Pharmaceutical Research (v.29, #7)

Bottlenecks Caused by Software Gaps in miRNA and RNAi Research by Sean Ekins; Ron Shigeta; Barry A. Bunin (1717-1721).
Understanding the regulation of gene expression is critical to many areas of biology while control via RNAs has found considerable interest as a tool for scientific discovery and potential therapeutic applications. For example whole genome RNA interference (RNAi) screens and whole proteome scans provide views of how the entire transcriptome or proteome responds to biological, chemical or environmental perturbations of a gene’s activity. Small RNA (sRNA) or MicroRNA (miRNA) are known to regulate pathways and bind mRNA, while the function of miRNAs discovered in experimental studies is often unknown. In both cases, RNAi and miRNA require labor intensive studies to tease out their functions within gene networks. Available software to analyze relationships is currently an ad hoc and often a manual process that can take up to several hours to analyze a single candidate RNAi or miRNA. With experiments frequently highlighting tens to hundreds of candidates this represents a considerable bottleneck. We suggest there is a gap in miRNA and RNAi research caused by inadequate current software that could be improved. For example a new software application could be created that provides interactive, comprehensive target analysis that leverages past datasets to lead to statistically stronger analyses.
Keywords: informatics; microRNA; screening; siRNA; software

Camptothecin analogues are anticancer drugs effective when dosed in protracted schedules. Such treatment is best suited for oral formulations. AR-67 is a novel lipophilic analogue with potent efficacy in preclinical models. Here we assessed factors that may influence its oral bioavailability in rats.Plasma pharmacokinetic (PK) studies were conducted following administration of AR-67 lactone or carboxylate doses alone or after pre-dosing with inhibitors of the efflux transporters P-gp and Bcrp. A population PK model that simultaneously fitted to oral and intravenous data was used to estimate the bioavailability (F) and clearance of AR-67.An inverse Gaussian function was used as the oral input into the model and provided the best fits. Covariate analysis showed that the bioavailability of the lactone, but not its clearance, was dose dependent. Consistent with this observation, the bioavailability of AR-67 increased when animals were pretreated orally with GF120918 or Zosuquidar.Absorption of AR-67 is likely affected by solubility of its lactone form and interaction with efflux pumps in the gut. AR-67 appears to be absorbed as the lactone form, most likely due to gastric pH favoring its formation and predominance. F increased at higher doses suggesting saturation of efflux mechanisms.
Keywords: BCRP; camptothecin; carboxylate; lactone; P-gp

To elucidate the effect of sterols on the aggregation of amphotericin B (AmB) in PEG-phospholipid micelles and its consequences on the hemolytic activity of AmB.AmB-incorporated PEG-phospholipid micelles co-loaded with ergosterol, cholesterol, or 7-dehydrocholesterol were prepared at 4:1:1 and 20:5:1 ratios of polymer-to-sterol-to-AmB. The aggregation state of AmB was elucidated by UV–vis spectroscopy. AmB/sterol co-loaded PEG-phospholipid micelles were incubated with red blood cells and the hemolytic activity of AmB assessed by measurement of free hemoglobin.AmB in PEG-phospholipid micelles stayed mostly in a deaggregated state in the absence of sterol or with cholesterol, but aggregated in the presence of ergosterol or 7-dehydrocholesterol. The fraction of aggregated AmB in PEG-phospholipid micelles was lower at the 20:5:1 ratio. In an aggregated state or in the absence of sterol, AmB caused rapid and complete hemolysis. In contrast, deaggregated AmB co-loaded with cholesterol caused slower and incomplete hemolysis, especially at a 20:5:1 ratio.The aggregation state of AmB in PEG-phospholipid micelles was sterol dependant. AmB/cholesterol co-loaded PEG-phospholipid micelles caused low in vitro hemolysis due to deaggregation of AmB and micellar stability, presumably owing to cholesterol/phospholipid versus cholesterol/AmB interactions in the interior core region.
Keywords: amphotericin B; aggregation state; hemolysis; micelle; sterol

In drug discovery research the formation of soluble compound aggregates is a major cause of false positives, false negatives, and distorted values in High-Throughput Screening assays that measure either binding or functional activity. These aggregation-based artifacts lead to serious distortions in the SAR which are critical to successful lead optimization. In this work we introduce a new approach by which the “critical aggregation concentration” (CAC) is determined, thereby overcoming limitations inherent to traditional solubility methods and enabling estimation of small molecule monomer solubility.The theoretical and experimental basis of a new confocal Static Light Scattering plate reader assay is presented.Tests conducted with model systems, commercial compounds, and Abbott library compounds show that the CAC assay can measure aqueous monomer solubilities reproducibly and reliably, achieving a sensitivity of ~0.2 μm, which is an improvement of approximately two orders of magnitude over nephelometry.Determination of compound monomer solubilities in a screening format is possible for the first time with the cSLS-CAC methodology. It is currently in routine use in Abbott’s drug discovery program, and has enabled identification of many compound induced artifacts in binding or activity assays that are missed by traditional kinetic solubility measurements.
Keywords: aggregation; confocal; drug discovery; static light scattering; solubility

Drug Release Patterns and Cytotoxicity of PEG-poly(aspartate) Block Copolymer Micelles in Cancer Cells by Allison M. Eckman; Eleftheria Tsakalozou; Nayon Y. Kang; Andrei Ponta; Younsoo Bae (1755-1767).
To test physicochemical and biological properties of PEG-poly(aspartate) [PEG-p(Asp)] block copolymer micelles entrapping doxorubicin hydrochloride (DOX) through ionic interaction.PEG-p(Asp) was synthesized from 5 kDa PEG and 20 Asp units. Carboxyl groups of p(Asp) were present as benzyl ester [PEG-p(Asp/Bz)], sodium salt [PEG-p(Asp/Na)] or free acid [PEG-p(Asp/H)]. Block copolymers and DOX were mixed at various ratios to prepare polymer micelles, which were subsequently characterized to determine particle size, drug loading and release patterns, and cytotoxicity against prostate (PC3 and DU145) and lung (A549) cancer cell lines.PEG-p(Asp/Bz), Na- and H-micelles entrapped 1.1, 56.8 and 40.6 wt.% of DOX, respectively. Na- and H-micelles (<100 nm) showed time-dependent DOX release at pH 7.4, which was accelerated at pH 5.0. Na-micelles were most stable at pH 7.4, retaining 31.8% of initial DOX for 48 h. Cytotoxicity of Na-micelles was 23.2% (A549), 28.5% (PC3) and 45.9% (DU145) more effective than free DOX.Ionic interaction appeared to entrap DOX efficiently in polymer micelles from PEG-p(Asp) block copolymers. Polymer micelles possessing counter ions (Na) of DOX in the core were the most stable, releasing drugs for prolonged time in a pH-dependent manner, and suppressing cancer cells effectively.
Keywords: cytotoxicity; doxorubicin; drug delivery; intracellular drug uptake; polymer micelles

Sotalol Permeability in Cultured-Cell, Rat Intestine, and PAMPA System by Wei Liu; Hideaki Okochi; Leslie Z. Benet; Suo-Di Zhai (1768-1774).
To clarify sotalol’s classification in the BCS versus BDDCS systems through cellular, rat everted sac and PAMPA permeability studies.Studies were carried out in Madin Darby canine kidney (MDCK) and MDR1-transfected MDCK (MDCK-MDR1) cell lines, rat everted gut sacs and the Parallel Artificial Membrane Permeability Assay (PAMPA) system. Three-hour transport studies were conducted in MDCK cell lines (with apical pH changes) and MDCK-MDR1 cells (with and without the P-glycoprotein inhibitor GG918); male Sprague-Dawley rats (300~350 g) were used to prepare everted sacs. In the PAMPA studies, drug solutions at different pH’s were dosed in each well and incubated for 5 h. Samples were measured by LC-MS/MS, or liquid scintillation counting and apparent permeability (Papp) was calculated.Sotalol showed low permeability in all of the cultured-cell lines, everted sacs and PAMPA systems. It might be a border line P-glycoprotein substrate. The PAMPA study showed that sotalol’s permeability increased with a higher apical pH, while much less change was found in MDCK cells.The low permeability rate for sotalol correlates with its Class 3 BDDCS assignment and lack of in vivo metabolism.
Keywords: biopharmaceutics drug disposition classification system (BDDCS); MDCK cells; permeability; p-glycoprotein; solute transporters

Inclusion Complex of Novel Curcumin Analogue CDF and β-Cyclodextrin (1:2) and Its Enhanced In Vivo Anticancer Activity Against Pancreatic Cancer by Prasad R. Dandawate; Alok Vyas; Aamir Ahmad; Sanjeev Banerjee; Jyoti Deshpande; K. Venkateswara Swamy; Abeda Jamadar; Anne Catherine Dumhe-Klaire; Subhash Padhye; Fazlul H. Sarkar (1775-1786).
Several formulations have been proposed to improve the systemic delivery of novel cancer therapeutic compounds, including cyclodextrin derivatives. We aimed to synthesize and characterize of CDF-β-cyclodextrin inclusion complex (1:2) (CDFCD).The compound was characterized by Fourier transform infrared, differential scanning calorimetry, powder X-ray diffraction studies, H1 & C13 NMR studies and scanning electron microscopic analysis. Its activity was tested against multiple cancer cell lines, and in vivo bioavailability was checked.CDF-β-cyclodextrin was found to lower IC50 value by half when tested against multiple cancer cell lines. It preferentially accumulated in the pancreas, where levels of CDF-β-cyclodextrin in mice were 10 times higher than in serum, following intravenous administration of an aqueous CDF-β-cyclodextrin preparation.Novel curcumin analog CDF preferentially accumulates in the pancreas, leading to its potent anticancer activity against pancreatic cancer cells. Synthesis of such CDF-β-cyclodextrin self-assembly is an effective strategy to enhance its bioavailability and tissue distribution, warranting further evaluation for CDF delivery in clinical settings for treatment of human malignancies.
Keywords: CDF; curcumin analog; CDF-β-Cyclodextrin; pancreatic cancer

Linear Delivery of Verapamil via Nanofibrous Sheet-Based System by Ji Eun Lee; Chun Gwon Park; Byeong Moo An; Myung Hun Kim; Min Park; Seung Ho Lee; Young Bin Choy (1787-1796).
To achieve linear delivery of a highly water-soluble oral drug, verapamil, with a nanofibrous sheet-based system.The nanofibrous sheets made of poly (lactic-co-glycolic acid) were used as a diffusion barrier to cap a tablet containing verapamil. For controlled drug delivery, we varied the sheet thickness to 20 μm, 50 μm and 80 μm to give the capped drug tablets, 20CT, 50CT and 80CT, respectively.Drug release was more sustained as the sheet thickness increased. Thus, the periods for almost complete drug release could be extended up to 14 h with the 80 μm-thick sheets. As we assessed the linear least square fits to the in vitro drug release data from the capped tablets, 20CT and 50CT showed a fairly good correlation with linear release. The periods of linear release were 6 h and 8 h for 20CT and 50CT, respectively, both releasing more than 85% drug during this period.We conclude that a drug tablet capped with nanofibrous sheets is a promising system for linear delivery of a highly water-soluble oral drug.
Keywords: linear drug delivery; nanofibrous sheet; poly (lactic-co-glycolic acid); verapamil; water-soluble drug

Does the United States Pharmacopeia Throat Introduce De-agglomeration of Carrier-Free Powder from Inhalers? by Patricia Tang; Philip Chi Lip Kwok; Zhenbo Tong; Runyu Yang; Judy Agnes Raper; Hak-Kim Chan (1797-1807).
We hypothesize that the USP induction port may de-agglomerate carrier-free powder emitting from dry powder inhalers (DPIs).Aerosols emitting from a range of DPIs (Spinhaler®, Turbuhaler® and OsmohalerTM) and induction ports (USP throat, straight tube, Alberta idealized mouth-throat geometry (AG)) were sized by laser diffraction. Total drug recovery was obtained by HPLC and fine particle fraction computed. Air flow patterns were simulated using Computational Fluid Dynamics (CFD).The straight tube did not de-agglomerate emitted powder. However, the USP throat and AG further de-agglomerated powders from the Spinhaler, but not the Turbuhaler and Osmohaler. While budesonide powder deposited similarly in all induction ports, deposition was significantly higher in the AG for both DSCG and mannitol. CFD revealed agglomerates impacting on the USP throat with higher localized velocity compared with the straight tube. CFD further showed a more complex flow pattern with high-velocity air jets in the AG, which explains the higher FPF for DSCG and the lower FPF for mannitol using the AG.The USP throat further de-agglomerated the emitted powder from the DPI when it did not sufficiently disperse the powder. Other tools such as laser diffraction may be used for cross-examining to avoid artifacts in the results.
Keywords: de-agglomeration; dry powder inhaler; powder aerosol; USP throat

Comparison of Open-Flow Microperfusion and Microdialysis Methodologies When Sampling Topically Applied Fentanyl and Benzoic Acid in Human Dermis Ex Vivo by R. Holmgaard; E. Benfeldt; J. B. Nielsen; C. Gatschelhofer; J. A. Sorensen; C. Höfferer; M. Bodenlenz; T. R. Pieber; F. Sinner (1808-1820).
The purpose of this study is to compare two sampling methods—dermal Open-Flow Microperfusion (dOFM) and dermal Microdialysis (dMD) in an international joint experiment in a single-laboratory setting. We used human ex-vivo skin and sampled topically administered Fentanyl and Benzoic Acid. The second purpose was to provide guidance to researchers in choosing the most efficient method for a given penetrant and give suggestions concerning critical choices for successful dermal sampling.The dOFM and dMD techniques are compared in equal set-ups using three probe-types (one dOFM probe and two dMD probe-types) in donor skin (n = 9) - 27 probes of each type sampling each penetrant in solutions applied in penetrationchambers glued to the skin surface over a time range of 20 h.Pharmacokinetic results demonstrated concordance between dOFM and dMD sampling technique under the given experimental conditions. The methods each had advantages and limitations in technical, practical and hands-on comparisons.When planning a study of cutaneous penetration the advantages and limitations of each probe-type have to be considered in relation to the scientific question posed, the physico-chemical characteristics of the substance of interest, the choice of experimental setting e.g. ex vivo/in vivo and the analytical skills available.
Keywords: dermal; ex vivo ; human; microdialysis; open-flow microperfusion

To explore the involvement of transmembrane domain (TM) 7 of the human apical sodium-dependent bile acid transporter (hASBT) on bile acid (BA) binding/translocation, using two electrophilic BA derivatives as molecular probes.Two electrophilic derivatives of chenodeoxycholic acid (CDCA) were designed, synthesized and evaluated for their ability to inactivate hASBT, and the human organic cation/carnitine transporter (hOCTN2) as a control (i.e. a non-BA transporting model). The ability of electrophilic derivatives to interact with hASBT was evaluated by 2-aminoethyl-methanethiosulfonate (MTSEA)-biotin labeling of thiol groups in TM7 cysteine mutants.Unlike native BAs, the electrophilic CDCA derivatives specifically inactivated hASBT, but not hOCTN2, and inhibited hASBT in a time- and concentration-dependent fashion. Preincubation of hASBT Cys-mutants in the exofacial half of TM7 with reactive electrophilic probes blocked transporter biotinylation by MTSEA-biotin, similar to 2-(trimethylammonium)ethyl-methanethiosulfonate (MTSET) blocking. This blocking pattern differed from that produced by native BAs, which exposed exofacial TM7 residues, thereby increasing staining.Kinetic and biochemical data indicate these novel electrophilic BAs are potent and specific irreversible inhibitors of hASBT and offer new evidence about the role of TM7 in binding/translocation of bile acids.
Keywords: bile acid; hASBT; irreversible inhibitor; MTS-reagent

Mass Spectrometry-Based Quantification of CYP Enzymes to Establish In Vitro/In Vivo Scaling Factors for Intestinal and Hepatic Metabolism in Beagle Dog by Aki T. Heikkinen; Arno Friedlein; Jens Lamerz; Peter Jakob; Paul Cutler; Stephen Fowler; Tara Williamson; Roberto Tolando; Thierry Lave; Neil Parrott (1832-1842).
Physiologically based models, when verified in pre-clinical species, optimally predict human pharmacokinetics. However, modeling of intestinal metabolism has been a gap. To establish in vitro/in vivo scaling factors for metabolism, the expression and activity of CYP enzymes were characterized in the intestine and liver of beagle dog.Microsomal protein abundance in dog tissues was determined using testosterone-6β-hydroxylation and 7-hydroxycoumarin-glucuronidation as markers for microsomal protein recovery. Expressions of 7 CYP enzymes were estimated based on quantification of proteotypic tryptic peptides using multiple reaction monitoring mass spectrometry. CYP3A12 and CYP2B11 activity was evaluated using selective marker reactions.The geometric mean of total microsomal protein was 51 mg/g in liver and 13 mg/cm in intestine, without significant differences between intestinal segments. CYP3A12, followed by CYP2B11, were the most abundant CYP enzymes in intestine. Abundance and activity were higher in liver than intestine and declined from small intestine to colon.CYP expression in dog liver and intestine was characterized, providing a basis for in vitro/in vivo scaling of intestinal and hepatic metabolism.
Keywords: beagle dog; CYP; intestinal metabolism; in vitro/in vivo scaling; MRM

Fluorescence Imaging of the Lymph Node Uptake of Proteins in Mice after Subcutaneous Injection: Molecular Weight Dependence by Fang Wu; Suraj G. Bhansali; Wing Cheung Law; Earl J. Bergey; Paras N. Prasad; Marilyn E. Morris (1843-1853).
To use noninvasive fluorescence imaging to investigate the influence of molecular weight (MW) of proteins on the rate of loss from a subcutaneous (SC) injection site and subsequent uptake by the draining lymph nodes in mice.Bevacizumab (149 kDa), bovine serum albumin (BSA, 66 kDa), ovalbumin (44.3 kDa) or VEGF-C156S (23 kDa), labeled with the near infrared dye IRDye 680, were injected SC into the front footpad of SKH-1 mice. Whole body non-invasive fluorescence imaging was performed to quantitate the fluorescence signal at the injection site and in axillary lymph nodes.The half-life values, describing the times for 50% loss of proteins from the injection site, were 6.81 h for bevacizumab, 2.85 h for BSA, 1.57 h for ovalbumin and 0.31 h for VEGF-C156S. The corresponding axillary lymph node exposure, represented as the area of the % dose versus time curve, was 6.27, 5.13, 4.06 and 1.54% dose ∙ h, respectively.Our results indicate that the rate of loss of proteins from a SC injection site is inversely related to MW of proteins, while lymph node exposure is proportionally related to the MW of proteins in a mouse model.
Keywords: fluorescence imaging; lymphatic uptake; molecular weight; protein; subcutaneous injection

To be fully exploitable in both formulation and manufacturing, a drug cocrystal needs to demonstrate simultaneous improvement of multiple key pharmaceutical properties over the pure drug crystal. The present work was aimed at investigating such feasibility with two model profen-nicotinamide cocrystals.Phase pure 1:1 ibuprofen-nicotinamide and flurbiprofen-nicotinamide cocrystals were prepared from solutions through rapid solvent removal using rotary evaporation,and characterized by DSC, PXRD, FTIR, phase solubility measurements, equilibrium moisture sorption analysis, dissolution testing and tabletability analysis.Temperature-composition phase diagrams constructed from DSC data for each profen and nicotinamide crystal revealed the characteristic melting point of the 1:1 cocrystal as well as the eutectic temperatures and compositions. Both cocrystals exhibited higher intrinsic dissolution rates than the corresponding profens. The cocrystals also sorbed less moisture and displayed considerably better tabletability than the individual profens and nicotinamide.Phase behaviors of 1:1 profen-nicotinamide cocrystal systems were delineated by constructing their temperature-composition phase diagrams. Cocrystallization with nicotinamide can simultaneously improve tableting behavior, hygroscopicity, and dissolution performance of ibuprofen and flurbiprofen. This could pave the way for further development of such cocrystal systems into consistent, stable, efficacious and readily manufacturable drug products.
Keywords: crystal engineering; dissolution; pharmaceutical cocrystal; solubility; tabletability

Nanocrystalline drug-polymer dispersions are of significant interest in pharmaceutical delivery. The purpose of this work is to demonstrate the applicability of methods based on two-dimensional (2D) and multinuclear solid-state NMR (SSNMR) to a novel nanocrystalline pharmaceutical dispersion of ebselen with polyvinylpyrrolidone-vinyl acetate (PVP-VA), after initial characterization with other techniques.A nanocrystalline dispersion of ebselen with PVP-VA was prepared and characterized by powder X-ray diffraction (PXRD), confocal Raman microscopy and mapping, and differential scanning calorimetry (DSC), and then subjected to detailed 1D and 2D SSNMR analysis involving 1H, 13C, and 77Se isotopes and 1H spin diffusion.PXRD was used to show that dispersion contains nanocrystalline ebselen in the 35–60 nm size range. Confocal Raman microscopy and spectral mapping were able to detect regions where short-range interactions may occur between ebselen and PVP-VA. Spin diffusion effects were analyzed using 2D SSNMR experiments and are able to directly detect interactions between ebselen and the surrounding PVP-VA.The methods used here, particularly the 2D SSNMR methods based on spin diffusion, provided detailed structural information about a nanocrystalline polymer dispersion of ebselen, and should be useful in other studies of these types of materials.
Keywords: confocal Raman microscopy; dipolar spin diffusion; nanocrystalline polymer dispersion; powder X-ray diffraction; solid-state NMR

Novel Surfactants with Diglutamic Acid Polar Head Group: Drug Solubilization and Toxicity Studies by Nathalie Ménard; Nicolas Tsapis; Cécile Poirier; Thomas Arnauld; Laurence Moine; Claire Gignoux; François Lefoulon; Jean-Manuel Péan; Elias Fattal (1882-1896).
Novel surfactants made of diglutamic acid (DG) polar head linked to lithocholic, arachidonic, linoleic or stearic acids were designed for drug solubilization.Surfactants 3-D conformer and packing parameter were determined by molecular modelling and self-assembling properties by pyrene fluorescence measurements. Cytotoxicity was assessed on Human Umbilical Vein Endothelial Cells (HUVEC) and haemolyitic activity on rat red blood cells. Drug solubilization was quantified and its interaction with hydrophobic moieties was characterized using differential scanning calorimetry and X-ray diffraction. Self organisation of stearoyl-DG was observed by cryogenic transmission electron microscopy. Toxicity after repeated injections of stearoyl-DG was investigated in Wistar rats.DG-based surfactants self-assemble into water and their critical micellar concentrations are comprised between 200 and 920 μg/mL. Cytotoxicity and haemolysis were lower than for polysorbate 80. At best, stearoyl-DG solubilized the drug up to 22% (w/w). Solid-state characterization evidenced drug/lipid interactions leading to the formation of a new complex. Stearoyl-DG formed spherical micelles of 20 nm, as predicted by packing parameter calculation. However, it induced a possible liver toxicity after intravenous administration in rats.Among the surfactants tested, stearoyl-DG is the more efficient for drug solubilization but its use is limited by its possible liver toxicity.
Keywords: insoluble drug; micelle; self-assembly; solubilization; surfactant; toxicity

Vaginal Film Drug Delivery of the Pyrimidinedione IQP-0528 for the Prevention of HIV Infection by Anthony S. Ham; Lisa Cencia Rohan; Ashlee Boczar; Lu Yang; Karen W. Buckheit; Robert W. Buckheit Jr. (1897-1907).
Polymeric quick-dissolving films were developed as a solid dosage topical microbicide formulation for the vaginal delivery of the highly potent and non-toxic, dual-acting HIV nonnucleoside reverse transcriptase inhibitor (NNRTI) pyrimidinedione, IQP-0528.Formulated from approved excipients, a polyvinyl alcohol (PVA) based film was manufactured via solvent casting methods. The film formulations were evaluated based upon quantitative physicochemical evaluations defined by a Target Product Profile (TPP)Films dosed with 0.1% (w/w) of IQP-0528 disintegrated within 10 min with over 50% of drug released and near 100% total drug released after 30 min. The IQP-0528 films were found to be non-toxic in in vitro CEM-SS and PBMC cell-based assays and biologically active with sub-nanomolar efficacy against HIV-1 infection. In a 12 month stability protocol, the IQP-0528 films demonstrated no significant degradation at International Conference on Harmonization (ICH) recommended standard (25°C/65% relative humidity (R.H.)) and accelerated (40°C/75% R.H.) environmental conditions.Based on the above evaluations, a vaginal film formulation has been identified as a potential solid dosage form for the vaginal delivery of the topical microbicide candidate IQP-0528.
Keywords: films; formulation; HIV; microbicides; pyrimidinedione

Relationship between the Affinity of PEO-PPO-PEO Block Copolymers for Biological Membranes and Their Cellular Effects by Martin Redhead; Giuseppe Mantovani; Selina Nawaz; Paola Carbone; Dariusz C. Gorecki; Cameron Alexander; Cynthia Bosquillon (1908-1918).
The interactions of poly(ethylene oxide)-co-poly(propylene oxide) tri-block copolymers (PEO-PPO-PEO block copolymers, Pluronics®, Synperonics®, Poloxamers) of differing chemical composition with cell membranes were systematically investigated in order to clarify the mechanisms behind their previously reported various cellular responses.Relationships between the structural components of a defined series of PEO-PPO-PEO block copolymers and i) their interactions with biological membranes; ii) their cytotoxic potential were probed using a combination of haemolysis studies and cytotoxicity assays in the Caco-2 and HMEC-1 cell lines.The length of the PPO block as well as the PEO/PPO ratio were determinants of their membrane binding constant and cytotoxicity endpoints measured in the MTS and LDH assays. Similar 2D parabolic relationships were found between polymer composition and their affinity for membranes or their cytotoxicity potential. Cytotoxicity was related to the ability of the copolymers to form ion transversable pores within the cell membrane.The data suggest a link between the affinity of certain Pluronics for biological membranes and their cellular adverse effects. This first cell-based investigation of the interactions of Pluronics with biological membranes is an important step towards unravelling the complex mechanisms which govern the biological effects of widely used amphiphilic materials.
Keywords: biocompatibility; drug delivery; haemolysis; membrane; pluronics

To encapsulate a large amount of protein (superoxide dismutase, SOD) into unilamellar liposomes using a simple process and to investigate the lipid-protein interaction.To achieve protein encapsulation, preformed unilamellar empty liposomes were mixed with SOD and subjected to freeze-thaw cycling. To investigate the lipid-protein interaction, a novel light scattering technique was used.Up to 50% protein encapsulation was achieved at ∼150 nm. There was no significant change in particle size following the freeze-thaw cycling. SOD had a strong interaction with DPPC liposomes containing high concentration of cholesterol. Light scattering data revealed that in some cases the SOD molecules were present inside the lipid bilayer.The method reported here allows great flexibility in the manufacturing process as the liposome preparation and protein-loading operations can be separated. Accordingly, empty liposomes can be prepared without concern about protein stability, making the manufacturing process more flexible and easy to control and ultimately leading to improved product quality. To explain the SOD-lipid interaction, a “pocket-embedding” theory was proposed. The encapsulation method reported here can be applied to hydrophilic small molecules as well as most hydrophilic proteins to achieve high encapsulation efficiency.
Keywords: freeze-and-thaw unilamellar vesicles (FAT-ULV); high performance liquid chromatography (HPLC); light scattering; liposome; superoxide dismutase

Pharmacokinetic-Pharmacodynamic Modeling of the D2 and 5-HT2A Receptor Occupancy of Risperidone and Paliperidone in Rats by Magdalena Kozielska; Martin Johnson; Venkatesh Pilla Reddy; An Vermeulen; Cheryl Li; Sarah Grimwood; Rik de Greef; Geny M. M. Groothuis; Meindert Danhof; Johannes H. Proost (1932-1948).
A pharmacokinetic-pharmacodynamic (PK-PD) model was developed to describe the time course of brain concentration and dopamine D2 and serotonin 5-HT2A receptor occupancy (RO) of the atypical antipsychotic drugs risperidone and paliperidone in rats.A population approach was utilized to describe the PK-PD of risperidone and paliperidone using plasma and brain concentrations and D2 and 5-HT2A RO data. A previously published physiology- and mechanism-based (PBPKPD) model describing brain concentrations and D2 receptor binding in the striatum was expanded to include metabolite kinetics, active efflux from brain, and binding to 5-HT2A receptors in the frontal cortex.A two-compartment model best fit to the plasma PK profile of risperidone and paliperidone. The expanded PBPKPD model described brain concentrations and D2 and 5-HT2A RO well. Inclusion of binding to 5-HT2A receptors was necessary to describe observed brain-to-plasma ratios accurately. Simulations showed that receptor affinity strongly influences brain-to-plasma ratio pattern.Binding to both D2 and 5-HT2A receptors influences brain distribution of risperidone and paliperidone. This may stem from their high affinity for D2 and 5-HT2A receptors. Receptor affinities and brain-to-plasma ratios may need to be considered before choosing the best PK-PD model for centrally active drugs.
Keywords: dopamine D2 receptor occupancy; mechanism-based PK-PD; paliperidone; risperidone; serotonin 5-HT2A receptor occupancy

Treatment of Experimental Brain Metastasis with MTO-Liposomes: Impact of Fluidity and LRP-Targeting on the Therapeutic Result by Andrea Orthmann; Reiner Zeisig; Regine Süss; Dorothea Lorenz; Margit Lemm; Iduna Fichtner (1949-1959).
To test targeted liposomes in an effort to improve drug transport across cellular barriers into the brain.Therefore we prepared Mitoxantrone (MTO) entrapping, rigid and fluid liposomes, equipped with a 19-mer angiopeptide as ligand for LDL lipoprotein receptor related protein (LRP) targeting.Fluid, ligand bearing liposomes showed in vitro the highest cellular uptake and transcytosis and were significantly better than the corresponding ligand-free liposomes and rigid, ligand-bearing vesicles. Treatment of mice, transplanted with human breast cancer cells subcutaneously and into the brain, with fluid membrane liposomes resulted in a significant reduction in the tumor volume by more than 80% and in a clear reduction in drug toxicity. The improvement was mainly depended on liposome fluidity while the targeting contributed only to a minor degree. Pharmacokinetic parameters were also improved for liposomal MTO formulations in comparison to the free drug. So the area under the curve was increased and t1/2 was extended for liposomes.Our data show that it is possible to significantly improve the therapy of brain metastases if MTO-encapsulating, fluid membrane liposomes are used instead of free MTO. This effect could be further enhanced by fluid, ligand bearing liposomes.
Keywords: brain metastases; LRP; targeting; transcytosis; uptake

Drug–Drug Interaction Potential of Marketed Oncology Drugs: In Vitro Assessment of Time-Dependent Cytochrome P450 Inhibition, Reactive Metabolite Formation and Drug–Drug Interaction Prediction by Jane R. Kenny; Sophie Mukadam; Chenghong Zhang; Suzanne Tay; Carol Collins; Aleksandra Galetin; S. Cyrus Khojasteh (1960-1976).
To evaluate 26 marketed oncology drugs for time-dependent inhibition (TDI) of cytochrome P450 (CYP) enzymes. Evaluate TDI-positive drugs for potential to generate reactive intermediates. Assess clinical drug–drug interaction (DDI) risk using static mechanistic models.Human liver microsomes and CYP-specific probes were used to assess TDI in a dilution shift assay followed by generation of KI and kinact. Reactive metabolite trapping studies were performed with stable label probes. Static mechanistic model was used to predict DDI risk using a 1.25-fold AUC increase as a cut-off for positive DDI.Negative TDI across CYPs was observed for 13/26 drugs; the rest were time-dependent inhibitors of, predominantly, CYP3A. The kinact/KI ratios for 11 kinase inhibitors ranged from 0.7 to 42.2 ml/min/μmol. Stable label trapping agent–drug conjugates were observed for ten kinase inhibitors. DDI predictions gave no false negatives, one true negative, four false positives and three true positives. The magnitude of DDI was overestimated irrespective of the inhibitor concentration selected.13/26 oncology drugs investigated showed TDI potential towards CYP3A, formation of reactive metabolites was also observed. An industry standard static mechanistic model gave no false negative predictions but did not capture the modest clinical DDI potential of kinase inhibitors.
Keywords: drug-drug interaction; kinase inhibitors; prediction; reactive metabolites; time-dependent CYP inhibition

Stability Influences the Biodistribution, Toxicity, and Anti-tumor Activity of Doxorubicin Encapsulated in PEG-PE Micelles in Mice by Xiuli Wei; Yiguang Wang; Wenfeng Zeng; Feng Huang; Lei Qin; Chunling Zhang; Wei Liang (1977-1989).
To investigate the influences of stability of doxorubicin (DOX) retained in PEG-PE/HSPC micelles on its biodistribution, toxicity and anti-tumor activity in mice.We incorporated HSPC into PEG-PE micelles at various molar ratios by a self-assembly procedure. Micelles were characterized by dynamic light scattering, transmission electron microscope, atomic force microscopy. Agarose gel electrophoresis assay was used to detect stable retention of DOX in micellar preparations. Biodistribution, toxicity and anti-tumor activity of doxorubicin encapsulated in PEG-PE/HSPC micelles in mice were investigated.HSPC incorporation not only changed the size and shape of PEG-PE micelles, but also decreased the ability of DOX stable retained in PEG-PE micelles, resulting in a great discrepancy in biodistribution, toxicity and anti-tumor activity among micellar DOX preparations. DOX encapsulated in PEG-PE micelles (M1-DOX), with narrower size distribution and greater stability, demonstrated better cytotoxicity in vitro and low systemic toxicity with superior anti-tumor metastasis activity in vivo.Encapsulation of DOX into PEG-PE micelles showed the best therapeutic activity and lowest systemic toxicity compared to other HSPC-incorporated PEG-PE micellar preparations. Stable retention of drugs within micelles is important and is determined by compatibility between drugs and polymer blocks.
Keywords: anti-tumor metastasis; biodistribution; PEG-PE/HSPC micelles; stability; toxicity

Reversine, a 2,6-disubstituted Purine, as an Anti-cancer Agent in Differentiated and Undifferentiated Thyroid Cancer Cells by Shih-Che Hua; Tien-Chun Chang; Hau-Ren Chen; Chieh-Hsiang Lu; Yi-Wen Liu; Shu-Hsin Chen; Hui-I Yu; Yi-Ping Chang; Ying-Ray Lee (1990-2005).
A novel and effective treatment is urgently needed to deal with the current treatment dilemma in incurable differentiated thyroid cancer (DTC), poorly differentiated thyroid cancer (PDTC) and anaplastic thyroid cancer (ATC). Reversine, a small synthetic purine analogue (2,6-disubstituted purine), has been shown to be effective in tumor suppression.We performed in vitro evaluation of anti-tumor effects of reversine on proliferation, cell cycle, and apoptosis in human PDTC, ATC, and follicular thyroid cancer cell lines, respectively.Treatment of these three lines with reversine inhibited proliferation in a time- and dose-dependent manner. G2/M accumulation was demonstrated in cell cycle analysis. Reversine induced apoptosis in PDTC cells with caspase-3 and caspase-8 activation, but not caspase-9. Use of a pan-caspase inhibitor before treatment with reversine attenuated cell death. Reversine also showed in vivo growth inhibitory effects on ATC cells in a xenograft nude mice model.Data demonstrated that reversine is effective in inhibiting the growth of thyroid cancer cells by cell cycle arrest or apoptosis, especially with the more aggressive ATC and PDTC. Apoptosis was induced by the mitochondria-independent pathway. Reversine is therefore worthy of further investigation in clinical therapeutics.
Keywords: reversine; thyroid cancer; cell cycle; apoptosis; xenograft nude mice

Multiple proteins are involved in activation and inactivation of 2′,2′-difluorodeoxycytidine (gemcitabine, dFdC). We aimed to clarify the mechanism of dFdC resistance in a pancreatic cancer cell line by applying a combination of targeted proteomic and metabolomic analyses.Twenty-five enzyme and transporter proteins and 6 metabolites were quantified in sensitive and resistant pancreatic cancer cell lines, PK9 and RPK9, respectively.The protein concentration of deoxycytidine kinase (dCK) in RPK9 cells was less than 0.02-fold (2 %) compared with that in PK9 cells, whereas the differences (fold) were within a factor of 3 for other proteins. Targeted metabolomic analysis revealed that phosphorylated forms of dFdC were reduced to less than 0.2 % in RPK9 cells. The extracellular concentration of 2′,2′-difluorodeoxyuridine (dFdU), an inactive metabolite of dFdC, reached the same level as the initial dFdC concentration in RPK9 cells. However, tetrahydrouridine treatment did not increase phosphorylated forms of dFdC and did not reverse dFdC resistance in RPK9 cells, though this treatment inhibits production of dFdU.Combining targeted proteomics and metabolomics suggests that acquisition of resistance in RPK9 cells is due to attenuation of dFdC phosphorylation via suppression of dCK.
Keywords: drug resistance; gemcitabine; metabolomics; proteomics; pancreatic cancer

Enhanced Topical Delivery of Small Hydrophilic or Lipophilic Active Agents and Epidermal Growth Factor by Fractional Radiofrequency Microporation by Jaekwan Kim; Ji-Hye Jang; Ji Hae Lee; Jin Kyu Choi; Woo-Ram Park; Il-Hong Bae; Joonho Bae; Jin Woo Park (2017-2029).
To evaluate the ability of a novel radiofrequency (RF) microporation technology based on ablation of the skin barrier to enhance topical delivery of active ingredientsThe influence of RF fluence and the molecular size of the absorbent on the permeation enhancement was confirmed by in vitro skin permeation study using Franz diffusion cells. The improved skin rejuvenation effects, such as depigmentation and anti-wrinkle effects, by enhanced topical delivery of α-bisabolol and epidermal growth factor (EGF) through the RF microchannels were investigated in photo-damaged skin.The cumulative amounts of active ingredients through the RF microporated skin were significantly increased. Topically applied α-bisabolol after RF microporation induced rapid onset of skin whitening and significantly increased the ΔL-value of UVB-induced hyperpigmented melanin hairless mouse skin. In addition, wrinkle formation after topical application of EGF with RF microporation was significantly reduced and prevented after 12 weeks, and all parameters involving wrinkles in a replica analysis were similar to those in the negative control.RF microporation enhances the topical delivery of active ingredients with high molecular weight or of small hydrophilic or lipophilic molecules. Thus, this technology can effectively improve photo-induced hyperpigmentation and wrinkle formation by enhancing topical delivery of active agents.
Keywords: ablation; epidermal growth factor; microporation; radiofrequency; topical delivery

Erratum to: A Nebulized Gelatin Nanoparticle-Based CpG Formulation is Effective in Immunotherapy of Allergic Horses by John Klier; Sebastian Fuchs; Anna May; Ulrike Schillinger; Christian Plank; Gerhard Winter; Conrad Coester; Heidrun Gehlen (2030-2030).

AAPS Connection (2031-2033).