyte quantitation using Gyrolab to derive plasma concentrations. Additional studies were conducted to understand matrix and sampling site effects on drug concentrations.The drug concentration profiles, irrespective of biological matrix, and PK parameters using both sampling methods were not significantly different. There were no sampling site effects on drug concentration measurements except that concentrations were slightly lower in sodium citrated plasma than other matrices.We recommend the application of mouse serial sampling, particularly with limiting drug supply or specialized animal models. Overall the efficiencies gained by serial sampling were 40–80% savings in study cost, animal usage, study length and drug conservation while inter-subject variability across PK parameters was less than 30%.
Keywords: gyrolab; ligand-binding assay (LBA); mouse serial sampling; pharmacokinetics (PK)

Biodistribution and Pharmacokinetics of Dapivirine-Loaded Nanoparticles after Vaginal Delivery in Mice by José das Neves; Francisca Araújo; Fernanda Andrade; Mansoor Amiji; Maria Fernanda Bahia; Bruno Sarmento (1834-1845).
To assess the potential of polymeric nanoparticles (NPs) to affect the genital distribution and local and systemic pharmacokinetics (PK) of the anti-HIV microbicide drug candidate dapivirine after vaginal delivery.Dapivirine-loaded, poly(ethylene oxide)-coated poly(epsilon-caprolactone) (PEO-PCL) NPs were prepared by a nanoprecipitation method. Genital distribution of NPs and their ability to modify the PK of dapivirine up to 24 h was assessed after vaginal instillation in a female mouse model. Also, the safety of NPs upon daily administration for 14 days was assessed by histological analysis and chemokine/cytokine content in vaginal lavages.PEO-PCL NPs (180–200 nm) were rapidly eliminated after administration but able to distribute throughout the vagina and lower uterus, and capable of tackling mucus and penetrate the epithelial lining. Nanocarriers modified the PK of dapivirine, with higher drug levels being recovered from vaginal lavages and vaginal/lower uterine tissues as compared to a drug suspension. Systemic drug exposure was reduced when NPs were used. Also, NPs were shown safe upon administration for 14 days.Dapivirine-loaded PEO-PCL NPs were able to provide likely favorable genital drug levels, thus attesting the potential value of using this vaginal drug delivery nanosystem in the context of HIV prophylaxis.
Keywords: HIV/AIDS; microbicides; poly(ε-caprolactone); pre-exposure prophylaxis; vaginal drug delivery

Novel Hollow Microneedle Technology for Depth-Controlled Microinjection-Mediated Dermal Vaccination: A Study with Polio Vaccine in Rats by Koen van der Maaden; Sebastiaan J. Trietsch; Heleen Kraan; Eleni Maria Varypataki; Stefan Romeijn; Raphäel Zwier; Heiko J. van der Linden; Gideon Kersten; Thomas Hankemeier; Wim Jiskoot; Joke Bouwstra (1846-1854).
The aim of the study was to develop a cheap and fast method to produce hollow microneedles and an applicator for injecting vaccines into the skin at a pre-defined depth and test the applicability of the system for dermal polio vaccination.Hollow microneedles were produced by hydrofluoric acid etching of fused silica capillaries. An electromagnetic applicator was developed to control the insertion speed (1–3 m/s), depth (0–1,000 μm), and angle (10°–90°). Hollow microneedles with an inner diameter of 20 μm were evaluated in ex vivo human skin and subsequently used to immunize rats with inactivated poliovirus vaccine (IPV) by an intradermal microinjection of 9 μL at a depth of 300 μm and an insertion speed of 1 m/s. Rat sera were tested for IPV-specific IgG and virus-neutralizing antibodies.Microneedles produced from fused silica capillaries were successfully inserted into the skin to a chosen depth, without clogging or breakage of the needles. Intradermal microinjection of IPV induced immune responses comparable to those elicited by conventional intramuscular immunization.We successfully developed a hollow microneedle technology for dermal vaccination that enables fundamental research on factors, such as insertion depth and volume, and insertion angle, on the immune response.
Keywords: intradermal immunization; microinjections; microneedles; polio vaccine; vaccine delivery

The blood–brain barrier (BBB) represents a target for therapeutic intervention and an obstacle for brain drug delivery. Targeting endocytic receptors on brain endothelial cells (ECs) helps transport drugs and carriers into and across this barrier. While most receptors tested are associated with clathrin-mediated pathways, clathrin-independent routes are rather unexplored. We have examined the potential for one of these pathways, cell adhesion molecule (CAM)-mediated endocytosis induced by targeting intercellular adhesion molecule -1 (ICAM-1), to transport drug carriers into and across BBB models.Model polymer nanocarriers (NCs) coated with control IgG or antibodies against ICAM-1 (IgG NCs vs. anti-ICAM NCs; ~250-nm) were incubated with human brain ECs, astrocytes (ACs), or pericytes (PCs) grown as monocultures or bilayered (endothelial+subendothelial) co-cultures.ICAM-1 was present and overexpressed in disease-like conditions on ECs and, at a lesser extent, on ACs and PCs which are BBB subendothelial components. Specific targeting and CAM-mediated uptake of anti-ICAM NCs occurred in these cells, although this was greater for ECs. Anti-ICAM NCs were transported across endothelial monolayers and endothelial+subendothelial co-cultures modeling the BBB.CAM-mediated transport induced by ICAM-1 targeting operates in endothelial and subendothelial cellular components of the BBB, which may provide an avenue to overcome this barrier.
Keywords: blood–brain barrier transport; brain endothelial and subendothelial cell layers; CAM-mediated endocytosis; clathrin- and caveolae-independent transport; ICAM-1-targeted nanocarriers

Biorelevant In Vitro Performance Testing of Orally Administered Dosage Forms—Workshop Report by Christos Reppas; Horst-Dieter Friedel; Amy R. Barker; Lucinda F. Buhse; Todd L. Cecil; Susanne Keitel; Johannes Kraemer; J. Michael Morris; Vinod P. Shah; Mary P. Stickelmeyer; Chikako Yomota; Cynthia K. Brown (1867-1876).
Biorelevant in vitro performance testing of orally administered dosage forms has become an important tool for the assessment of drug product in vivo behavior. An in vitro performance test which mimics the intraluminal performance of an oral dosage form is termed biorelevant. Biorelevant tests have been utilized to decrease the number of in vivo studies required during the drug development process and to mitigate the risk related to in vivo bioequivalence studies. This report reviews the ability of current in vitro performance tests to predict in vivo performance and generate successful in vitro and in vivo correlations for oral dosage forms. It also summarizes efforts to improve the predictability of biorelevant tests. The report is based on the presentations at the 2013 workshop, Biorelevant In Vitro Performance Testing of Orally Administered Dosage Forms, in Washington, DC, sponsored by the FIP Dissolution/Drug Release Focus Group in partnership with the American Association of Pharmaceutical Scientists (AAPS) and a symposium at the AAPS 2012 Annual meeting on the same topic.
Keywords: biorelevant; dissolution; in vitro testing; oral dosage forms; quality control

Erratum to: Nanobubble Ultrasound Contrast Agents for Enhanced Delivery of Thermal Sensitizer to Tumors Undergoing Radiofrequency Ablation by Reshani H. Perera; Luis Solorio; Hanping Wu; Mihika Gangolli; Eric Silverman; Christopher Hernandez; Pubudu M. Peiris; Ann-Marie Broome; Agata A. Exner (1877-1877).

AAPS Connection (1878-1880).