Pharmaceutical Research (v.32, #12)

Predicting Clearance Mechanism in Drug Discovery: Extended Clearance Classification System (ECCS) by Manthena V. Varma; Stefanus J. Steyn; Charlotte Allerton; Ayman F. El-Kattan (3785-3802).
Early prediction of clearance mechanisms allows for the rapid progression of drug discovery and development programs, and facilitates risk assessment of the pharmacokinetic variability associated with drug interactions and pharmacogenomics. Here we propose a scientific framework – Extended Clearance Classification System (ECCS) – which can be used to predict the predominant clearance mechanism (rate-determining process) based on physicochemical properties and passive membrane permeability. Compounds are classified as: Class 1A – metabolism as primary systemic clearance mechanism (high permeability acids/zwitterions with molecular weight (MW) ≤400 Da), Class 1B – transporter-mediated hepatic uptake as primary systemic clearance mechanism (high permeability acids/zwitterions with MW >400 Da), Class 2 – metabolism as primary clearance mechanism (high permeability bases/neutrals), Class 3A –renal clearance (low permeability acids/zwitterions with MW ≤400 Da), Class 3B – transporter mediated hepatic uptake or renal clearance (low permeability acids/zwitterions with MW >400 Da), and Class 4 – renal clearance (low permeability bases/neutrals). The performance of the ECCS framework was validated using 307 compounds with single clearance mechanism contributing to ≥70% of systemic clearance. The apparent permeability across clonal cell line of Madin − Darby canine kidney cells, selected for low endogenous efflux transporter expression, with a cut-off of 5 × 10−6 cm/s was used for permeability classification, and the ionization (at pH7) was assigned based on calculated pKa. The proposed scheme correctly predicted the rate-determining clearance mechanism to be either metabolism, hepatic uptake or renal for ~92% of total compounds. We discuss the general characteristics of each ECCS class, as well as compare and contrast the framework with the biopharmaceutics classification system (BCS) and the biopharmaceutics drug disposition classification system (BDDCS). Collectively, the ECCS framework is valuable in early prediction of clearance mechanism and can aid in choosing the right preclinical tool kit and strategy for optimizing drug exposure and evaluating clinical risk of pharmacokinetic variability caused by drug interactions and pharmacogenomics.
Keywords: extended clearance classification system (ECCS); hepatic uptake; metabolism; permeability; renal clearance

Quantitative Correlation between Viscosity of Concentrated MAb Solutions and Particle Size Parameters Obtained from Small-Angle X-ray Scattering by Masakazu Fukuda; Chifumi Moriyama; Tadao Yamazaki; Yoshimi Imaeda; Akiko Koga (3803-3812).
To investigate the relationship between viscosity of concentrated MAb solutions and particle size parameters obtained from small-angle X-ray scattering (SAXS).The viscosity of three MAb solutions (MAb1, MAb2, and MAb3; 40–200 mg/mL) was measured by electromagnetically spinning viscometer. The protein interactions of MAb solutions (at 60 mg/mL) was evaluated by SAXS. The phase behavior of 60 mg/mL MAb solutions in a low-salt buffer was observed after 1 week storage at 25°C.The MAb1 solutions exhibited the highest viscosity among the three MAbs in the buffer containing 50 mM NaCl. Viscosity of MAb1 solutions decreased with increasing temperature, increasing salt concentration, and addition of amino acids. Viscosity of MAb1 solutions was lowest in the buffer containing histidine, arginine, and aspartic acid. Particle size parameters obtained from SAXS measurements correlated very well with the viscosity of MAb solutions at 200 mg/mL. MAb1 exhibited liquid–liquid phase separation at a low salt concentration.Simultaneous addition of basic and acidic amino acids effectively suppressed intermolecular attractive interactions and decreased viscosity of MAb1 solutions. SAXS can be performed using a small volume of samples; therefore, the particle size parameters obtained from SAXS at intermediate protein concentration could be used to screen for low viscosity antibodies in the early development stage.
Keywords: MAb; SAXS; Viscosity

Effect of siRNA pre-Exposure on Subsequent Response to siRNA Therapy by Hamidreza Montazeri Aliabadi; Parvin Mahdipoor; Cezary Kucharsky; Nicole Chan; Hasan Uludağ (3813-3826).
An alternative cancer therapy based on RNA interference (RNAi) has shown considerable promise but the possibility of resistance development is not known. This study explored the possibility of therapeutic resistance against siRNA nanoparticles in human cancer cells.Two approaches to siRNA treatment were undertaken using lipid-modified polyethylenimines, a single high concentration (shock) and repeated increasing concentrations (gradual). The targets were Mcl-1, RPS6KA5 and KSP in MDA-MB-435 cells.There was no evidence of resistance development in shock-treated cells, while the decrease in mRNA levels of targeted proteins was not as robust in naïve cells in gradual treatment. However, silencing efficiency was restored after a 7-day recovery period when expression of suppressed proteins returned to normal levels. Cellular uptake of siRNA was not affected by pre-treatments. Other mediators involved in cell survival and proliferation were altered in siRNA-treated cells, but only JUN silencing led to a heightened loss of viability. In vivo experiments demonstrated similar silencing efficiency at mRNA level after repeat doses.Human cancer cells responded to repeat siRNA nanoparticles in a similar fashion after a temporary initial alteration and little, if any, resistance was evident against repeated siRNA treatments.
Keywords: siRNA; cancer therapy; resistance; polymeric carriers

Potent Functional Immunogenicity of Plasmodium falciparum Transmission-Blocking Antigen (Pfs25) Delivered with Nanoemulsion and Porous Polymeric Nanoparticles by Rajesh Kumar; Grace Ledet; Richard Graves; Dibyadyuti Datta; Shana Robinson; Geetha P. Bansal; Tarun Mandal; Nirbhay Kumar (3827-3836).
To evaluate functional immunogenicity of CHrPfs25. a malaria transmission blocking vaccine antigen, using nanoemulsion and porous polymeric PLGA nanoparticles.CHrPfs25 was formulated with nanoemulsions (NE) and poly(D,L-lactide-co-glycolide) nanoparticles (PLGA-NP) and evaluated via IM route in mice. Transmission blocking efficacy of antibodies was evaluated by standard mosquito membrane feeding assay using purified IgG from immune sera. Physicochemical properties and stability of various formulations were evaluated by measuring poly-dispersity index, particle size and zeta potential.Mice immunized with CHrPfs25 using alum via IP and IM routes induced comparable immune responses. The highest antibody response was obtained with CHrPfs25 formulated in 4% NE as compared to 8% NE and PLGA-NP. No further increases were observed by combining NE with MPL-A and chitosan. One hundred percent transmission blocking activity was demonstrated at 400 μg/ml of IgG for alum groups (both routes IP and IM), 4% NE and NE-MPL-A. Purified IgG from various adjuvant groups at lower doses (100 μg/mL) still exhibited >90% transmission blocking activity, while 52–81% blocking was seen at 50 μg/mL.Results suggest that CHrPfs25 delivered in various adjuvants / nanoparticles elicited strong functional immunogenicity in pre-clinical studies in mice. We are now continuing these studies to develop effective vaccine formulations for further evaluation of immune correlates of relative immunogenicity of CHrPfs25 in various adjuvants and clinical trials.
Keywords: malaria; nanoemulsion; PLGA nanoparticles; vaccine

Intranasal H102 Peptide-Loaded Liposomes for Brain Delivery to Treat Alzheimer’s Disease by Xiaoyao Zheng; Xiayan Shao; Chi Zhang; Yuanzhen Tan; Qingfeng Liu; Xu Wan; Qizhi Zhang; Shumei Xu; Xinguo Jiang (3837-3849).
H102, a novel β-sheet breaker peptide, was encapsulated into liposomes to reduce its degradation and increase its brain penetration through intranasal administration for the treatment of Alzheimer’s disease (AD).The H102 liposomes were prepared using a modified thin film hydration method, and their transport characteristics were tested on Calu-3 cell monolayers. The pharmacokinetics in rats’ blood and brains were also investigated. Behavioral experiments were performed to evaluate the improvements on AD rats’ spatial memory impairment. The neuroprotective effects were tested by detecting acetylcholinesterase (AchE), choline acetyltransferase (ChAT) and insulin degrading enzyme (IDE) activity and conducting histological assays. The safety was evaluated on rats’ nasal mucosa and cilia.The liposomes prepared could penetrate Calu-3 cell monolayers consistently. After intranasal administration, H102 could be effectively delivered to the brain, and the AUC of H102 liposomes in the hippocampus was 2.92-fold larger than that of solution group. H102 liposomes could excellently ameliorate spatial memory impairment of AD model rats, increase the activities of ChAT and IDE and inhibit plaque deposition, even in a lower dosage compared with H102 intranasal solution. H102 nasal formulations showed no toxicity on nasal mucosa.The H102-loaded liposome prepared in this study for nasal administration is stable, effective and safe, which has great potential for AD treatment.
Keywords: H102 peptide; Liposome; Intranasal administration; Brain delivery; Alzheimer’s disease (AD)

Inhalable Clarithromycin Microparticles for Treatment of Respiratory Infections by Frantiescoli Dimer; Cristiane de Souza Carvalho-Wodarz; Jörg Haupenthal; Rolf Hartmann; Claus-Michael Lehr (3850-3861).
The aim of this work was to develop clarithromycin microparticles (CLARI-MP) and evaluate their aerodynamic behavior, safety in bronchial cells and anti-bacterial efficacy.Microparticles containing clarithromycin were prepared as dry powder carrier for inhalation, using leucine and chitosan. CLARI-MP were deposited on Calu-3 grown at air-interface condition, using the pharmaceutical aerosol deposition device on cell cultures (PADDOCC). Deposition efficacy, transport across the cells and cytotoxicity were determined. Anti-antibacterial effect was evaluated against Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus.Microparticles were of spherical shape, smooth surface and size of about 765 nm. Aerosolization performance showed a fine particle fraction (FPF) of 73.3%, and a mass median aerodynamic diameter (MMAD) of 1.8 μm. Deposition on Calu-3 cells using the PADDOCC showed that 8.7 μg/cm2 of deposited powder were transported to the basolateral compartment after 24 h. The safety of this formulation is supported by the integrity of the cellular epithelial barrier and absence of toxicity, and the antimicrobial activity demonstrated for Gram positive and Gram negative bacteria.The appropriate aerodynamic properties and the excellent deposition on Calu-3 cells indicate that clarithromycin microparticles are suitable for administration via pulmonary route and are efficient to inhibit bacteria proliferation.
Keywords: aerosol; dry powder inhaler; lung infection; safety

Enhanced Specificity and Drug Delivery in Tumors by cRGD - Anchoring Thermosensitive Liposomes by Bilyana M. Dicheva; Timo L. M. ten Hagen; Ann L. B. Seynhaeve; Mohamadreza Amin; Alexander M. M. Eggermont; Gerben A. Koning (3862-3876).
To develop RGD-targeted thermosensitive liposomes with increased tumor retention, improving drug release efficiency upon mild hyperthermia (HT) in both tumor and angiogenic endothelial cells.Standard termosensitive liposomes (TSL) and TSL containing a cyclic Arg-Gly-Asp (cRGD) pentapeptide with the sequence Arg-Cys-D-Phe-Asp-Gly (RGDf[N-Met]C) were synthetized, loaded with Dox and characterized. Temperature- and time-dependent drug release profiles were assessed by fluorometry. Intracellular Dox delivery was studied by flow cytometry and confocal microscopy. Cytotoxic effect of TSL and RGD-TSL was studied on B16Bl6 melanoma, B16F10 melanoma and HUVEC. Intravital microscopy was performed on B16Bl6 tumors implanted in dorsal-skin fold window-bearing mice. Pharmacokinetic and biodistribution of Dox-TSL and Dox-RGD-TSL were followed in B16Bl6 tumor bearing mice upon normothermia or initial hyperthermia conditions.DLS and cryo-TEM revealed particle homogeneity and size of around 85 nm. Doxorubicin loading efficiency was >95%as assessed by spectrofluorometry. Flow cytometry and confocal microscopy showed a specific uptake of RGD-TSL by melanoma and endothelial cells when compared to TSL and an increased doxorubicin delivery. High resolution intravital microscopy demonstrated specific accumulation of RGD-TSL to the tumor vasculature. Moreover, application of hyperthermia resulted in massive drug release from RGD-TSL. Biodistribution studies showed that initial hyperthermia increases Dox uptake in tumors from TSL and RGD-TSL.RGD-TSL have potency to increase drug efficacy due to higher uptake by tumor and angiogenic endothelial cells in combination with heat-triggered drug release.
Keywords: Doxorubicin; drug delivery; hyperthermia; RGD - thermosensitive liposomes

Histological Quantification of Gene Silencing by Intratracheal Administration of Dry Powdered Small-Interfering RNA/Chitosan Complexes in the Murine Lung by Daisuke Ihara; Noboru Hattori; Yasushi Horimasu; Takeshi Masuda; Taku Nakashima; Tadashi Senoo; Hiroshi Iwamoto; Kazunori Fujitaka; Hirokazu Okamoto; Nobuoki Kohno (3877-3885).
The use of small-interfering RNA (siRNA) as an inhalation therapy has recently received much attention. Some reports have confirmed the suppression of gene expression in whole lungs following intratracheal administration of dry powdered siRNA; however, the anatomical location in the lung where gene silencing occurs has not been precisely identified. Here, we aimed to histologically evaluate gene silencing efficacy in murine lungs by intratracheal administration of an siRNA/chitosan complex as a dry powder.Enhanced green fluorescence protein (EGFP)-specific siRNA (EGFP-siRNA)/chitosan powder was prepared and administered intratracheally to EGFP transgenic mice or mice carrying metastatic lung tumors consisting of Lewis lung carcinoma (LLC) cells stably expressing EGFP (EGFP-LLCs). Thereafter, green fluorescence intensities were quantified in the airways, parenchyma, and lung tumors.Intratracheal administration of the EGFP-siRNA/chitosan powder suppressed EGFP expression in the bronchi, bronchioles, and alveolar walls of EGFP transgenic mice. Additionally, EGFP-siRNA/chitosan effectively silenced EGFP expression in lung tumors consisting of EGFP-LLC cells.Pulmonary administration of siRNA/chitosan powder suppressed gene expression throughout the lung and in lung tumors. Therefore, this may become a powerful strategy to target genes expressed in a wide range of respiratory diseases involving the airways, parenchyma, and lung tumors.
Keywords: airway epithelium; alveolar wall; dry powdered siRNA/chitosan complex; intratracheal administration; metastatic lung tumor

PLGA-Based Nanoparticles: a Safe and Suitable Delivery Platform for Osteoarticular Pathologies by Mathieu Riffault; Jean-Luc Six; Patrick Netter; Pierre Gillet; Laurent Grossin (3886-3898).
Despite the promising applications of PLGA based particles, studies examining the fate and consequences of these particles after intra-articular administration in the joint are scanty. This study was carried out to evaluate the neutrality of the unloaded delivery system on different articular cell types. To facilitate tracking, we have thus developed a fluorescent core of particles, combined to a hyaluronate shell for cell recognition.Fluorescence pictures were taken at time intervals to assess the internalization and the corresponding inflammatory response was monitored by RT-qPCR and biochemical measurements. After NPs pre-treatment, mesenchymal stem cells (MSCs) were cultured into chondrogenic, adipogenic or osteogenic differentiation media, to investigate if NPs exposure interferes with differentiation ability. Finally, intra-articular injections were performed in healthy rat knees and joint’s structure analysed by histological studies.Particles were detected in cytoplasm 8 h after exposure. Internalization led to a slight and reversible increase of inflammatory markers, but lower than in inflammatory conditions. We have confirmed particles exposure minimal neutrality on MSCs pluripotency. Histological exams of joint after intra-articular injections do not demonstrate any side effects of NPs.Our findings suggest that such a delivery platform is well tolerated locally and could be used to deliver active molecules to the joint.
Keywords: hyaluronate; nanoparticles; osteoarticular; PLGA; vectorization

Elucidation of Molecular Mechanisms Behind the Self-Assembly Behavior of Chitosan Amphiphilic Derivatives Through Experiment and Molecular Modeling by Mohammad Mahmoudzadeh; Afshin Fassihi; Farid Dorkoosh; Reyhaneh Heshmatnejad; Karim Mahnam; Hassan Sabzyan; Amir Sadeghi (3899-3915).
Chitosan-based polymeric micelles (CBPMs) are considered as promising carriers for delivery of anticancer drugs, imaging agents and genes. To optimize the physicochemical, pharmaceutical and biological properties of CBPMs, the molecular mechanisms behind the self-assembly behavior of chitosan (CHI) amphiphilic derivatives are elucidated.This study has two stages. In the experimental stage, dexamethasone (DEX) as a hydrophobic group is grafted to CHI in three degrees of substitution in order to obtain CHI derivatives with different degrees of hydrophobicity. These new CHI amphiphilic derivatives (CHI_DEXs) form micelles in water where their critical aggregation concentration (CAC), size and zeta potential are measured. Through comparing the results of these measurements, the change of self-assembly behavior of CHI_DEXs in response to increasing their hydrophobicity is evaluated. Correlating this evaluation with the results of the 13 MD simulations conducted on CHI_DEXs in atomistic molecular dynamics (MD) simulation stage, reveals the molecular mechanisms behind the self-assembly behavior of CHI_DEXs.Our evaluation of the experimental results reveals that increasing hydrophobicity of a CHI amphiphilic derivative would not necessarily cause it to form micelles with lower CAC value, smaller size and lower zeta potential. The MD simulations reveal that there exists a balance between intra- and inter-chain interactions which is responsible for the self-assembly behavior of CHI amphiphilic derivatives.An increase in DS of the hydrophobic group triggers a cascade of molecular events that shifts the balance between intra- and inter-chain interactions leading to changes in the CAC, size and zeta potential of the CBPMs.
Keywords: chitosan; molecular dynamics simulation; molecular mechanisms; polymeric micelles; self-assembly

Functional Expression of PEPT2 in the Human Distal Lung Epithelial Cell Line NCl-H441 by Mikihisa Takano; Natsumi Sugimoto; Carsten Ehrhardt; Ryoko Yumoto (3916-3926).
The peptide transporter PEPT2 is expressed in alveolar type II epithelial cells. So far, however, no appropriate alveolar epithelial cell line for studying PEPT2 function has been known. In this study, we examined the functional expression of PEPT2 in the human distal lung epithelial cell line NCl-H441 (H441).Expression of PEPT2 mRNA and protein was examined in H441 cells. Transport function of PEPT2 was studied using glycylsarcosine (Gly-Sar) as a substrate.Lamellar bodies were well developed in H441 cells and mRNA expression of type II cell markers and PEPT2 increased during time in culture. PEPT2 protein expression was confirmed in H441 cells, but not in A549 cells, by immunostaining and Western blotting. The uptake of Gly-Sar in H441 cells was inhibited by cefadroxil, and the cefadroxil-sensitive uptake was pH-dependent and peaked at pH 6.5. Gly-Sar uptake in H441 cells showed saturation kinetics with a K m value of 112.5 μM. In addition, apical-to-basal, but not basal-to-apical, transport of cephalexin across H441 cell monolayers was sensitive to cefadroxil.PEPT2 is functionally expressed in H441 cells, making the cell line a good in vitro model to study PEPT2 function and its regulation in human distal lung.
Keywords: alveolar epithelial cells; Gly-Sar uptake; proton-coupled oligopeptide transporter; pulmonary drug disposition; transcellular transport

The Pharmacokinetics of the CYP3A Substrate Midazolam in Morbidly Obese Patients Before and One Year After Bariatric Surgery by Margreke J. Brill; Anne van Rongen; Eric P. van Dongen; Bert van Ramshorst; Eric J. Hazebroek; Adam S. Darwich; Amin Rostami-Hodjegan; Catherijne A. Knibbe (3927-3936).
Bariatric surgery is nowadays commonly applied as treatment for morbid obesity (BMI > 40 kg/m2). As information about the effects of this procedure on a drug’s pharmacokinetics is limited, we aimed to evaluate the pharmacokinetics of CYP3A probe substrate midazolam after oral and intravenous administration in a cohort of morbidly obese patients that was studied before and 1 year post bariatric surgery.Twenty morbidly obese patients (aged 26–58 years) undergoing bariatric surgery participated in the study of which 18 patients returned 1 year after surgery. At both occasions, patients received 7.5 mg oral and 5 mg intravenous midazolam separated by 160 ± 48 min. Per patient and occasion, a mean of 22 blood samples were collected. Midazolam concentrations were analyzed using population pharmacokinetic modeling.One year after bariatric surgery, systemic clearance of midazolam was higher [0.65 (7%) versus 0.39 (11%) L/min, mean ± RSE (P < 0.01), respectively] and mean oral transit time (MTT) was faster [23 (20%) versus 51 (15%) minutes (P < 0.01)], while oral bioavailability was unchanged (0.54 (9%)). Central and peripheral volumes of distribution were overall lower (P < 0.05).In this cohort study in morbidly obese patients, systemic clearance was 1.7 times higher 1 year after bariatric surgery, which may potentially result from an increase in hepatic CYP3A activity per unit of liver weight. Although MTT was found to be faster, oral bioavailability remained unchanged, which considering the increased systemic clearance implies an increase in the fraction escaping intestinal first pass metabolism.
Keywords: bariatric surgery; midazolam; pharmacokinetics; CYP3A

In Vitro Assessment of Uptake and Lysosomal Sequestration of Respiratory Drugs in Alveolar Macrophage Cell Line NR8383 by Ayşe Ufuk; Graham Somers; J. Brian Houston; Aleksandra Galetin (3937-3951).
To assess accumulation and lysosomal sequestration of 9 drugs used in respiratory indications (plus imipramine as positive control) in the alveolar macrophage (AM) cell line NR8383.For all drugs, uptake at 5 μM was investigated at 37 and 4°C to delineate active uptake and passive diffusion processes. Accumulation of basic clarithromycin, formoterol and imipramine was also assessed over 0.1–100 μM concentration range. Lysosomal sequestration was investigated using ammonium chloride (NH4Cl), monensin and nigericin. Impact of lysosomal sequestration on clarithromycin accumulation kinetics was investigated.Both cell-to-medium concentration ratio (Kp) and uptake clearance (CLuptake) ranged > 400-fold for the drugs investigated. The greatest Kp was observed for imipramine (391) and clarithromycin (82), in contrast to no accumulation seen for terbutaline. A concentration-dependent accumulation was evident for the basic drugs investigated. Imipramine and clarithromycin Kp and CLuptake were reduced by 59–85% in the presence of NH4Cl and monensin/nigericin, indicating lysosomal accumulation, whereas lysosomal sequestration was not pronounced for the other 8 respiratory drugs. Clarithromycin uptake rate was altered by NH4Cl, highlighting the impact of subcellular distribution on accumulation kinetics.This study provides novel evidence of the utility of NR8383 for investigating accumulation and lysosomal sequestration of respiratory drugs in AMs.
Keywords: alveolar macrophages; lysosomal sequestration; NR8383; respiratory drugs

Comparative Evaluation of Two Methods for Preparative Fractionation of Proteinaceous Subvisible Particles—Differential Centrifugation and FACS by Björn Boll; Emilien Folzer; Christof Finkler; Jörg Huwyler; Hanns-Christian Mahler; Roland Schmidt; Atanas V. Koulov (3952-3964).
The goal of this study was to compare and evaluate two preparative techniques for fractionation of proteinaceous subvisible particles. This work enables future studies to address the potential biological consequences of proteinaceous subvisible particles in protein therapeutic products.Particles were generated by heat stress and separated by size using differential centrifugation and FACS (Fluorescence-activated cell sorter). Resulting fractions were characterized by size-exclusion chromatography, light obscuration, flow imaging microscopy and resonant mass measurement.Here we report the optimization and comprehensive evaluation of two methods for preparative fractionation of subvisible proteinaceous particles into distinct size fractions in the range between 0.25 and 100 μm: differential centrifugation and FACS. Using these methods, well-defined size fractions were prepared and characterized in detail. Critical assessment and comparison of the two techniques demonstrated their complementarity and for the first time—their relative advantages and drawbacks.FACS and differential centrifugation are valuable tools to prepare well-defined size-fractions of subvisible proteinaceous particles. Both techniques possess unique and advantageous attributes and will likely find complementary application in future research on the biological consequences of proteinaceous subvisible particles.
Keywords: particle fractionation; particle size; protein aggregation; proteinaceous particles; subvisible particles

In Silico Estimation of Skin Concentration Following the Dermal Exposure to Chemicals by Tomomi Hatanaka; Shun Yoshida; Wesam R. Kadhum; Hiroaki Todo; Kenji Sugibayashi (3965-3974).
To develop an in silico method based on Fick’s law of diffusion to estimate the skin concentration following dermal exposure to chemicals with a wide range of lipophilicity.Permeation experiments of various chemicals were performed through rat and porcine skin. Permeation parameters, namely, permeability coefficient and partition coefficient, were obtained by the fitting of data to two-layered and one-layered diffusion models for whole and stripped skin. The mean skin concentration of chemicals during steady-state permeation was calculated using the permeation parameters and compared with the observed values.All permeation profiles could be described by the diffusion models. The estimated skin concentrations of chemicals using permeation parameters were close to the observed levels and most data fell within the 95% confidence interval for complete prediction. The permeability coefficient and partition coefficient for stripped skin were almost constant, being independent of the permeant’s lipophilicity.Skin concentration following dermal exposure to various chemicals can be accurately estimated based on Fick’s law of diffusion. This method should become a useful tool to assess the efficacy of topically applied drugs and cosmetic ingredients, as well as the risk of chemicals likely to cause skin disorders and diseases.
Keywords: dermal exposure; Fick’s law of diffusion; in silico ; skin concentration; skin permeation

Self-Interaction Chromatography of mAbs: Accurate Measurement of Dead Volumes by S. H. M. Hedberg; J. Y. Y. Heng; D. R. Williams; J. M. Liddell (3975-3985).
Measurement of the second virial coefficient B22 for proteins using self-interaction chromatography (SIC) is becoming an increasingly important technique for studying their solution behaviour. In common with all physicochemical chromatographic methods, measuring the dead volume of the SIC packed column is crucial for accurate retention data; this paper examines best practise for dead volume determination.SIC type experiments using catalase, BSA, lysozyme and a mAb as model systems are reported, as well as a number of dead column measurements.It was observed that lysozyme and mAb interacted specifically with Toyopearl AF-Formyl dead columns depending upon pH and [NaCl], invalidating their dead volume usage. Toyopearl AF-Amino packed dead columns showed no such problems and acted as suitable dead columns without any solution condition dependency. Dead volume determinations using dextran MW standards with protein immobilised SIC columns provided dead volume estimates close to those obtained using Toyopearl AF-Amino dead columns.It is concluded that specific interactions between proteins, including mAbs, and select SIC support phases can compromise the use of some standard approaches for estimating the dead volume of SIC columns. Two other methods were shown to provide good estimates for the dead volume.
Keywords: dead volume; mAbs; protein-protein interactions; second virial coefficient; self-interaction chromatography

Drug Release and Targeting: the Versatility of Polymethacrylate Nanoparticles for Peroral Administration Revealed by Using an Optimized In Vitro-Toolbox by Susanne Beyer; Aline Moosmann; Astrid S. Kahnt; Thomas Ulshöfer; Michael J. Parnham; Nerea Ferreirós; Sylvia Wagner; Matthias G. Wacker (3986-3998).
The contribution of permeability and drug release to drug targeting were investigated in the course of development of a nanosized formulation of the anti-inflammatory compound TMP-001, for the local treatment in the gastrointestinal tract.TMP-001 was encapsulated by nanoprecipitation into Eudragit® RS 100. The permeability of these carriers was investigated in an Ussing chamber model and the release rate was determined under biorelevant conditions. Formulation toxicity and particle-cell-interaction were investigated by flow cytometry, fluorescence and electron microscopy. Furthermore, spray drying was performed.Effective internalization of Eudragit®-nanoparticles into cancer cells was demonstrated. A burst release of the nanoparticles implied poor interaction of TMP-001 with Eudragit®. A sustained release (70.5% release after 30 min compared to 98.0% for the API) was accomplished after spray drying yielded an increased particle size. Recovery rate of TMP-001 after spray drying was 94.2 ± 5.9%.The release of API from polymeric nanoparticles contributes profoundly to the in vivo-performance of drug delivery devices in the gastrointestinal tract. The impact of drug-polymer interaction and particle size was analyzed. Sustained release of TMP-001 could only be achieved by increasing particle size. Therefore, biorelevant release testing has been demonstrated to be a valid tool for nanoformulation design.
Keywords: biorelevant release; Eudragit® RS 100; nanoparticles; peroral drug delivery; Ussing chamber

Solid Phase Extraction as an Innovative Separation Method for Measuring Free and Entrapped Drug in Lipid Nanoparticles by Alexis Guillot; Anne-Claude Couffin; Xavier Sejean; Fabrice Navarro; Markus Limberger; Claus-Michael Lehr (3999-4009).
Contrary to physical characterization techniques for nanopharmaceuticals (shape, size and zeta-potential), the techniques to quantify the free and the entrapped drug remain very few and difficult to transpose in routine analytical laboratories. The application of Solid Phase Extraction (SPE) technique was investigated to overcome this challenge.The separation of free and entrapped drug by SPE was quantitatively validated by High Performance Liquid Chromatography. The developed protocol was implemented to characterize cyclosporine A-loaded 120 nm-sized lipid nanoparticles (LNPs, Lipidot®) dispersed in aqueous buffer. The colloidal stability was assessed by Dynamic Light Scattering (DLS).Validation experiments demonstrated suitable linearity, repeatability, accuracy and specificity to quantify residual free, entrapped and total drug. For the investigated LNPs, the method revealed a very limited shelflife of the formulation when stored in an aqueous buffer at 5°C and even more at elevated temperature. Nevertheless, the DLS measurements confirmed the stability of nanoparticles during SPE in a suitable concentration range.SPE, when successfully validated, represents a valuable tool for drug development and quality control purposes of lipid-based nanopharmaceuticals in an industrial environment.
Keywords: cyclosporine; lipid nanoemulsion; nanoparticles; separation techniques; solid-phase extraction

AAPS Connection (4010-4012).