Pharmaceutical Research (v.25, #2)

Matrix Metalloproteinase Gene Delivery for Liver Fibrosis by Yuji Iimuro; David A. Brenner (249-258).
The resolution of advanced liver fibrosis has been recently recognized to be possible, if the causative stimuli are successfully removed. However, whether complete resolution from cirrhosis, the end stage of liver fibrosis, can be achieved is still questionable. Delivery of interstitial collagenases, such as matrix metalloproteinase (MMP)-1, in the liver could be an attractive strategy to treat advanced hepatic fibrosis from the view point that the imbalance between too few interstitial collagenases and too many of their inhibitors is the main obstacle to the resolution from fibrosis. Remodeling of hepatic extracellular matrix by delivered interstitial collagenases also facilitates the disappearance of activated hepatic stellate cells, the main matrix-producing cells in the liver, and promotes the proliferation of hepatocytes. This review will focus on the impact of the gene delivery of MMPs for the treatment of advanced liver fibrosis while discussing other current therapeutic strategies for liver fibrosis, and on the need for the development of a safe and effective delivery system of MMPs.
Keywords: gene delivery; hepatic stellate cells; liver fibrosis; MMP; TIMP

Excipients are added to lyophilized protein drug formulations to protect the protein during processing and storage, but the mechanisms are poorly understood. Here, hydrogen/deuterium (H/D) exchange with mass spectrometry was used to assess protein conformation and excipient interactions in lyophilized solids.Calmodulin (CaM, 17 kD) was co-lyophilized with carbohydrate excipients (sucrose, mannitol, trehalose, raffinose, dextran 5,000, dextran 12,000) or guanidine hydrochloride (negative control) and exposed to D2O vapor at 33% RH and RT. Samples were then dissolved and analyzed by mass spectrometry (+ESI/MS). Peptic digestion provided additional, site-specific information on H/D exchange. Solids were further characterized by powder x-ray diffraction (PXRD), differential scanning calorimetry (DSC), infrared spectroscopy (FTIR) and water vapor sorption.Excipients protected CaM from H/D exchange, increasing in the order guanidine hydrochloride < no excipient, mannitol < dextran 5,000, dextran 12,000 < sucrose < raffinose < trehalose. Effects were exerted primarily in the protein’s α-helical segments.The effects of carbohydrate excipients on protein conformation in lyophilized solids are not exhibited uniformly along the protein sequence, but instead are exerted in a site-specific manner. The results also demonstrate the utility of H/D exchange with ESI/MS for protein structure characterization in lyophilized samples.
Keywords: ESI mass spectrometry; excipient; FTIR; hydrogen/deuterium exchange; lyophilization; protein

Combination Therapy of Heparin–Deoxycholic Acid Conjugate and Doxorubicin against Squamous Cell Carcinoma and B16F10 Melanoma by Kyeongsoon Park; Gee Young Lee; Rang-Woon Park; In-San Kim; Sang Yoon Kim; Youngro Byun (268-276).
Our previous study confirmed that heparin–deoxycholic acid conjugate (HD) had a potent antiangiogenic effect and safety to use for long-term treatment. Herein, the combined therapeutic effect of HD and doxorubicin (DOX) was evaluated against squamous cell carcinoma (SCC7) and B16F10 melanoma.The inhibitory effect of cell proliferation and cellular uptake of HD was studied in SCC7 and B16F10. The combination effects of HD and DOX were evaluated by measuring cytotoxicity and apoptosis as well as tumor growth and apoptosis in vivo against SCC7 and B16F10 tumor-bearing mice.HD displayed potent inhibitory effect on SCC7 and B16F10 cell proliferation, but it showed a low cytotoxic effect. Concurrent treatment of HD and DOX displayed enhanced cytotoxic effects and apoptosis on SCC7 and B16F10. The cellular uptake of HD and DOX was affected by the collective cytotoxic effects of these two drugs: each drug suppressed the tumor growth, and their combined treatment enhanced apoptosis and collectively inhibited the tumor growth of SCC7 and B16F10 in vivo.These results demonstrated that HD with cytostatic and antiangiogenetic activities, enhanced the antitumor activity of DOX against SCC7 and B16F10, and the combined treatment of these two drugs might have enhanced therapeutic efficacy.
Keywords: apoptosis; B16F10; doxorubicin; heparin–deoxycholic acid conjugate; SCC

At present, there is no published data examining the effect of relative humidity on the electrostatic charges of dry powder inhaler aerosols. The charging behaviour of two commercial products, Pulmicort® and Bricanyl® Turbuhalers®, were investigated using an electrical low pressure impactor (ELPI).ELPI was successfully modified to disperse the aerosols at 60 l/min. Four doses from each new inhaler were sampled at 15, 40, 65, and 90% RH. Particles deposited on the impactor stages according to their aerodynamic diameters and their charges were measured simultaneously by the electrometers. The drug in each size fraction was quantified using HPLC.Both products generated bipolar charges. The charging behaviour of the two types of inhaler showed different humidity dependence although the mass output was not significantly affected. The absolute specific charge of budesonide fine particles from Pulmicort® was the lowest at 40% RH but increased at lower and higher RHs. In contrast, the terbutaline sulfate fine particles from Bricanyl® followed the expected trend of charge reduction with increasing RH.The distinct trends of charging of aerosols from Pulmicort® and Bricanyl® Turbuhalers® was explained by differences in hygroscopicity and other physicochemical factors between the two drugs.
Keywords: aerosol; dry powder inhaler; electrostatic charge; ELPI; relative humidity

Core–shell Particles for the Dispersion of Small Polar Drugs and Biomolecules in Hydrofluoroalkane Propellants by Libo Wu; Balaji Bharatwaj; Jayanth Panyam; Sandro R. P. da Rocha (289-301).
Demonstrate the applicability of a novel particle-based technology for the development of suspensions of small polar drugs and biomolecules in hydrofluoroalkane (HFA) propellants for pressurized metered-dose inhalers (pMDIs).Emulsification diffusion was used to prepare core–shell particles. The shell consisted of oligo(lactide) grafts attached onto a short chitosan backbone. The active drug was arrested within the particle core. Colloidal Probe Microscopy (CPM) was used to determine the cohesive forces between particles in a model HFA propellant. The aerosol characteristics of the formulations were determined using an Anderson Cascade Impactor (ACI). Cytotoxicity studies were performed on lung epithelial and alveolar type II cells.CPM results indicate that particle cohesive forces in liquid HFA are significantly screened in the presence of the polymeric shell and correlate well with the physical stability of suspensions in propellant HFA. The proposed formulation showed little or no cytotoxic effects on both Calu-3 and A549 cells.Core–shell particles with a shell containing the lactide moiety as the HFA-phile showed excellent dispersion stability and aerosol characteristics in HFA-based pMDIs. This is a general strategy that can be used for developing novel suspension pMDIs of both small polar drugs and large therapeutic molecules.
Keywords: biomolecules; colloidal probe microscopy; pressurized metered-dose inhaler; pulmonary drug delivery; salbutamol sulfate

To determine the effects of vial packing density in a laboratory freeze dryer on drying rate profiles of crystalline and amorphous formulations.The Christ freeze-drying balance measured cumulative water loss, m(t), and instantaneous drying rate, $$mathop mlimits^ cdot left( t ight)$$ , of water, mannitol, sucrose and sucrose/BSA formulations in commercial vials.Crystalline mannitol shows drying rate behaviour indicative of a largely homogeneous dried-product layer. The drying rate behaviour of amorphous sucrose indicates structural heterogeneity, postulated to come from shrinkage or microcollapse. Trehalose dries more slowly than sucrose. Addition of BSA to either disaccharide decreases primary drying time. Higher vial packing density greatly reduces drying rate because of effects of radiation heat transfer from chamber walls to test vial.Plots of m(t) versust and $$mathop mlimits^ cdot left( t ight)$$ versus layer thickness (either ice or dried-product) allow interpretation of changes in internal cake morphology during drying. Vial packing density greatly influences these profiles.
Keywords: drying rate; freeze-drying; product morphology; product resistance

Novel Lipid and Preservative-free Propofol Formulation: Properties and Pharmacodynamics by François Ravenelle; Sandra Gori; Dorothée Le Garrec; David Lessard; Laibin Luo; Dana Palusova; J. Robert Sneyd; Damon Smith (313-319).
Propofol is a water-insoluble intravenous anesthetic agent that is actually formulated as a water-in-oil emulsion with known drawbacks such as pain on injection, microorganism growth support and stability. We report on the properties of formulations of propofol in poly (N-vinyl-2-pyrrolidone)-block-poly(d,l-lactide), PVP–PLA, polymeric micelles (Propofol-PM).Microbial growth in these formulations was evaluated with Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 25922) and Candida albicans (ATCC 10231). Sleep-recovery studies in female Sprague–Dawley rats, at a dose of 10mg/kg were performed to compare pharmacodynamic profiles of the new Propofol-PM formulations with those of Diprivan®, a commercially available lipid based propofol formulation.Growth of microorganisms was not supported in the Propofol-PM formulations tested. No significant differences in times to unconsciousness, awakening, recovery of righting reflex and full recovery were observed between Propofol-PM formulations and Diprivan®.Propofol loaded in PVP–PLA micelles (Propofol-PM) is not significantly different in terms of pharmacodynamic but demonstrates no microorganism growth support and improved stability that opens up the door to pain on injection reduction strategy.
Keywords: anesthesia; micelle; microbial growth; propofol; PVP–PLA

The United States Pharmacopoeia (USP) imposes strict requirements on the geometry and operating conditions of the USP Dissolution Testing Apparatus II. A previously validated Computational Fluid Dynamics (CFD) approach was used here to study the hydrodynamics of USP Apparatus II when the impeller was placed at four different locations, all within the limits specified by USP.CFD was used to predict the velocity profiles, energy dissipation rates, and strain rates when the impeller was placed in the reference location (centrally mounted, 25 mm off the vessel bottom), 2 mm off-center, 2 mm higher, and 2 mm lower than the reference location.Small changes in impeller location, especially if associated with loss of symmetry, produced extensive changes in velocity profiles and shear rates. Centrally located impellers, irrespective of their off-bottom clearance, produced non-uniform but nearly symmetric strain rates. The off-center impeller produced a more uniform but slightly asymmetric strain rate distribution.The system hydrodynamics depends strongly on small differences in equipment configurations and operating conditions, which are likely to affect significantly the flow field and shear rate experienced by the oral dosage form being tested, and hence the solid–liquid mass transfer and dissolution rate.
Keywords: CFD; computational fluid dynamics; hydrodynamics; off-center impeller; USP dissolution testing apparatus

An Investigation into the Dispersion Mechanisms of Ternary Dry Powder Inhaler Formulations by the Quantification of Interparticulate Forces by Matthew D. Jones; Jennifer C. Hooton; Michelle L. Dawson; Alan R. Ferrie; Robert Price (337-348).
To investigate the dispersion mechanism(s) of ternary dry powder inhaler (DPI) formulations by comparison of the interparticulate adhesions and in vitro performance of a number of carrier–drug–fines combinations.The relative levels of adhesion and cohesion between a lactose carrier and a number of drugs and fine excipients were quantified using the cohesion–adhesion balance (CAB) approach to atomic force microscopy. The in vitro performance of formulations produced using these materials was quantified and the particle size distribution of the aerosol clouds produced from these formulations determined by laser diffraction.Comparison between CAB ratios and formulation performance suggested that the improvement in performance brought about by the addition of fines to which the drug was more adhesive than cohesive might have been due to the formation of agglomerates of drug and fines particles. This was supported by aerosol cloud particle size data. The mechanism(s) underlying the improved performance of ternary formulations where the drug was more cohesive than adhesive to the fines was unclear.The performance of ternary DPI formulations might be increased by the preferential formation of drug–fines agglomerates, which might be subject to greater deagglomeration forces during aerosolisation than smaller agglomerates, thus producing better formulation performance.
Keywords: adhesion; agglomeration; atomic force microscope; fines; ternary interactive mixture

To study how polymeric additives interact with the crystal surface of acetaminophen, and why they have different effects on drug crystallization and dissolution.The effects of different polymers on the etching patterns, crystallization and intrinsic dissolution rate (IDR) of acetaminophen have been studied.Some polymers have shown clear consistency in their effects on the etching patterns, crystallization and IDR of acetaminophen. For example, hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidone (PVP), not only changed the etching patterns of the acetaminophen (010) face, but also inhibited acetaminophen crystallization significantly. Some polymers, like 2-hydroxyethyl cellulose (HEC), poly(vinyl alcohol) (PVA) and poly(ethylene glycol) (PEG) only had limited effects on the IDR and etching patterns, and no significant inhibitory effects on crystallization.Even though some polymeric additives have no structural similarity to acetaminophen, they still can affect dissolution and crystallization of acetaminophen due to the synergic effects of their neighboring subunits during surface adsorption. The effects of polymeric additives on crystallization and dissolution of acetaminophen are affected not only by the specific interactions between adsorbed polymer molecules and crystal surface, but also by the mobility of the functional groups involved in the specific interactions.
Keywords: crystallization; dissolution; etching pattern; mobility of functional groups; surface adsorption

Population Pharmacokinetic Data Analysis of Cilobradine, an If Channel Blocker by Gabriele Fliss; Alexander Staab; Christiane Tillmann; Dirk Trommeshauser; Hans G. Schaefer; Charlotte Kloft (359-368).
To evaluate the population pharmacokinetic characteristics of cilobradine including a covariate analysis based on six phase I trials and to assess the predictive performance of the model developed.Single or multiple doses of cilobradine were administered as solution, capsule or infusion. Two thousand, seven hundred and thirty-three plasma samples (development data set) were used for model development in NONMEM. Model evaluation was performed using also an external data set.Data were best described by a linear three-compartment model. Typical Vss was large (∼100 l) and CL was 21.5 l/h. Covariate analysis revealed a statistically significant but clinically irrelevant relation between KA and dose. Inter-individual variability was moderate (15–46%); imprecision of estimates was generally low. The final model was successfully applied to the external data set revealing its robustness and general applicability. Its final estimates resembled those of the development data set except for the covariate relation not being supported. When excluding the covariate relation, all observations were well predicted.A robust population PK model has been developed for cilobradine predicting plasma concentrations from a different study design well. Therefore, the model can serve as a tool to simulate and evaluate different dosing regimens for further clinical trials.
Keywords: cilobradine; If channel blocker; NONMEM; population pharmacokinetics

Pharmacodynamics of Recombinant Human Erythropoietin in Murine Bone Marrow by Peter J. Bugelski; Thomas Nesspor; Amy Volk; Joanne O’Brien; Dorie Makropoulos; Kim Shamberger; Paul W. Fisher; Ian James; Danielle Graden; Renold J. Capocasale (369-378).
Originally approved for three times/week dosing, recombinant human erythropoietin (rhEPO) is now often used at weekly intervals. We have studied rhEPO in mice to better understand why the extended dosing interval retains efficacy.C57Bl/6 mice received a single sc. dose of rhEPO (3,000 IU/kg). Bone marrow and blood were collected at 8 h and 1, 2, 5 and 7 days. Staining for TER-119 and CD71, pulse labeling with bromodeoxyuridine, annexin-V binding and vital staining with 7-aminoactinomycin d were used cell cycle and apoptosis in erythroblasts by four color flow cytometry.A wave of proliferation and/or maturation progressed through all erythroblasts, resulting in the emigration of immature reticulocytes into the periphery. An increase in the fraction of erythroblasts in S and G2M was found, but suppression of apoptosis was not.Most of the effects of rhEPO occurred 48 h after dosing, when the concentration of rhEPO was less than 1% of Cmax, suggesting that the processes set in motion by rhEPO can continue after rhEPO concentrations fall. Our observation of apoptosis in erythroblasts even when rhEPO concentrations were high suggests that regulatory mechanisms which down-regulate erythropoiesis are also engaged.
Keywords: cytometry; erythropoiesis; erythropoietin; hematology; pharmacodynamics

Locoregional recurrence is the most common complication after adenocarcinoma resection in the colon, despite adjuvant chemotherapy. Therapy efficacy could be improved if designed to target malignant cells by incorporating specific recognition factors in the drugs or the drug vehicles. The aim of this study was to elucidate whether the overexpression of sialic acid (SA) on colonic malignant tissues could be utilized for drug targeting by cationic polymers.Cell lines (IEC-6, SW-480 and SW-620) and colon polyps and normal adjacent tissues harvested from dimethylhydrazine (DMH) induced rats were used as in vitro and in vivo models of different metastatic stages of colon cancer. SA expression was identified by fluorescent wheat germ agglutinin (WGA), and verified by pretreatment with neuraminidase. The role of mucus in the mucosal binding experiments was explored by pretreatment with dithiothreitol (DTT). The binding of FITC labeled cationic polymers of various degrees of cationization to normal and malignant colonic cells and tissue was measured.SA was overexpressed on malignant colonic cells and tissues, and its expression correlated to the metastatic stage in vitro. The binding of the cationic copolymers to the cell lines and tissues correlated with the charge density of the polymer and with the metastatic stage of the cell line. The interaction between the malignant colonic cells and tissues with the polymers was SA dependent and increased after mucus removal.Cationic polymers could be used as a targeting tool to colonic malignant epithelium, to be implemented in drug delivery and diagnosis.
Keywords: cationic polymers; colon cancer; drug targeting; rat model; sialic acid

Synergistic Effects of a Combination of Dietary Factors Sulforaphane and (−) Epigallocatechin-3-gallate in HT-29 AP-1 Human Colon Carcinoma Cells by Sujit Nair; Vidya Hebbar; Guoxiang Shen; Avantika Gopalakrishnan; Tin Oo Khor; Siwang Yu; Changjiang Xu; Ah-Ng Kong (387-399).
The objective of this study was to investigate combinations of two chemopreventive dietary factors: EGCG 20 μM (or 100 μM) and SFN (25 μM) in HT-29 AP-1 human colon carcinoma cells.After exposure of HT-29 AP-1 cells to SFN and EGCG, individually or in combination, we performed AP-1 luciferase reporter assays, cell viability assays, isobologram analyses, senescence staining, quantitative real-time PCR (qRT-PCR) assays, Western blotting, and assays for HDAC activity and hydrogen peroxide. In some experiments, we exposed cells to superoxide dismutase (SOD) or Trichostatin A (TSA) in addition to the treatment with dietary factors.The combinations of SFN and EGCG dramatically enhanced transcriptional activation of AP-1 reporter in HT-29 cells (46-fold with 25 μM SFN and 20 μM EGCG; and 175-fold with 25 μM SFN and 100 μM EGCG). Isobologram analysis showed synergistic activation for the combinations with combination index, CI < 1. Interestingly, co-treatment with 20units/ml of SOD, a free radical scavenger, attenuated the synergism elicited by the combinations (2-fold with 25 μM SFN and 20 μM EGCG; and 15-fold with 25 μM SFN and 100 μM EGCG). Cell viability assays showed that the low-dose combination decreased cell viability to 70% whereas the high-dose combination decreased cell viability to 40% at 48 h, with no significant change in cell viability at 24 h as compared to control cells. In addition, 20 μM and 100 μM EGCG, but not 25 μM SFN, showed induction of senescence in the HT-29 AP-1 cells subjected to senescence staining. However, both low- and high-dose combinations of SFN and EGCG attenuated the cellular senescence induced by EGCG alone. There was no significant change in the protein levels of phosphorylated forms of ERK, JNK, p38, and Akt-Ser473 or Akt-Thr308. Besides, qRT-PCR assays corroborated the induction of the luciferase gene seen with the combinations in the reporter assay. Relative expression levels of transcripts of many other genes known to be either under the control of the AP-1 promoter or involved in cell cycle regulation or cellular influx–efflux such as cyclin D1, cMyc, ATF-2, Elk-1, SRF, CREB5, SLCO1B3, MRP1, MRP2 and MRP3 were also quantified by qRT-PCR in the presence and absence of SOD at both 6 and 10 h. In addition, pre-treatment with 100 ng/ml TSA, a potent HDAC inhibitor, potentiated (88-fold) the synergism seen with the low-dose combination on the AP-1 reporter transcriptional activation. Cytoplasmic and nuclear fractions of treated cells were tested for HDAC activity at 2 and 12 h both in the presence and absence of TSA, however, there was no significant change in their HDAC activity. In addition, the H2O2 produced in the cell system was about 2 μM for the low-dose combination which was scavenged to about 1 μM in the presence of SOD.Taken together, the synergistic activation of AP-1 by the combination of SFN and EGCG that was potentiated by HDAC inhibitor TSA and attenuated by free radical scavenger SOD point to a possible multifactorial control of colon carcinoma that may involve a role for HDACs, inhibition of cellular senescence, and SOD signaling.
Keywords: AP-1; colon cancer; dietary factors; EGCG; isothiocyanate; sulforaphane

Lysosomal Enzyme Replacement of the Brain with Intravenous Non-Viral Gene Transfer by Yun Zhang; Yuntao Wang; Ruben J. Boado; William M. Pardridge (400-406).
The delivery of non-viral plasmid DNA to brain across the blood-brain barrier (BBB) with intravenous administration of non-viral plasmid DNA encoding a lysosomal enzyme, β-glucuronidase (GUSB), was examined in GUSB null mice, a model of type VII mucopolysaccharidosis.The plasmid, designated pCMV-GUSB, is encapsulated in Trojan horse liposomes, which are targeted across the BBB, and the brain cell membrane, with a monoclonal antibody to the mouse transferrin receptor.The GUSB enzyme activity was increased >50-fold in cell culture of fibroblasts obtained from GUSB null mice, following application of the antibody-targeted liposomes carrying the pCMV–GUSB, and enzyme activity remained high for >2 weeks. Adult GUSB null mice were treated with a single intravenous administration of 0.2 ml of Trojan horse liposomes carrying the pCMV–GUSB at a dose of 10 μg/mouse of plasmid DNA. The GUSB enzyme activity was increased greater than tenfold in brain, liver, spleen, lung, and kidney, but not in heart.Intravenous Trojan horse liposome administration increased brain GUSB enzyme activity to the therapeutic range of brain GUSB enzyme activity. These studies show it is possible to deliver non-viral plasmid DNA encoding lysosomal enzymes to the brain following intravenous administration of receptor-specific Trojan horse liposomes.
Keywords: blood-brain barrier; liposomes; lysosomal storage disorders; transferrin receptor

Gene Delivery to the Epidermal Cells of Human Skin Explants Using Microfabricated Microneedles and Hydrogel Formulations by Marc Pearton; Chris Allender; Keith Brain; Alexander Anstey; Chris Gateley; Nicolle Wilke; Anthony Morrissey; James Birchall (407-416).
Microneedles disrupt the stratum corneum barrier layer of skin creating transient pathways for the enhanced permeation of therapeutics into viable skin regions without stimulating pain receptors or causing vascular damage. The cutaneous delivery of nucleic acids has a number of therapeutic applications; most notably genetic vaccination. Unfortunately non-viral gene expression in skin is generally inefficient and transient. This study investigated the potential for improved delivery of plasmid DNA (pDNA) in skin by combining the microneedle delivery system with sustained release pDNA hydrogel formulations.Microneedles were fabricated by wet etching silicon in potassium hydroxide. Hydrogels based on Carbopol polymers and thermosensitive PLGA-PEG-PLGA triblock copolymers were prepared. Freshly excised human skin was used to characterise microneedle penetration (microscopy and skin water loss), gel residence in microchannels, pDNA diffusion and reporter gene (β-galactosidase) expression.Following microneedle treatment, channels of approximately 150–200 μm depth increased trans-epidermal water loss in skin. pDNA hydrogels were shown to harbour and gradually release pDNA. Following microneedle-assisted delivery of pDNA hydrogels to human skin expression of the pCMVβ reporter gene was demonstrated in the viable epidermis proximal to microchannels.pDNA hydrogels can be successfully targeted to the viable epidermis to potentially provide sustained gene expression therein.
Keywords: DNA; human skin; hydrogel; microneedles; thermosensitive

Decreased Biosynthesis of Lung Surfactant Constituent Phosphatidylcholine Due to Inhibition of Choline Transporter by Gefitinib in Lung Alveolar Cells by Naoki Ishiguro; Masanobu Oyabu; Toshihiro Sato; Tomoji Maeda; Hironobu Minami; Ikumi Tamai (417-427).
We investigated whether gefitinib, an anticancer agent, inhibits phosphatidylcholine (PC) biosynthesis and choline uptake by alveolar epithelial type II cells.Uptake of choline and PC biosynthesis were examined in vitro, using human alveolar epithelia-derived cell line A549 and rat alveolar type (AT) II cells as models.Gefitinib reduced the incorporation of [3H]choline into PC in A549 and rat ATII cells. The uptake of [3H]choline by A549 and rat ATII cells was concentration-dependent, and the Km values were 15.0 and 10–100 μM, respectively. The uptake of [3H]choline by A549 and rat ATII cells was weakly Na+-dependent, and inhibited by hemicholinium-3. RT-PCR revealed expression of choline transporter-like protein (CTL)1 and organic cation transporter (OCT)3 mRNAs in both cells. The choline uptake by A549 and rat ATII cells was strongly inhibited by gefitinib with the IC50 value of 6.77 μM and 10.5 μM, respectively.Our results demonstrate that gefitinib reduces PC biosynthesis via inhibition of cellular choline uptake by A549 and rat ATII cells, which is mainly mediated by CTL1, resulting in abnormality of lung surfactant that can be one of mechanisms of the interstitial lung disease associated with gefitinib.
Keywords: alveolar type II; choline; gefitinib; lung toxicity; phosphatidylcholine

Anti-tumor Effect of All-Trans Retinoic Acid Loaded Polymeric Micelles in Solid Tumor Bearing Mice by Narin Chansri; Shigeru Kawakami; Masayuki Yokoyama; Tatsuhiro Yamamoto; Pensri Charoensit; Mitsuru Hashida (428-434).
All-trans retinoic acid (ATRA) polymeric micelles were developed for parenteral administration. The distribution characteristics and antitumor activities of ATRA polymeric micelles were evaluated after intravenous administration to mice bearing CT26 solid tumors.ATRA incorporated in poly(ethylene glycol)-poly(benzyl aspartate) block copolymer was prepared by the evaporation method. The levels of [3H]ATRA in blood and tissue including tumor were determined by measuring the radioactivity after injection into mice. The tumor volume and the survival of the mice were determined to assess the anticancer activity.The delivery of ATRA by polymeric micelles prolonged the blood circulation and enhanced the accumulation of ATRA in the tumor tissue compared with the administration of free ATRA. Tumor growth was significantly delayed and the survival time of mice was prolonged following the treatment by ATRA polymeric micelles demonstrating the improved anticancer activity of ATRA.Polymeric micelles are a promising and effective carrier of ATRA in order to enhance tumor delivery and they have a promising potential application in the treatment of solid tumors.
Keywords: all-trans retinoic acid; antitumor activity; biodistribution; drug targeting; nanomedicine; polymeric micelles

Novel Beads Made of Alpha-cyclodextrin and Oil for Topical Delivery of a Lipophilic Drug by Laury Trichard; M. Begoña Delgado-Charro; Richard H. Guy; Elias Fattal; Amélie Bochot (435-440).
To investigate the potential of a novel lipid carrier, comprising beads of alpha-cyclodextrin and soybean oil, for topical drug delivery. Adapalene was chosen as a model drug to explore the ability of the beads to encapsulate and release a highly lipophilic compound.Adapalene-loaded beads were prepared and characterised. Skin tolerance to unloaded beads was tested on human volunteers, while drug release and delivery into stratum corneum, was evaluated in pig skin ex vivo.The preparation and physical characteristics of the beads were not dependent on whether adapalene had been previously dissolved or dispersed in soybean oil. Drug encapsulation efficiency was high (>96%) and drug loading on the order of a therapeutic level could be achieved in freeze-dried beads prepared from an oily dispersion of adapalene. After application to human skin, unloaded beads induced no adverse reaction and were better tolerated than an alcoholic gel. Tape-stripping the stratum corneum from treated pig skin showed that adapalene release and penetration from the beads was comparable to that from gel and cream formulations available on the market.These novel beads may offer a well-tolerated and efficient system for the encapsulation and topical delivery of lipophilic drugs.
Keywords: adapalene; bead; cyclodextrin; lipid carrier; topical formulation

Membrane Transporters in Drug Disposition by Guofeng You (441-443).
received her undergraduate education in Peking Medical University, P.R. China and her Ph.D. in Biochemistry from the Department of Chemistry, Clark University, Worcester, MA, USA. She then worked as a postdoctoral fellow in the Department of Medicine, Harvard Medical School, where she pursued her research in the cloning and characterization of several novel transporter proteins. In 1997, she accepted a position as Assistant Professor in the Department of Medicine, the Mount Sinai School of Medicine, to further her research in the field of drug transport. She is now an Associate Professor in the Department of Pharmaceutics, the Ernest Mario School of Pharmacy, Rutgers-the State University of New Jersey. She has authored many peer-reviewed papers in drug transport and is currently on the editorial Board of Pharmaceutical Research. She has recently co-edited with Professor Marilyn Morris a book entitled “Drug Transporters” published by Wiley & Sons, Inc. The book is the first to cover the basic transport mechanisms, clinical implications of transporters in human physiology and disease, and their role in drug therapy.

Hormonal Regulation of BCRP Expression in Human Placental BeWo Cells by Honggang Wang; Jashvant D. Unadkat; Qingcheng Mao (444-452).
We investigated whether the pregnancy-related hormones, estriol (E3), testosterone, human placental lactogen (hPL), human prolactin (hPRL), and human chorionic gonadotropin (hCG) affect BCRP expression in human placental BeWo cells.The effects of these hormones on BCRP protein and mRNA expression in BeWo cells were determined by immunoblotting and quantitative real-time RT-PCR, respectively. The effects of these hormones on membrane localization of BCRP in BeWo cells were examined by immunofluorescent confocal microscopy.E3, hPL, and hPRL significantly increased BCRP protein and mRNA approximately two to threefold at physiological concentrations. Induction of BCRP by E3 was abrogated by the estrogen receptor (ER) antagonist ICI-182,780. However, knock-down of ERα by RNA interference did not abolish the inductive effect of E3. Testosterone by itself did not affect BCRP expression at physiological concentrations. However, testosterone together with 17β-estradiol (E2) increased BCRP protein and mRNA approximately twofold, and this induction was abolished by ICI-182,780 or the testosterone receptor (TR) antagonist flutamide or knock-down of ERα expression. Further analysis revealed that E2 increased TR mRNA approximately 5.9-fold, suggesting that testosterone in combination with E2 increases BCRP expression, possibly through E2-mediated up-regulation of TR. hCG at physiological concentrations had no effect on BCRP expression.E3, hPL, hPRL, and testosterone in combination with E2 may up-regulate BCRP expression in the placenta during pregnancy.
Keywords: BCRP; BeWo cells; hormones; pregnancy; regulation

To elucidate the role of the renal basolateral transporter, Oat3, in the disposition of methotrexate.Chinese hamster ovary cells expressing mouse Oat3 were used to determine kinetics and specificity of inhibition of methotrexate transport. Methotrexate clearance was then examined in vivo in wildtype and Oat3 knockout mice.NSAIDs, ß-lactams, and uremic toxins inhibited mOat3-mediated methotrexate uptake by 70–100%, while folate, leucovorin, and 5-methyltetrahydrofolate inhibited transport by 25–50%. A K m of 60.6 ± 9.3 μM for methotrexate transport was determined. Oat3 knockout mice exhibited reduced methotrexate-to-inulin clearance ratios versus wildtype. Male wildtype mice, but not knockouts or females, demonstrated significantly accelerated methotrexate clearance in response to reduced folates. Reduced folates also markedly inhibited hepatic methotrexate accumulation in males, but not females, and the response was independent of Oat3 function.Oat3 contributes to methotrexate clearance, but represents only one component responsible for methotrexate’s elimination. Therefore, in patients, dysfunctional hOAT3 polymorphisms or drug competition for hOAT3 transport may severely impact methotrexate elimination only when redundant means of methotrexate removal are also compromised. Furthermore, the present findings suggest that reduced-folate administration only influences methotrexate disposition in males, with the renal reduced-folate response influenced by OAT3 function.
Keywords: cancer; carrier; chemotherapy; polymorphisms; renal secretion

Membrane Trafficking of the Human Organic Anion-Transporting Polypeptide C (hOATPC) by An-Qiang Sun; Vijaya M. Ponamgi; James L. Boyer; Frederick J. Suchy (463-474).
The human organic anion transporting polypeptide C (OATPC) is one of the major transport proteins involved in the enterohepatic circulation of bile salts and plays an important role in vectorial transport of organic anions and drugs across hepatocytes.In this study, the effects of biological reagents on the membrane localization of OATPC were investigated by confocal microscopy and estrone-3-sulfate transport.Our results demonstrated that the functional membrane expression of fluorescent chimera OATPC-GFP was achieved in non-polarized (COS7 and HEK293) and polarized (MDCK) cells. Both brefeldin A (a Golgi complex disruptor) and bafilomycin A1 (an inhibitor of vacuolar H+-ATPase) treatment significantly decreased the polarized membrane trafficking and markedly reduced the uptake of estrone-3-sulfate (∼40–90%) in OATPC-GFP transfected cells, suggesting that membrane sorting of hOATPC-GFP was mediated by Golgi complex and vacuolar H+-ATPase-related vesicle transport pathways. Treatment with 8-Br-cAMP (a cAMP analog) stimulated OATPC-GFP membrane localization and enhanced estrone-3-sulfate uptake by ∼20%. The protein kinase A (PKA) inhibitors (H89 and KT5720), but not a PKG inhibitor, blocked the polarized membrane expression of OATPC-GFP and reduced estrone-3-sulfate transport activity. The simultaneous treatment of cells with PKA activator/inhibitor and bafilomycin A1 demonstrated that bafilomycin A1 did not change the effects of 8-Br-cAMP and H89 on the membrane localization of OATPC-GFP compared with the use of 8-Br-cAMP and H89 alone.These data suggest that a cAMP-PKA sensitive membrane sorting pathway for OATPC-GFP is independent of the vacuolar H+-ATPase associated (bafilomycin A1 sensitive) vesicle mediated membrane sorting pathway. In contrast, with combined treatment with brefeldin A, neither the PKA-activator (8-Br-cAMP) nor the inhibitor (H89) further altered the plasma membrane expression and transport activity of OATPC-GFP compared with brefeldin A treatment alone. These data suggest that the cAMP-PKA regulation of OATPC membrane expression involves the Golgi complex. When the Golgi apparatus was disrupted by brefeldin A treatment, the effects of cAMP-PKA on the Golgi-to-basolateral surface sorting process of OATPC was also diminished. In summary, the plasma membrane localization of human OATPC is mediated by Golgi complex and vacuolar H+-ATPase vesicle mediated membrane sorting pathways. cAMP-PKA regulates sorting process through the Golgi complex but not the vacuolar H+-ATPase associated vesicular pathway.
Keywords: human OATPC; membrane trafficking; organic anion transporter; protein kinase A

To compare the interaction of human organic anion transporter hOAT4 with PDZ proteins between kidney cells and placental cells.PDZ proteins PDZK1 and NHERF1 were transfected into kidney LLC-PK1 cells and placental BeWo cells expressing hOAT4 or hOAT4-Δ, which lacks the PDZ consensus binding site. The interaction of PDZK1 and NHERF1 with hOAT4 and hOAT4-Δ was investigated by measurement of [3H] estrone sulfate uptake, cell surface and total cell expression of hOAT4.PDZK1 and NHERF1 enhanced hOAT4 activity in LLC-PK1 cells by increasing the cell surface expression of the transporter. In contrasts, these two PDZ proteins had no effect on hOAT4 activity in BeWo cells.The interaction of PDZ proteins with hOAT4 may be cell-specific. In placenta, a different set of interacting proteins from PDZK1 and NHERF1 may be required to modulate hOAT4 activity.
Keywords: cell surface biotinylation; co-immunoprecipitation.; drug transporter; estrone sulfate transport; organ-specific interaction; PDZ proteins

AAPS Update (481-482).