Pharmaceutical Research (v.27, #10)

Chemical Space: Missing Pieces in Cheminformatics by Sean Ekins; Rishi R. Gupta; Eric Gifford; Barry A. Bunin; Chris L. Waller (2035-2039).
Cheminformatics is at a turning point, the pharmaceutical industry benefits from using the various methods developed over the last twenty years, but in our opinion we need to see greater development of novel approaches that non-experts can use. This will be achieved by more collaborations between software companies, academics and the evolving pharmaceutical industry. We suggest that cheminformatics should also be looking to other industries that use high performance computing technologies for inspiration. We describe the needs and opportunities which may benefit from the development of open cheminformatics technologies, mobile computing, the movement of software to the cloud and precompetitive initiatives.
Keywords: ADME/Tox; cheminformatics; open chemistry development kit; pharmacophores; QSAR

The Mucosal Immune Response to Plant-Derived Vaccines by Kathleen Laura Hefferon (2040-2042).
Transgenic plants present enormous potential as one of the most cost-effective and safe systems for large-scale production of proteins for industrial, pharmaceutical, veterinary and agricultural uses. Heat-stable plant-derived vaccines that are administered orally could in effect enhance vaccine coverage in children and infants, particularly in developing countries. Here we discuss the current status of plant-derived vaccines and their potential to champion the battle against infectious diseases in the least developed countries.
Keywords: plant-derived biopharmaceuticals; mucosal immunity; plant biotechnology

Biodegradable Implants for Sustained Drug Release in the Eye by Susan S. Lee; Patrick Hughes; Aron D. Ross; Michael R. Robinson (2043-2053).
The safety and effectiveness of systemic and topical medical therapies for ocular disorders are limited due to poor ocular drug uptake, nonspecificity to target tissues, systemic side effects, and poor adherence to therapy. Intravitreal injections can enhance ocular drug delivery, but the need for frequent retreatment and potential injection-related side effects limit the utility of this technique. Sustained-release drug delivery systems have been developed to overcome these limitations; such systems can achieve prolonged therapeutic drug concentrations in ocular target tissues while limiting systemic exposure and side effects and improving patient adherence to therapy. A critical factor in the development of safe and effective drug delivery systems has been the development of biocompatible polymers, which offer the versatility to tailor drug release kinetics for specific drugs and ocular diseases. Ocular implants include nonbiodegradable and biodegradable designs, with the latter offering several advantages. The polymers most commonly used in biodegradable delivery systems are synthetic aliphatic polyesters of the poly-α-hydroxy acid family including polylactic acid, polyglycolic acid, and polylactic-co-glycolic acid. The characteristics of these polymers for medical applications as well as the pharmacological properties, safety, and clinical effectiveness of biodegradable drug implants for the treatment of ocular diseases are reviewed herein.
Keywords: biodegradable polymer; ocular implant; polylactic acid; polylactic-co-glycolic acid; sustained release

Vectorial Ligand Transport Through Mammalian Choroid Plexus by Reynold Spector; Conrad E. Johanson (2054-2062).
In the last decade, there has been substantial progress in understanding vectorial ligand transport through rodent and human choroid plexus (CP), the locus of the blood-CSF interface. In this Review, we enumerate the experimental data required to establish vectorial transport through CP and describe transporters involved in vectorial transport across CP. We also note how these transporters differ from those at the blood-brain barrier. The ligand (substrate) examples presented are methyltetrahydrofolate, methotrexate, leukotriene C4, nucleosides, thiamine monophosphate, prostaglandins, and digoxin. Our focus is on more definitive experiments, including animal and human transporter “knock-outs.” Finally, we discuss the neurochemical implications of vectorial transport through CP and the clinical implications of transporter polymorphisms and knockouts. Examples include descriptions of how vectorial transport through the CP for several micronutrients (e.g., methyltetrahydrofolate) nourishes the brain and how knowledge of CP vectorial transport can lead to important treatments.
Keywords: brain nourishment; cerebrospinal fluid; multidrug resistance proteins; neuroprotection; organic acid transporting polypeptides

Development of PLGA-Based Injectable Delivery Systems For Hydrophobic Fenretinide by Christian Wischke; Ying Zhang; Sachin Mittal; Steven P. Schwendeman (2063-2074).
Although efficient in vitro, fenretinide has not been successful clinically for either of the targeted indications—cancer prevention and dry age-related macular degeneration—because of various issues, such as low oral bioavailability. Therefore, controlled release carriers for parenteral delivery of fenretinide were developed.After examining the solubility profile of fenretinide, the drug was encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles at 20% drug loading by an s/o/w methodology as well as into in situ-forming PLGA implants. The carrier morphology and drug release kinetics in an elevated polysorbate 80-containing release medium were studied.Preformulation studies revealed increased fenretinide solubility in various PLGA solvents including N-methylpyrrolidone (NMP) and 1:9 v/v methanol:methylene chloride. Co-solvent emulsion methods resulted in low encapsulation efficiency. With a s/o/w method, fenretinide release rates from injectable microparticles were adjusted by the o-phase concentration of end-capped PLGA, the drug particle size, and the particle porosity. In situ implants from non-capped PLGA in NMP exhibited a continuous release of ~70% drug over 1 month.Injectable carriers for fenretinide were successfully prepared, exhibiting excellent drug stability. Based on the in vitro release properties of the different carriers, the preferred injection sites and in vivo release rates will be determined in future preclinical studies.
Keywords: controlled release; fenretinide; hydrophobic drug; in situ implant; s/o/w PLGA microparticle

Hypoxia-Inducible Vascular Endothelial Growth Factor Gene Therapy Using the Oxygen-Dependent Degradation Domain in Myocardial Ischemia by Hyun Ah Kim; Soyeon Lim; Hyung-Ho Moon; Sung Wan Kim; Ki-Chul Hwang; Minhyung Lee; Sun Hwa Kim; Donghoon Choi (2075-2084).
A hypoxia-inducible VEGF expression system with the oxygen-dependent degradation (ODD) domain was constructed and tested to be used in gene therapy for ischemic myocardial disease.Luciferase and VEGF expression vector systems were constructed with or without the ODD domain: pEpo-SV-Luc (or pEpo-SV-VEGF) and pEpo-SV-Luc-ODD (or pEpo-SV-VEGF-ODD). In vitro gene expression efficiency of each vector type was evaluated in HEK 293 cells under both hypoxic and normoxic conditions. The amount of VEGF protein was estimated by ELISA. The VEGF expression vectors with or without the ODD domain were injected into ischemic rat myocardium. Fibrosis, neovascularization, and cardiomyocyte apoptosis were assessed using Masson’s trichrome staining, α-smooth muscle actin (α-SMA) immunostaining, and the TUNEL assay, respectively.The plasmid vectors containing ODD significantly improved the expression level of VEGF protein in hypoxic conditions. The enhancement of VEGF protein production was attributed to increased protein stability due to oxygen deficiency. In a rat model of myocardial ischemia, the pEpo-SV-VEGF-ODD group exhibited less myocardial fibrosis, higher microvessel density, and less cardiomyocyte apoptosis compared to the control groups (saline and pEpo-SV-VEGF treatments).An ODD-mediated VEGF expression system that facilitates VEGF-production under hypoxia may be useful in the treatment of ischemic heart disease.
Keywords: gene therapy; hypoxia-inducible expression; ischemic myocardium; oxygen-dependent degradation domain; vascular endothelial growth factor

Silibinin Exerts Sustained Growth Suppressive Effect against Human Colon Carcinoma SW480 Xenograft by Targeting Multiple Signaling Molecules by Balaiya Velmurugan; Subhash Chander Gangar; Manjinder Kaur; Alpna Tyagi; Gagan Deep; Rajesh Agarwal (2085-2097).
Earlier, we reported the strong preventive efficacy of silibinin against colorectal cancer (CRC), but its usefulness against established CRC or effect of its withdrawal on CRC growth remained unknown. Present study focused on these important issues by employing two different treatment protocols in advanced human CRC SW480 xenograft in nude mice.In the first treatment protocol, silibinin was fed for 28 days (200 mg/kg body weight, 5 days/week) to mice with growing SW480 xenograft; thereafter, tumor growth was monitored for additional 3 weeks without silibinin treatment. In the second protocol, silibinin treatment was started after 25 days of SW480 cells injection (established tumors), and tumor growth was studied 4 days, 8 days and 16 days after silibinin treatment.In both treatment protocols, silibinin had strong and sustained inhibitory effect on xenograft growth. Detailed xenograft analyses showed that silibinin, in both treatment protocols, exerts anti-proliferative, pro-apoptotic and anti-angiogenic effects. Further, silibinin reduced the expression of β-catenin and phospho-GSK3β in xenograft tissues. Silibinin also targeted signaling molecules involved in CRC proliferation and survival (cyclin D1, c-Myc and survivin) as well as angiogenesis regulators (VEGF and iNOS).Collectively, these findings substantiate silibinin’s therapeutic efficacy against CRC, advocating its translational potential.
Keywords: angiogenesis; β-catenin; colorectal cancer; silibinin; SW480 cells

In Vivo Inhibition of BCRP/ABCG2 Mediated Transport of Nitrofurantoin by the Isoflavones Genistein and Daidzein: A Comparative Study in Bcrp1 −/− Mice by Gracia Merino; Miriam Perez; Rebeca Real; Estefania Egido; Julio G. Prieto; Ana I. Alvarez (2098-2105).
The aim of this study was to determine in vivo inhibition by the isoflavones genistein and daidzein of nitrofurantoin (NTF), a well-known substrate of the ABC transporter BCRP/ABCG2.MDCKII cells and their human BCRP- and murine Bcrp1-transduced subclones were used to establish inhibition in transepithelial transport assays. Bcrp1−/− and wild-type mice were coadministered with nitrofurantoin (20 mg/kg) and a mixture of genistein (100 mg/kg) and daidzein (100 mg/kg).Transepithelial NFT transport was inhibited by the isoflavones. Plasma concentration of NTF at 30 min was 1.7-fold higher (p ≤ 0.05) in wild-type mice after isoflavone administration. AUC values were not significantly different. BCRP/ABCG2-mediated secretion into milk was inhibited since milk/plasma ratios were lower in wild-type mice with isoflavones (7.1 ± 4.2 vs 4.2 ± 1.6, p ≤ 0.05). NTF bile levels were significantly decreased by isoflavone administration in wild-type animals (8.8 ± 3.4 μg/ml with isoflavones vs 3.7 ± 3.3 μg/ml without isoflavones).Our data showed that in vivo interaction of high doses of soy isoflavones with BCRP substrates may affect plasma levels but the main effect occurs in specific target organs, in our case, liver and mammary glands.
Keywords: BCRP/ABCG2; Bcrp1 −/− ; isoflavones; MDCKII cells; nitrofurantoin

Polymethacrylate Microparticles Gel for Topical Drug Delivery by Hagar Ibrahim Labouta; Labiba K. El-Khordagui (2106-2118).
Evaluating the potentials of particulate delivery systems in topical drug delivery.Polymethacrylate microparticles (MPs) incorporating verapamil hydrochloride (VRP) as a model hydrophilic drug with potential topical clinical uses, using Eudragit RS100 and Eudragit L100 were prepared for the formulation of a composite topical gel. The effect of initial drug loading, polymer composition, particularly the proportion of Eudragit L100 as an interacting polymer component and the HLB of the dispersing agent on MPs characteristics was investigated. A test MPs formulation was incorporated in gel and evaluated for drug release and human skin permeation.MPs showed high % incorporation efficiency and % yield. Composition of the hybrid polymer matrix was a main determinant of MPs characteristics, particularly drug release. Factors known to influence drug release such as MPs size and high drug solubility were outweighed by strong VRP-Eudragit L100 interaction. The developed MPs gel showed controlled VRP release and reduced skin retention compared to a free drug gel.Topical drug delivery and skin retention could be modulated using particulate delivery systems. From a practical standpoint, the VRP gel developed may offer advantage in a range of dermatological conditions, in response to the growing off-label topical use of VRP.
Keywords: polymethacrylates; topical drug delivery; microparticles gel; verapamil

Predicting Intestinal Precipitation—A Case Example for a Basic BCS Class II Drug by Sara Carlert; Anna Pålsson; Gunilla Hanisch; Christian von Corswant; Catarina Nilsson; Lennart Lindfors; Hans Lennernäs; Bertil Abrahamsson (2119-2130).
To investigate the prediction accuracy of in vitro and in vitro/in silico methods for in vivo intestinal precipitation of basic BCS class II drugs in humans.Precipitation rate of a model drug substance, AZD0865 (pKa = 6.1; log KD = 4.2), was investigated in vitro using simulated intestinal media, and calculations of the crystallization rates were made with a theoretical model. Human intestinal precipitation was estimated by analysis of pharmacokinetic data from clinical studies at different doses.All in vitro models predicted rapid drug precipitation, where the intestinal concentration of dissolved AZD0865 at the highest dose tested was expected to decrease to half after less than 20 min. However, there was no indication of precipitation in vivo in humans as there was a dose proportional increase in drug plasma exposure. The theoretical model predicted no significant precipitation within the range of expected in vivo intestinal concentrations.This study indicated that simple in vitro methods of in vivo precipitation of orally administered bases overpredict the intestinal crystalline precipitation in vivo in humans. Hydrodynamic conditions were identified as one important factor that needs to be better addressed in future in vivo predictive methods.
Keywords: absorption-biopharmaceutics classification system; gastrointestinal; in vitro/in vivo correlations (IVIVC); precipitation

Inhalation Performance of Physically Mixed Dry Powders Evaluated with a Simple Simulator for Human Inspiratory Flow Patterns by Daiki Hira; Tomoyuki Okuda; Daisuke Kito; Kazunori Ishizeki; Toyoko Okada; Hirokazu Okamoto (2131-2140).
To construct a simple simulator reproducing human inspiratory flow patterns and use it to evaluate the inhalation performance of active ingredient particle-carrier particle systems (physically mixed dry powders).Inspiratory flow patterns were collected and analyzed using a flow recorder. The simulator was constructed using an airtight container, a valve, and a connecting tube. Several of the patterns reproduced by the simulator were compared with those recorded. In addition, the influence of inspiratory flow on the inhalation performance of physically mixed dry powders composed of salbutamol sulfate (SS) and coarse lactose monohydrate was investigated using a twin-stage liquid impinger (TSLI) equipped with the simulator.Human inspiratory flow patterns could be characterized by three parameters: inspiratory flow volume (area under the flow rate-time curve (AUC)), flow increase rate (FIR), and peak flow rate (PFR). The patterns could be reproduced using the simulator. Testing with the simulator in vitro revealed that PFR, but not FIR or AUC, greatly affected the inhalation performance of physically mixed dry powders.The simulator is simple to construct and can schematically reproduce human inspiratory flow patterns. Testing with a TSLI and the simulator is useful to evaluate dry powder formulations for clinical application.
Keywords: dry powder inhaler; inhaler testing; inspiratory flow pattern; physically mixed dry powders; twin-stage liquid impinger (TSLI)

Intestinal Absorption of HMG-CoA Reductase Inhibitor Pravastatin Mediated by Organic Anion Transporting Polypeptide by Yoshiyuki Shirasaka; Kensuke Suzuki; Takeo Nakanishi; Ikumi Tamai (2141-2149).
The purpose of this study is to clarify the impact of organic anion transporting polypeptide (Oatp) on intestinal absorption of the HMG-CoA reductase inhibitor, pravastatin.OATP/Oatp-mediated pravastatin uptake was evaluated with Xenopus oocytes systems. Intestinal permeability was measured by an in situ closed loop method in rats. In vivo pravastatin absorption was kinetically assessed by measuring plasma concentration after oral administration in rats.Uptake of pravastatin by Oatp1a5, Oatp2b1, OATP1A2, and OATP2B1 cRNA-injected Xenopus oocytes was significantly increased compared with that by water-injected oocytes. Naringin, a potent inhibitor of Oatps and Mdr1, decreased the Oatp1a5-mediated uptake of pravastatin with IC 50 value of 30.4 μM. Rat intestinal permeability of pravastatin was significantly reduced in the presence of 1,000 μM naringin. Similar results were obtained in in vivo absorption studies in rats. Furthermore, no significant change in the permeability was observed in the presence of elacridar, a potent inhibitor of both Mdr1 and Bcrp, though the permeability was significantly decreased in the presence of both elacridar and naringin, suggesting that Mdr1 and Bcrp are not involved in intestinal absorption of pravastatin.Oatp, but not by Mdr1 or Bcrp, contributes to the intestinal absorption of pravastatin in rats.
Keywords: intestinal absorption; Mdr1; naringin; Oatp; pravastatin

To provide a definitive assessment of prediction of in vivo CL int from human liver in vitro systems for assessment of typical underprediction.A database of published predictions of clearance from human hepatocytes and liver microsomes was compiled, including only intravenous CL b. The influence of liver model (well-stirred (WS) or parallel tube (PT)), plasma protein binding and clearance level on the relationship between in vitro and in vivo CL int was examined.Average prediction bias was about 5- and 4-fold for microsomes and hepatocytes, respectively. Reduced bias using the PT model, in preference to the popular WS model, was only marginal across a wide range of clearance with a consequential minor impact on prediction. Increasing underprediction with decreasing fu b, or increasing CL int, was found only for hepatocytes, suggesting fundamental in vitro artefacts rather than failure to model potentially unequilibrated binding during rapid extraction.In contrast to microsomes, hepatocytes give a disproportionate prediction with increasing clearance suggesting limitations either at the active site, such as cofactor exhaustion, or with intracellular concentration equilibrium, such as rate-limiting cell permeability. A simple log linear empirical relationship can be used to correct hepatocyte predictions.
Keywords: clearance; hepatocytes; human; microsomes; prediction

Using human lung cancer cells, we evaluated the involvement of plasminogen activator inhibitor-1 (PAI-1) in the anti-invasive action of cannabidiol, a non-psychoactive cannabinoid.Invasion was quantified by a modified Boyden chamber assay. PAI-1 protein in cell culture media and PAI-1 mRNA were determined by immunoblotting and RT-PCR, respectively.Cannabidiol caused a profound inhibition of A549 cell invasion, accompanied by a decreased expression and secretion of PAI-1. Cannabidiol's effects on PAI-1 secretion and invasion were suppressed by antagonists to CB1 and CB2 receptors as well as to transient receptor potential vanilloid 1. Recombinant human PAI-1 and PAI-1 siRNA led to a concentration-dependent up- and down-regulation of invasiveness, respectively, suggesting a crucial role of PAI-1 in A549 invasiveness. Evidence for a causal link between cannabidiol's effects on PAI-1 and invasion was provided by experiments showing a reversal of its anti-invasive action by addition of recombinant PAI-1 at non-proinvasive concentrations. Key data were confirmed in two other human lung cancer cell lines (H460, H358). In vivo, a significant downregulation of PAI-1 protein by cannabidiol was demonstrated in A549 xenografts.Our data provide evidence for a hitherto unknown mechanism underlying the anti-invasive action of cannabidiol on human lung cancer cells.
Keywords: cannabidiol; lung cancer cells; matrigel invasion; plasminogen activator inhibitor-1

To develop a novel oral drug delivery system comprising a hydrophobic chemotherapeutic drug entrapped within beta casein (β–CN), a major milk protein, which self-associates into micelles in aqueous solutions. The efficient gastric digestibility of β–CN suggests possible targeting to gastric cancers.Antitumor drug entrapment was performed by stirring its dimethyl-sulfoxide solution into a phosphate-buffered saline containing β–CN. The association of drugs to β–CN was characterized by spectrophotometry and Trp143 fluorescence quenching; particle-size by dynamic light scattering, and colloidal stability by zeta potential.The optimal drug-to-β–CN molar loading-ratios for paclitaxel and vinblastine at 1 mg/ml β–CN were found to be 7.3 ± 1.2 and 5.3 ± 0.6 and the association constants were (6.3 ± 1.0)·103 M−1 and (2.0 ± 0.3)·104 M−1, respectively. Zeta potential analysis suggested that nanoencapsulation by β–CN stabilized all studied drugs in aqueous solution. The initial drug-β–CN association was apparently governed by hydrophobic interactions and at higher drug concentrations, also by electrostatic interactions. Up to the optimal drug:β–CN loading-ratio, ~80% of the particles were below 100 nm in diameter. At higher drug concentrations, particle diameter increased, and bi- or tri-modal particle distributions were observed.Beta–CN forms colloidally-stable nanovehicles of hydrophobic anticancer drugs, and may be used for oral-delivery of chemotherapeutics.
Keywords: β-casein micelles; cancer; gastrointestinal tract; hydrophobic chemotherapeutics; targeted oral delivery

Biorelevant Media to Simulate Fluids in the Ascending Colon of Humans and Their Usefulness in Predicting Intracolonic Drug Solubility by Maria Vertzoni; Amalia Diakidou; Manos Chatzilias; Erik Söderlind; Bertil Abrahamsson; Jennifer B. Dressman; Christos Reppas (2187-2196).
To develop media simulating human colonic fluids (HCFs), to evaluate their use in predicting intracolonic solubility of ketoconazole, danazol and felodipine and to compare solubilities in HCFs with previously determined solubilities in gastric (HGFs) and small intestinal (HIFs) fluids.Fasted state simulated colonic fluid (FaSSCoF) and fed state simulated colonic fluid (FeSSCoF) were designed to reflect fluids previously collected from the ascending colon in healthy adults. Solubilities of the three model compounds were determined in HCFs, simulated HCFs, and plain buffers.For ketoconazole, solubilities in FaSSCoF and FeSSCoF were closer than those in the corresponding plain buffers to the solubility in HCFs. For danazol and felodipine, solubilities in FaSSCoF and FeSSCoF predicted solubilities in HCFs. In the fasted state, solubilities of danazol and felodipine in HCFs were higher than or similar to in HGFs or HIFs, while the ketoconazole solubility was lower. In the fed state, solubilities of all three model compounds in HCFs were lower than in HGFs or HIFs.FaSSCoF and FeSSCoF more closely predict solubility of poorly soluble compounds in HCFs than plain buffers. In most cases, solubility in HCFs differs from those in HGFs and HIFs.
Keywords: ascending colon; drug solubility; FaSSCoF; FeSSCoF; simulated colonic fluids

Mass Spectrometric Analysis of Intact Human Monoclonal Antibody Aggregates Fractionated by Size-Exclusion Chromatography by Başak Kükrer; Vasco Filipe; Esther van Duijn; Piotr T. Kasper; Rob J. Vreeken; Albert J. R. Heck; Wim Jiskoot (2197-2204).
The aim of this study was to develop a method to characterize intact soluble monoclonal IgG1 antibody (IgG) oligomers by mass spectrometry.IgG aggregates (dimers, trimers, tetramers and high-molecular-weight oligomers) were created by subjecting an IgG formulation to several pH jumps. Protein oligomer fractions were isolated by high performance size exclusion chromatography (HP-SEC), dialyzed against ammonium acetate pH 6.0 (a mass spectrometry-compatible volatile buffer), and analyzed by native electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS).Monomeric and aggregated IgG fractions in the stressed IgG formulation were successfully isolated by HP-SEC. ESI-TOF MS analysis enabled us to determine the molecular weight of the monomeric IgG as well as the aggregates, including dimers, trimers and tetramers. HP-SEC separation and sample preparation proved to be necessary for good quality signal in ESI-TOF MS. Both the HP-SEC protocol and the ESI-TOF mass spectrometric technique were shown to leave the IgG oligomers largely intact.ESI-TOF MS is a useful tool complementary to HP-SEC to identify and characterize small oligomeric protein aggregates.
Keywords: HP-SEC; IgG1; monoclonal antibody; native ESI-TOF mass spectrometry; protein aggregation

Holmium Nanoparticles: Preparation and In Vitro Characterization of a New Device for Radioablation of Solid Malignancies by Wouter Bult; Rosanne Varkevisser; Fouad Soulimani; Peter R. Seevinck; Hendrik de Leeuw; Chris J. G. Bakker; Peter R. Luijten; Alfred D. van het Schip; Wim E. Hennink; J. Frank W. Nijsen (2205-2212).
The present study introduces the preparation and in vitro characterization of a nanoparticle device comprising holmium acetylacetonate for radioablation of unresectable solid malignancies.HoAcAc nanoparticles were prepared by dissolving holmium acetylacetonate in chloroform, followed by emulsification in an aqueous solution of a surfactant and evaporation of the solvent. The diameter, surface morphology, holmium content, and zeta potential were measured, and thermal behavior of the resulting particles was investigated. The stability of the particles was tested in HEPES buffer. The r2* relaxivity of protons and mass attenuation coefficient of the nanoparticles were determined. The particle diameter and surface morphology were studied after neutron activation.Spherical particles with a smooth surface and diameter of 78 ± 10 nm were obtained, and the particles were stable in buffer. Neutron irradiation did not damage the particles, and adequate amounts of activity were produced for nuclear imaging and radioablation of malignancies through intratumoral injections.The present study demonstrates that HoAcAc nanoparticles were prepared using a solvent evaporation process. The particle diameter can easily be adapted and can be optimized for specific therapeutic applications and tumor types.
Keywords: ablation; characterization; holmium; nanoparticles; tumor

Microneedle Pre-treatment of Human Skin Improves 5-Aminolevulininc Acid (ALA)- and 5-Aminolevulinic Acid Methyl Ester (MAL)-Induced PpIX Production for Topical Photodynamic Therapy Without Increase in Pain or Erythema by Patrycja Mikolajewska; Ryan F. Donnelly; Martin J. Garland; Desmond I. J. Morrow; Thakur Raghu Raj Singh; Vladimir Iani; Johan Moan; Asta Juzeniene (2213-2220).
To determine the impact of skin pretreatment with microneedles (MNs) on ALA- and MAL-induced protoporphyrin IX (PpIX) production, as well as MN impact on pain sensations during light exposure and erythema after PDT.The skin of 14 healthy volunteers was preteated with MNs. Equal amounts of creams containing 2%, 8% and 16% (w/w) ALA and MAL were applied on 1 cm2 areas for 4 h. Additionally, 16% ALA and MAL creams were applied for 24 h. Afterwards, PpIX fluorescence spectra were measured. Sixteen percent ALA and MAL spots were exposed to red light (632 nm, 77 mW/cm2). Time for pain to occur was measured in seconds, and erythemal response was monitored up to 6 h after the end of the light exposure.Use of MNs increased the PpIX fluorescence after 4 h incubation time with 2% and 8% ALA or MAL, but not with 16% ALA or MAL. Pretreatment with MNs did not increase the pain sensations during light exposure, nor did it influence erythema occurrence.MNs are a promising tool for improving the efficiency of topical PDT by improving the cutaneous delivery of ALA and MAL, without increase in side effects.
Keywords: aminolevulinic acid (ALA); aminolevulinic acid methyl ester (MAL); erythema; microneedles (MNs); photodynamic therapy (PDT)

Anisotropic Porous Structure of Pharmaceutical Compacts Evaluated by PGSTE-NMR in Relation to Mechanical Property Anisotropy by Patrice Porion; Virginie Busignies; Vincent Mazel; Bernard Leclerc; Pierre Evesque; Pierre Tchoreloff (2221-2233).
The pore space anisotropy of pharmaceutical compacts was evaluated in relation to the mechanical property anisotropy.The topology and the pore space anisotropy were characterized by PGSTE-NMR measurements. Parallelepipedical compacts of anhydrous calcium phosphate (aCP) and microcrystalline cellulose (MCC) were tested on top, bottom and side faces. A microindentation and three-point single beam tests were used to measure Brinell hardness, tensile strength and Young’s modulus. All the data were submitted to a statistical analysis to test for significance.The porous structure of MCC compacts was anisotropic, contrary to those of aCP. The analysis of the pore space by PGSTE-NMR method showed that its structural anisotropy was controlled by the behaviour under compaction of the excipients. At the same time, the Young’s modulus and the tensile strength were the same whatever the direction of testing. For the aCP compacts, all the faces had the same Brinell hardness. With MCC compacts, only the bottom face showed a lower Brinell hardness.Except for Brinell hardness measured on MCC compacts, the tested samples were characterized by anisotropic mechanical properties when its porous structures were sometimes anisotropic. Then, there is not a straight link between porosity anisotropy and mechanical properties.
Keywords: anisotropy; compaction; mechanical property anisotropy; porous structure; pulsed-gradient stimulated-echo NMR

To investigate the use of thermally stimulated current (TSC) to characterize disorder resulting from micronization of a crystalline drug substance. Samples processed at different milling energies are characterized, and annealing studied.Molecular mobility in micronized drug substance was studied using TSC and compared to results from differential scanning calorimetry (DSC). The micronized drug substance TSC spectra are compared to crystalline and amorphous references.TSC shows distinct relaxation modes for micronized material in comparison to a single weak exotherm observed with DSC. Molecular mobility modes are unique for micronized material compared to the amorphous reference indicating physically distinct disorder compared to phase-separated amorphous material. Signals are ascribed as arising from crystal defects. TSC differentiates material processed at different milling energies showing reasonable correlation between the AUC of the α-relaxation and micronization energy. The annealing process of crystal defects in micronized drug appears to proceed differently for α and β relaxations.TSC proves sensitive to the crystal defects in the micronized drug substance studied here. The technique is able to differentiate distinct types of disorder and can be used to characterize noncrystalline regions arising from milling processes which are physically distinct from amorphous material.
Keywords: annealing of surface disorder; crystal defects; micronization energy; non-crystalline content; thermally stimulated current (TSC)

Simultaneous Delivery of Chemotherapeutic and Thermal-Optical Agents to Cancer Cells by a Polymeric (PLGA) Nanocarrier: An In Vitro Study by Yuan Tang; Tingjun Lei; Romila Manchanda; Abhignyan Nagesetti; Alicia Fernandez-Fernandez; Supriya Srinivasan; Anthony J. McGoron (2242-2253).
To test the effectiveness of a dual–agent-loaded PLGA nanoparticulate drug delivery system containing doxorubicin (DOX) and indocyanine green (ICG) in a DOX-sensitive cell line and two resistant cell lines that have different resistance mechanisms.The DOX-sensitive MES-SA uterine sarcoma cell line was used as a negative control. The two resistant cell lines were uterine sarcoma MES-SA/Dx5, which overexpresses the multidrug resistance exporter P-glycoprotein, and ovarian carcinoma SKOV-3, which is less sensitive to doxorubicin due to a p53 gene mutation. The cellular uptake, subcellular localization and cytotoxicity of the two agents when delivered via nanoparticles (NPs) were compared to their free-form administration.The cellular uptake and cytotoxicity of DOX delivered by NPs were comparable to the free form in MES-SA and SKOV-3, but much higher in MES-SA/Dx5, indicating the capability of the NPs to overcome P-glycoprotein resistance mechanisms. NP-encapsulated ICG showed slightly different subcellular localization, but similar fluorescence intensity when compared to free ICG, and retained the ability to generate heat for hyperthermia delivery.The dual-agent-loaded system allowed for the simultaneous delivery of hyperthermia and chemotherapy, and this combinational treatment greatly improved cytotoxicity in MES-SA/Dx5 cells and to a lesser extent in SKOV-3 cells.
Keywords: doxorubicin; indocyanine green; hyperthermia; P-glycoprotein; p53

News (2254-2255).