Pharmaceutical Research (v.25, #6)

Knowledge concerning transport of maternally administered drugs across the placental barrier is essential for determining potential toxicity of drugs to the fetus and the value of drug therapy during pregnancy. An important determinant for fetal drug exposure is the expression of efflux transporters in the placenta. Among human tissues, the ATP-binding cassette efflux transporter BCRP (gene symbol ABCG2) is most abundantly expressed in the apical membrane of placental syncytiotrophoblasts. Although the precise physiological role of BCRP in the placenta is still unclear, existing data strongly suggest that BCRP plays an important role in protecting the fetus against the potential toxicity of drugs, xenobiotics, and metabolites by expelling them across the placental barrier. In this review, we summarize the current knowledge with respect to the expression, function, and polymorphisms of BCRP, as well as transcriptional and posttranscriptional regulation of the transporter in the placenta. Finally, clinical significance of BCRP in the placenta for drug therapy in pregnant women is discussed.
Keywords: ABC transporter; BCRP/ABCG2 ; fetus; placenta; pregnancy

Development of Stable Influenza Vaccine Powder Formulations: Challenges and Possibilities by J-P. Amorij; A. Huckriede; J. Wilschut; H. W. Frijlink; W. L. J. Hinrichs (1256-1273).
Influenza vaccination represents the cornerstone of influenza prevention. However, today all influenza vaccines are formulated as liquids that are unstable at ambient temperatures and have to be stored and distributed under refrigeration. In order to stabilize influenza vaccines, they can be brought into the dry state using suitable excipients, stabilizers and drying processes. The resulting stable influenza vaccine powder is independent of cold-chain facilities. This can be attractive for the integration of the vaccine logistics with general drug distribution in Western as well as developing countries. In addition, a stockpile of stable vaccine formulations of potential vaccines against pandemic viruses can provide an immediate availability and simple distribution of vaccine in a pandemic outbreak. Finally, in the development of new needle-free dosage forms, dry and stable influenza vaccine powder formulations can facilitate new or improved targeting strategies for the vaccine compound. This review represents the current status of dry stable inactivated influenza vaccine development. Attention is given to the different influenza vaccine types (i.e. whole inactivated virus, split, subunit or virosomal vaccine), the rationale and need for stabilized influenza vaccines, drying methods by which influenza vaccines can be stabilized (i.e. lyophilization, spray drying, spray-freeze drying, vacuum drying or supercritical fluid drying), the current status of dry influenza vaccine development and the challenges for ultimate market introduction of a stable and effective dry-powder influenza vaccine.
Keywords: analytical challenges; influenza vaccine; lyophilization; needle-free dosage forms; spray drying; spray-freeze drying; stability; stock piling for pandemics; virosomes

The purpose of this study was to increase the dissolution rate of the poorly water soluble, antifungal drug Itraconazole.Itraconazole was successfully micronized using both the gas antisolvent (GAS) and aerosol solvent extraction systems (ASES) using Acetone as the solvent. The affects of operating conditions such as temperature, pressure and solvent choice on variables such as morphology, particle size and dissolution were investigated. The influence of temperature in the range 25 to 40°C and pressure between 90 and 190 bar were investigated.Solvent choice was found to have the largest affect on particle production, with acetone found to be the optimal solvent choice when compared with dimethyl formamide (DMF), tetrahydrofuran (THF) and dichloromethane (DCM). Itraconazole particles with an average particle size of 6.9 μm were formed at the optimal ASES processing conditions of 40°C and 190 bar. More significantly, in the first 100 minutes of dissolution 71.1% of the dense gas processed itraconazole was dissolved compared with 52.5% of Sporonox (the commercially available formulation) and 14.6% of the unprocessed material. Additional studies demonstrated that the formation of an itraconazole/PEG composite resulted in a 6-fold increase in dissolution rate in the first 100 min, to 89.8%, when compared to the unprocessed material.Using ASES, microparticles of itraconazole were produced with an increased dissolution rate compared with raw material and commercially available product.
Keywords: dissolution rate; gas antisolvent; itraconazole; polyethylene glycol; supercritical fluids

Increased Intestinal Delivery of Viable Saccharomyces boulardii by Encapsulation in Microspheres by Sandrine Graff; Sajjad Hussain; Jean-Claude Chaumeil; Christine Charrueau (1290-1296).
Although probiotics are of a major potential therapeutic interest, their efficacy is usually limited by poor bioavailability of viable microorganisms on site. The aim of this study was to protect the probiotic Saccharomyces boulardii from degradation in order to ensure a greater number of viable yeast in the colon.Alginate microspheres coated with or not with chitosan were used to encapsulate the yeast by an extrusion method. The efficiency of encapsulation was assessed both in vitro and in vivo. In vitro, less than 1% of the non-encapsulated probiotic survived after 120 min at pH 1.1, whereas the majority of encapsulated yeast cells remained entrapped within both types of microspheres. Further exposure to a pH 6.8 allowed the release of about 35% of viable yeasts. In vivo, the percentage of viable yeast excreted over 96 h after a single oral dose of 2 × 108 cfu/100 g in rats was 2.5% for non-encapsulated yeast and reached 13.3 and 9.0% of the dose administered for the uncoated and chitosan-coated microspheres, respectively.Given the dose-dependent efficacy of S. boulardii and the efficiency of microencapsulation in protecting the yeast from degradation, alginate microspheres could be of great interest in therapeutic applications of the yeast.
Keywords: alginate; gastrointestinal transit; microspheres; probiotic; Saccharomyces boulardii

The purpose of the present study was to identify the biochemical mechanism(s) of the preventative and reversal effects of Chaetoglobosin K (ChK), a bioactive natural product, on inhibition of gap junction-mediated communication and connexin phosphorylation by the tumor promoting organochlorine compounds, lindane, and dieldrin.A fluorescent dye transfer assay was used to quantify gap junction-mediated communication and sensitivity to lindane and dieldrin. Analyses of connexin 43, PKC, ERK, GSK-3β, Raf, and Akt kinase phosphorylation were performed by Western blotting.Pre-incubation of astroglial cells with 10 μM ChK prevented inhibition of dye transfer by lindane and dieldrin, which correlates with stabilization of the connexin 43 P2 isoform, and addition of ChK after lindane or dieldrin reversed the inhibitory effect, which correlated with re-appearance of the P2 isoform. Using phosphorylation site-specific antibodies, we demonstrated that lindane, dieldrin, and ChK all activated p44/42 ERK, but only ChK activated Akt kinase. ChK also activated a downstream effector of Akt, GSK-3β, and activation of both kinases was inhibited by Wortmannin. Wortmannin also blocked ChK’s ability to prevent dieldrin-induced inhibition of gap junction-mediated communication between RG-2 cells.ChK’s protective effects, both preventative and reversal, on lindane and dieldrin inhibition of gap junction-mediated communication are associated with stabilization and reappearance of the connexin 43 P2 phosphoform and may be mediated by the Akt pathway.
Keywords: chaetoglobosin K; connexin; gap-junction; organochlorine

Toxicological Protein Biomarker Analysis—An Investigative One-week Single Dose Intravenous Infusion Toxicity and Toxicokinetic Study in Cynomolgus Monkeys using an Antibody–cytotoxic Conjugate against Ovarian Cancer by Frank Y. Hsieh; Elizabeth Tengstrand; Lily Y. Li; Yuling N. Huang; Mark N. Milton; Lee Silverman; Carl Alden; Gerald Miwa; Frank Lee (1309-1317).
Antibody—cytotoxic conjugates are complex novel therapeutic agents whose toxicological properties are not presently well understood. The objective of this study was to identify toxicological markers in serum that correlate with MLN8866 (an antibody—cytotoxic conjugate) exposure and related pathological events in monkeys.Cynomolgus monkeys were treated once with 5, 15, or 30 mg/kg MLN8866 via a 20 min intravenous infusion. MLN8866 exposure (Cmax and AUC0–4 day) was determined by quantifying MLN8866 levels in serum.The increase in MLN8866 exposure was approximately dose proportional. Two acute phase proteins in serum (serum amyloid A and haptoglobin) were correlated with MLN8866 exposure and toxicological outcomes (e.g., erythropoiesis and leucopoiesis).
Keywords: biologics; biomarker; predictive toxicity; toxicokinetics; toxicology

Aerodynamical, Immunological and Pharmacological Properties of the Anticancer Antibody Cetuximab Following Nebulization by Agnès Maillet; Nicolas Congy-Jolivet; Sandrine Le Guellec; Laurent Vecellio; Sophie Hamard; Yves Courty; Anthony Courtois; Francis Gauthier; Patrice Diot; Gilles Thibault; Etienne Lemarié; Nathalie Heuzé-Vourc’h (1318-1326).
Despite an increasing interest in the use of inhalation for local delivery of molecules for respiratory diseases and systemic disorders, methods to deliver therapy through airways has received little attention for lung cancer treatment. However, inhalation of anticancer drugs is an attractive alternative route to systemic administration which results in limited concentration of the medication in the lungs, and triggers whole-body toxicity. In this study, we investigated the feasibility of nebulization for therapeutic antibodies, a new class of fully-approved anticancer drugs in oncology medicine.Cetuximab, a chimeric IgG1 targeting the epidermal growth factor receptor (EGFR), was nebulized using three types of delivery devices: a jet nebulizer PARI LC+®, a mesh nebulizer AeronebPro® and an ultrasonic nebulizer SYST’AM® LS290. Aerosol size distribution was measured using a cascade impactor and aerosol droplets were observed under optical microscopy. The immunological and pharmacological properties of cetuximab were evaluated following nebulization using A431 cells.The aerosol particle clouds generated with the three nebulizers displayed similar aerodynamical characteristics, but the IgG formed aggregates in liquid phase following nebulization with both the jet and ultrasonic devices. Flow cytometry analyses and assays of EGFR-phosphorylation and cell growth inhibitions on A431 demonstrated that both the mesh and the jet nebulizers preserved the binding affinity to EGFR and the inhibitory activities of cetuximab.Altogether, our results indicate that cetuximab resists the physical constraints of nebulization. Thus, airway delivery represents a promising alternative to systemic administration for local delivery of therapeutic antibodies in lung cancer treatment.
Keywords: aerosol; aggregation; anticancer antibody; cetuximab; lung cancer; nebulizer

PPARα/γ Expression and Activity in Mouse and Human Melanocytes and Melanoma Cells by Linda L. Eastham; Caroline N. Mills; Richard M. Niles (1327-1333).
We examined the expression of PPARs and the effects of PPARα and PPARγ agonists on growth of mouse and human melanocytes and melanoma cells.PPARα,β, and PPARγ mRNA qualitative expression in melan-a mouse melanocytes, B16 mouse melanoma, human melanocytes, and A375 and SK-mel28 human melanoma cells was determined by RT-PCR, while quantitative PPARα mRNA levels were determined by QuantiGene assay. PPARα and PPARγ protein was assessed by Western blotting. The effect of natural and synthetic PPAR ligands on cell growth was determined by either hemocytometer counting or crystal violet assay. PPAR transcriptional activity was determined by a PPRE-reporter gene assay, while knockdown of PPARα expression was achieved by transient transfection of siRNA.Both mouse and human melanoma cells produced more PPARα and PPARγ protein compared to melanocytes. PPARα mRNA levels were elevated in human melanoma cells, but not in mouse melanoma cells relative to melanocytes. Silencing of PPARα in human melanoma cells did not alter cell proliferation or morphology. PPARγ-selective agonists inhibited the growth of both mouse and human melanoma cells, while PPARα-selective agonists had limited effects.Increased expression of PPARα in melanoma relative to melanocytes may be a common occurrence, however its biologic significance remains to be determined. PPARγ agonists may be useful for arresting the growth of some melanomas.
Keywords: growth inhibition; melanocytes; melanoma; PPAR expression

Formation of Stable Submicron Protein Particles by Thin Film Freezing by Joshua D. Engstrom; Edwina S. Lai; Baltej S. Ludher; Bo Chen; Thomas E. Milner; Robert O. Williams III; G. Barrie Kitto; Keith P. Johnston (1334-1346).
Highly stable, submicron lactate dehydrogenase (LDH) and lysozyme particles may be produced by thin film freezing (TFF) of aqueous solutions followed by lyophilization.The LDH activity was determined by measuring the decrease in absorbance of NADH over time for the reaction of pyruvate to lactate. For lysozyme the particle morphology was determined by scanning electron microscopy (SEM) and compared with the specific surface area (BET) and the particle size, as measured by laser light scattering,Protein particles with an average diameter of 300 nm and 100% enzyme activity upon reconstitution (for LDH) were formed by TFF. Droplets of protein solutions, 3.6 mm in diameter, spread upon impact with 223 and 133 K metal surfaces to form cylindrical disks with thicknesses of 200–300 μm. Calculated cooling rates of the disks of 102 K/s were confirmed experimentally with infrared measurements.The cooling rates of 102 K/s, intermediate to those in lyophilization (1 K/min) and spray freeze-drying (SFD) (106 K/s), were sufficiently fast to produce sub-micron protein particles with surface areas of 31–73 m2/g, an order of magnitude higher than in lyophilization. In addition, the low surface area/volume ratio (32–45 cm−1) of the gas–liquid interface led to minimal protein adsorption and denaturation relative to SFD.
Keywords: gas-liquid interface; intermediate cooling rate; lactate dehydrogenase; sub-micron protein particle; thin film freezing

In Vitro and In Vivo Drug Release from a Novel In Situ Forming Drug Delivery System by Heiko Kranz; Erol Yilmaz; Gayle A. Brazeau; Roland Bodmeier (1347-1354).
The objective of this work was to investigate the influence of various preparation and formulation parameters on the in vitro and in vivo release of bupivacaine hydrochloride from an injectable in situ forming microparticle system (ISM).The in vitro drug release of ISM was investigated as a function of various formulation and process parameters and was compared to the drug release from in situ forming implants and conventional microparticles. In vivo studies were carried out in male Sprague–Dawley rats.Upon contact with an aqueous medium, the internal polymer phase of the ISM system solidified and formed microparticles. The initial drug release from ISM systems was reduced with decreasing polymer phase/external oil phase ratio. An advantage of the ISM system compared to in situ implant systems was the significantly reduced burst effect, resulting in drug release profiles comparable to microparticles prepared by conventional methods. The in vivo drug release studies were in good agreement with the in vitro drug release. With the ISM system, the analgesic effect of the bupivacaine hydrochloride was prolonged when compared to the injection of a drug solution or drug-polymer solution.ISM are an attractive alternative for parenteral drug delivery systems.
Keywords: biodegradable polymers; in situ ; local anesthetic; microparticles; sustained-release depot

Eupatilin Protects Gastric Epithelial Cells from Oxidative Damage and Down-Regulates Genes Responsible for the Cellular Oxidative Stress by Eun-Ju Choi; Hyun-Mee Oh; Bo-Ra Na; T. P. Ramesh; Hyun-Ju Lee; Chang-Soo Choi; Suck-Chei Choi; Tae-Young Oh; Suck-Jun Choi; Jeong-Ryong Chae; Sang-Wook Kim; Chang-Duk Jun (1355-1364).
The formulated ethanol extract (DA-9601) of Artemisia asiatica has pronounced anti-inflammatory activities and exhibits cytoprotective effects against gastrointestinal damage. Here we investigated whether eupatilin, a major component of DA-9601, has a property of antioxidant activity and protects gastric epithelial cells from H2O2-induced damage.The protective effect of eupatilin against H2O2-induced damages was studied in gastric epithelial AGS cells by measuring wound healing, cell proliferation, and cell viability. Global gene expression profiling was obtained by high-density microarray.Hydrogen peroxide significantly delayed epithelial migration in wounded area. In contrast, eupatilin prevented the reduction of epithelial migration induced by H2O2. Eupatilin also ameliorated H2O2-induced actin disruption in AGS cells. Interestingly, treatment with eupatilin dramatically inhibited FeSO4-induced ROS production in a dose-dependent manner. In addition, eupatilin protected cells from FeSO4-induced F-actin disruption. With high-density microarray, we identified dozens of genes whose expressions were up-regulated in H2O2-treated cells. We found that eupatilin reduces the expression of such oxidative-responsible genes as HO-1, PLAUR and TNFRSF10A in H2O2-treated cells.These results suggest that eupatilin acts as a novel antioxidant and may play an important role in DA-9601-mediated effective repair of the gastric mucosa.
Keywords: anti-oxidant; eupatilin; microarray; reactive oxygen species; wound healing

Modulation of Tumor Necrosis Factor-mediated Cell Death by Fullerenes by Ljubica Harhaji; Aleksandra Isakovic; Ljubica Vucicevic; Kristina Janjetovic; Maja Misirkic; Zoran Markovic; Biljana Todorovic-Markovic; Nadezda Nikolic; Sanja Vranjes-Djuric; Zoran Nikolic; Vladimir Trajkovic (1365-1376).
The fullerene (C60/C70 mixture—C60/70) nanocrystalline suspension prepared by solvent exchange method using tetrahydrofyran (THF/nC60/70) and polyhydroxylated C60/70 [C60/70(OH) n ] were compared for their ability to modulate cytotoxicity of the proinflammatory cytokine tumor necrosis factor (TNF).TNF-induced cytotoxicity was assessed in L929 fibrosarcoma cells by crystal violet assay. The type of cell death (apoptosis/necrosis), production of reactive oxygen species, mitochondrial depolarization and caspase activation were determined by flow cytometry using the appropriate reporter dyes.THF/nC60/70 augmented, while C60/70(OH) n reduced the cytotoxicity of TNF. The numbers of cells undergoing apoptosis/necrosis, as well as of those displaying the activation of apoptosis-inducing enzymes of caspase family, were respectively increased or reduced by THF/nC60/70 or C60/70(OH) n . The antioxidant N-acetylcysteine and mitochondrial permeability transition inhibitor cyclosporin A each partly blocked the cytotoxic action of TNF, indicating the involvement of oxidative stress and mitochondrial dysfunction in the TNF cytotoxicity. Accordingly, THF/nC60/70 or C60/70(OH) n potentiated or suppressed, respectively, TNF-triggered oxidative stress and mitochondrial depolarization.The ability of different fullerene preparations to modulate TNF-induced oxidative stress and subsequent cell death suggests their potential value in the TNF-based cancer therapy or prevention of TNF-dependent tissue damage.
Keywords: apoptosis; fullerene; necrosis; oxidative stress; tumor necrosis factor

Myosin Light Chain Kinase Inhibition: Correction of Increased Intestinal Epithelial Permeability In Vitro by Linda M. Feighery; Sean W. Cochrane; Teresa Quinn; Alan W. Baird; Daniel O’Toole; Sian-Eleri Owens; Diarmuid O’Donoghue; Randall J. Mrsny; David J. Brayden (1377-1386).
To examine whether myosin light chain kinase (MLCK) inhibitors can reduce intestinal epithelial permeability increases in vitro.Isolated rat, mouse and human colonic tissue mucosae and Caco-2 monolayers were exposed to cytochalasin D (cD) and sodium caprate (C10), in the absence and presence of the MLCK inhibitors, ML-9 and D PIK. Transepithelial electrical resistance (TEER) and Papp of [14C]-mannitol or FITC-dextran 4000 (FD-4) were measured. Western blots were used to measure MLC phosphorylation.Increases in Papp of [14C]-mannitol and decreases in TEER were induced by tight junction openers. These changes were attenuated by ML-9. D-PIK offset the FD-4 Papp increase induced by C10 in Caco-2 only, while ML-9 and PIK inhibited MLC directly. cD induced constriction of peri-junctional actin in Caco-2 monolayers, but this was prevented by ML-9. Although mannitol fluxes across colonic mucosae from dextran-sulphate (DSS)-treated mice were higher than control, they were not ameliorated by either ML-9 or PIK in vitro.ML-9 inhibits paracellular permeability increases in several intestinal epithelial models. D-PIK reduced stimulated paracellular fluxes in Caco-2 monolayers, but not in tissue. Pre-established increases were not modified by two MLCK inhibitors in a mouse model of IBD.
Keywords: caco-2 monolayers; cytochalasins; dextran sodium sulphate; inflammatory bowel disease mouse models; mannitol fluxes; myosin light chain kinase; transepithelial electrical resistance

Long-term Stability and in vitro Release of hPTH(1–34) from a Multi-reservoir Array by Elizabeth R. Proos; James H. Prescott; Mark A. Staples (1387-1395).
Therapeutic peptides generally exhibit poor oral bioavailability and require alternative methods of delivery. Implanted microelectromechanical systems-based multi-reservoir devices enable programmable, chronic, pulsatile peptide delivery. This report describes parathyroid hormone fragment (hPTH(1–34)) formulations suitable for delivery from a multi-reservoir array.The stability of hPTH(1–34) lyophilizates obtained from aqueous acidic solutions was assessed by reverse phase high pressure liquid chromatography. An in vitro test device measured in vitro release kinetics.Novel, highly concentrated (>50 mg/mL) hPTH(1–34) solutions were dispensed as bulk samples (1–3 mg peptide) in vials and as individual doses (13–21 μg peptide) in reservoir arrays. Bulk and array samples were lyophilized and stored at 37°C. Bulk lyophilizate hPTH(1–34) purity after lyophilization, after 8 weeks, and after 26 weeks exceeded 96%, 90%, and 80%, respectively. The hPTH(1–34) stored in multi-reservoir arrays exhibited similar purity over 29 weeks at 37°C. Initially and over 29 weeks, over half of the peptide was consistently released from arrays into neutral, isotonic solution in less than 30 min with quantitative recoveries (>95%) within 3 h.Clinically relevant formulations of hPTH(1–34) for use with implantable multi-reservoir devices are achievable.
Keywords: drug delivery; microchip; osteoporosis; parathyroid hormone; protein and peptide formulation

Disaccharides such as trehalose are widely used as cryo-protectants to maintain the activity of proteinaceous drugs during freezing. One unresolved issue is the double transition that is observed very commonly in DSC experiments on disaccharide solutions in the frozen state; the assignment of these transitions remains disputed. Here we use calorimetry and two new techniques to shed light on the true nature of these transitions.Modulated Temperature DSC (MTDSC), cryo atomic force microscopy (AFM) and a novel DMA technique were used to study these transitions.MTDSC identified the two transitions Tr1 and Tr2 at −35.4 and −27.9°C respectively in the reversing heat flow signal, an exotherm and endotherm were observed in the non-reversing signal at circa −32 and −29°C respectively. It is shown for the first time that AFM images can be obtained of a softening and melting sample without damaging it. A force modulation imaging technique showed a softening at Tr1 and a loss of ice crystals at Tr2. These observations were supported by the DMA results.The results indicate Tr1 is associated with a glass transition while Tr2 is associated with the onset of loss of crystallinity.
Keywords: atomic force microscopy; freezing; microthermal analysis; modulated temperature differential scanning calorimetry; trehalose

Hydroxylation of the antidepressant and smoking deterrent drug bupropion is a clinically important bioactivation and elimination pathway. Bupropion hydroxylation is catalyzed selectively by cytochrome P4502B6 (CYP2B6). CYP2B6-catalyzed bupropion hydroxylation has been used as an in vitro and in vivo phenotypic probe for CYP2B6 activity and CYP2B6 drug interactions. Bupropion is chiral, used clinically as a racemate, and disposition is stereoselective. Nevertheless, it is unknown whether CYP2B6-catalyzed bupropion hydroxylation is stereoselective.Hydroxylation of racemic bupropion by recombinant CYP2B6 and human liver microsomes was evaluated using a stereoselective assay.At therapeutic concentrations, hydroxylation of (S)-bupropion was threefold and 1.5-greater than (R)-bupropion, respectively, by recombinant CYP2B6 and human liver microsomes. In vitro intrinsic clearances were likewise different for bupropion enantiomers.Stereoselective bupropion hydroxylation may have implications for the therapeutic efficacy of bupropion as an antidepressant or smoking cessation therapy, and for the use of bupropion as an in vivo phenotypic probe for CYP2B6 activity.
Keywords: bupropion; CYP2B6; cytochrome P4502B6; stereochemistry

Mechanistic Analysis of Chemical Permeation Enhancers for Oral Drug Delivery by Kathryn Whitehead; Samir Mitragotri (1412-1419).
Traditionally, the oral route cannot be employed for the delivery of macromolecular drugs such as proteins and peptides due, in large part, to limited transport across the epithelial membrane. This particular challenge can potentially be addressed through the use of chemical permeation enhancers, which affect transcellular and/or paracellular transport routes. Although certain permeation enhancers have been proposed for use in oral delivery, potential for application is often unclear when the route of enhancer action is unknown.A combination of theory and experiments was developed for determining mechanism of enhancer action. The effect of 51 enhancers on Caco-2 cells was studied using TEER, MTT, and LDH assays.The mechanistic details of intestinal permeability enhancement were uncovered for a broad set of enhancers in vitro. Understanding gained from enhancer mechanisms enabled the deduction of structure–function relationships for hydrophilic and hydrophobic permeation enhancers as well as the identification of a transcellular enhancer, 0.01% (w/v) palmityldimethyl ammonio propane sulfonate, which enabled the non-cytotoxic intracellular delivery of a model drug.The results presented here emphasize the importance of understanding enhancer mechanism and uncover a zwitterionic surfactant capable of safely and effectively achieving intraepithelial drug delivery in vitro.
Keywords: Caco-2; mechanism; oral drug delivery; permeation enhancer; transcellular

Stimulation of Phagocytic Activity of Alveolar Macrophages Toward Artificial Microspheres by Infection with Mycobacteria by Keiji Hirota; Keishiro Tomoda; Hiroyuki Inagawa; Chie Kohchi; Gen-Ichiro Soma; Kimiko Makino; Hiroshi Terada (1420-1430).
The purpose of this study is to know the effect of uptake of mycobacteria on the phagocytic activity of alveolar macrophage (Mφ) cells toward poly(lactic-co-glycolic) acid (PLGA) microspheres (MS) loaded with the anti-tuberculosis agent rifampicin (RFP-PLGA MS).Biological functions such as phagocytic activity toward PLGA MS loaded with fluorescent coumarin (cPLGA MS) and toward polystyrene latex MS (PSL MS), and generation of tumor necrosis factor-α (TNF-α) and nitric oxide (NO) were examined using alveolar Mφ cell NR8383 after they had phagocytosed Mycobacterium bovis Calmette-Guérin (BCG), heat-killed BCG (h-kBCG) or Escherichia coli.The ingestion of BCG, h-kBCG, and E. coli did not affect the viability of the Mφ cells within 2 days. The phagocytosis caused generation of TNF-α and NO, being more significant with E. coli than with both types of BCGs. The phagocytosis of both types of BCGs stimulated the phagocytic uptake of cPLGA and PSL MS’s, which took place prior to the generation of TNF-α or NO, but that of E. coli suppressed the uptake of both MS’s.Mycobacterial infection stimulated the phagocytic uptake toward cPLGA MS. These results suggest that RFP-PLGA MS is favorable for overcoming tuberculosis.
Keywords: alveolar macrophage; phagocytosis; polymer microspheres; pulmonary delivery; tuberculosis

A Physiological Model to Evaluate Drug Kinetics in Patients with Hemorrhagic Shock Followed by Fluid Resuscitation by Michel Tod; Franck Lagneau; Vincent Jullien; Olivier Mimoz (1431-1439).
To build a physiologically based pharmacokinetic model describing drug kinetics in interstitial fluid in case of hemorrhagic shock, and to propose a simple method to determine the subset of influential parameters that may be estimated with the data at hand.The model, which accounts for alterations of regional blood flows and body water distribution, was fitted to amoxicillin and clavulanate kinetic data, assessed in 12 trauma patients with hemorrhagic shock by comparison with 12 healthy volunteers. The predictions were the free concentrations of amoxicillin and clavulanate in 14 organs.In all tissues of trauma patients, the rate of distribution was lower, but the steady-state level was higher than those in healthy participants. Blood volume was reduced by 25% and blood flow in organs other than lung, brain, and heart were reduced by 18%. Compared with healthy subjects, the time that free amoxicillin concentration remained above 8 mg/L in the interstitial fluid of trauma patients was higher in blood and muscles, and lower in the tendon compartment.The results and predictions were consistent with the knowledge in this field. The model may be useful to optimize clinical trial designs and drug dosing regimens.
Keywords: amoxicillin; clavulanate; hemorrhagic shock; pharmacokinetics; physiological model

Effects of Moisture and Residual Solvent on the Phase Stability of Orthorhombic Paracetamol by Kyriakos Kachrimanis; Katharina Fucke; Michael Noisternig; Bernd Siebenhaar; Ulrich J. Griesser (1440-1449).
At high relative humidity (RH), orthorhombic paracetamol (form II) crystallized from ethanol transforms to monoclinic (form I) faster than such crystallized from the melt. The present study attempts to elucidate the reasons for this difference in stability.The transformation of form II was investigated by powder X-ray diffraction, optical microscopy, gravimetric moisture sorption, thermogravimetry, and vibrational spectroscopy.Solution-grown form II was found to be always contaminated with form I nuclei but still transforms much faster than corresponding physical mixtures of the pure forms in high RH, at a rate that is depending on the RH and the size of the crystals. A 0.1–0.6% w/w mass loss, inversely related to the initial monoclinic content, was observed during transformation of solution-grown form II, and was found to be due to residual ethanol that could not be removed by grinding, indicating incorporation by a solid solution mechanism.Moisture triggers the growth of existing form I nuclei but it exerts a weaker effect on nucleation, and the presence of residual ethanol greatly accelerates the transformation.
Keywords: crystal polymorphism; moisture-induced phase transition; orthorhombic paracetamol; residual solvent; solvent inclusion

Targeted Intestinal Delivery of Supersaturated Itraconazole for Improved Oral Absorption by Dave A. Miller; James C. DiNunzio; Wei Yang; James W. McGinity; Robert O. Williams III (1450-1459).
To investigate the use of Carbopol® 974P as a stabilizing agent for supersaturated levels of itraconazole (ITZ) in neutral pH aqueous media and the resultant effects on oral absorption of ITZ.Carbopol® 974P was incorporated into an EUDRAGIT® L 100-55 carrier matrix at concentrations of 20% and 40% based on polymer weight with the aim of prolonging supersaturated ITZ release from the enteric matrix. Amorphous solid dispersions of ITZ in EUDRAGIT® L 100-55 containing either 20% or 40% Carbopol® 974P were produced by hot-melt extrusion (HME). Solid state analysis of these compositions was performed using differential scanning calorimetry and qualitative energy dispersive X-ray spectroscopy. Dissolution analysis was conducted using a pH change method. Oral absorption of ITZ was evaluated in male Sprague–Dawley rats.Solid state analysis demonstrated that the extruded compositions were entirely amorphous and homogenous with respect to drug distribution in the polymer matrix. Dissolution analysis revealed that the addition of Carbopol® 974P to the EUDRAGIT® L 100-55 carrier system functioned to prolong the release of supersaturated levels of ITZ from the EUDRAGIT® L 100-55 matrix following an acidic-to-neutral pH transition. In vivo evaluation of ITZ absorption revealed that the addition of Carbopol® 974P substantially reduced the absorption variability seen with the EUDRAGIT® L 100-55 carrier system. In addition, the 20% Carbopol® 974P formulation exhibited a five-fold improvement in absorption over our initially reported ITZ particulate dispersion compositions that limited supersaturation of ITZ primarily to the stomach.The results of this study strongly suggest that substantial improvements in oral antifungal therapy with ITZ can be achieved via intestinal targeting and polymeric stabilization of supersaturation.
Keywords: hot-melt extrusion; intestinal targeting; itraconazole; oral absorption; supersaturation

Glucose- and Metabolically Regulated Hepatic Insulin Gene Therapy for Diabetes by Paul Yueh-Jen Hsu; Robert M. Kotin; Ya-Wun Yang (1460-1468).
The purpose of this study was to examine glucose- and metabolically modulation of insulin secretion by rAAV-mediated gene delivery in vitro and in vivo. A recombinant adeno-associated virus vector (rAAV) containing a furin-mutated human insulin gene, driven by the rat insulin I promoter, was used in this study. Glucose-responsive secretion of human insulin was determined by treating rAAV-transduced Huh7 human hepatoma cells with varying concentrations of glucose, with or without insulin secretagogues. Glucose- and metabolically modulated secretion of human insulin in the streptozotocin (STZ)-induced diabetic mice was assessed by intrahepatic administration of rAAV-polyethylenimine (PEI) complexes, followed by intraperitoneal glucose tolerance test (IPGTT), with or without theophylline.Glucose- and metabolically controlled human insulin secretion was obtained in the rAAV-transduced Huh7 cells. Treatment of STZ-induced diabetic animals with rAAV–polyethylenimine (rAAV-PEI) complexes resulted in production of human insulin and amelioration of hyperglycemia. Co-administration of glucose and theophylline in these animals augmented the secretion of human insulin, demonstrating metabolic modulation of insulin secretion in vivo. Immunohistochemical examination of the liver sections of rAAV-treated mice confirmed the production of human insulin.Glucose- and metabolically controlled hepatic insulin gene therapy was obtained both in vitro and in vivo.
Keywords: diabetes mellitus; insulin; polyethylenimine; recombinant adeno-associated virus; regulated gene therapy

Quantitative Atlas of Membrane Transporter Proteins: Development and Application of a Highly Sensitive Simultaneous LC/MS/MS Method Combined with Novel In-silico Peptide Selection Criteria by Junichi Kamiie; Sumio Ohtsuki; Ryo Iwase; Ken Ohmine; Yuki Katsukura; Kazunari Yanai; Yumi Sekine; Yasuo Uchida; Shingo Ito; Tetsuya Terasaki (1469-1483).
To develop an absolute quantification method for membrane proteins, and to construct a quantitative atlas of membrane transporter proteins in the blood–brain barrier, liver and kidney of mouse.Mouse tissues were digested with trypsin, and mixed with stable isotope labeled-peptide as a quantitative standard. The amounts of transporter proteins were simultaneously determined by liquid chromatography–tandem mass spectrometer (LC/MS/MS).The target proteins were digested in-silico, and target peptides for analysis were chosen on the basis of the selection criteria. All of the peptides selected exhibited a detection limit of 10 fmol and linearity over at least two orders of magnitude in the calibration curve for LC/MS/MS analysis. The method was applied to obtain the expression levels of 34 transporters in liver, kidney and blood–brain barrier of mouse. The quantitative values of transporter proteins showed an excellent correlation with the values obtained with existing methods using antibodies or binding molecules.A sensitive and simultaneous quantification method was developed for membrane proteins. By using this method, we constructed a quantitative atlas of membrane transporter proteins at the blood–brain barrier, liver and kidney in mouse. This technology is expected to have major implications for various fields of biomedical science.
Keywords: ABC transporter; LC/MS/MS; multiple reaction monitoring (MRM); pharmacoproteomics; SLC transporter