Pharmaceutical Research (v.30, #9)

Disease, Drug Metabolism, and Transporter Interactions by Jeffrey L. Staudinger (2171-2173).
is Professor in the Department of Pharmacology and Toxicology at the University of Kansas School of Pharmacy in Lawrence, Kansas. He received his B.S.(1987, Biology) from Nebraska Wesleyan University and his M.S. (1994, Biochemistry and Molecular Biology) and Ph.D. (1996, Biochemistry and Molecular Biology) from the University of Texas Graduate School of Biomedical Sciences. His professional experience includes Postdoctoral Research Fellow (1996–1999), Nuclear Receptor Group, GlaxoWellcome Research; Senior Postdoctoral Fellow (2000–2001), Department of Pharmacology, Toxicology and Experimental Therapeutics, University of Kansas Medical Center; Assistant Professor (2001–2006), Department of Pharmacology and Toxicology, University of Kansas School of Pharmacy; and Associate Professor (2006-present), Department of Pharmacology and Toxicology, University of Kansas School of Pharmacy. Dr. Staudinger’s laboratory is interested in basic regulatory mechanisms in toxicology. He works primarily with mice and tissue culture as model systems to investigate two major questions: (1) What role do ligand-activated transcription factors play in regulating xenobiotic homeostasis? (2) What signal transduction pathways interface with ligand-activated transcription factors in mediating xenobiotic homeostasis. Dr. Staudinger has been a member of the American Society for Pharmacology and Experimental Therapeutics (ASPET) since 2004 in the Drug Metabolism, Molecular Pharmacology, and Toxicology divisions. He is a member of the American Association for the Advancement of Science, American Society for Biochemistry and Molecular Biology, and International Society for the Study of Xenobiotics. He also serves on the Editorial Advisory Board for Molecular Pharmaceutics.

Acetaminophen (APAP) is one of the most widely used drugs. Though safe at therapeutic doses, overdose causes mitochondrial dysfunction and centrilobular necrosis in the liver. The first studies of APAP metabolism and activation were published more than 40 years ago. Most of the drug is eliminated by glucuronidation and sulfation. These reactions are catalyzed by UDP-glucuronosyltransferases (UGT1A1 and 1A6) and sulfotransferases (SULT1A1, 1A3/4, and 1E1), respectively. However, some is converted by CYP2E1 and other cytochrome P450 enzymes to a reactive intermediate that can bind to sulfhydryl groups. The metabolite can deplete liver glutathione (GSH) and modify cellular proteins. GSH binding occurs spontaneously, but may also involve GSH-S-transferases. Protein binding leads to oxidative stress and mitochondrial damage. The glucuronide, sulfate, and GSH conjugates are excreted by transporters in the canalicular (Mrp2 and Bcrp) and basolateral (Mrp3 and Mrp4) hepatocyte membranes. Conditions that interfere with metabolism and metabolic activation can alter the hepatotoxicity of the drug. Recent data providing novel insights into these processes, particularly in humans, are reviewed in the context of earlier work, and the effects of altered metabolism and reactive metabolite formation are discussed. Recent advances in the diagnostic use of serum adducts are covered.
Keywords: acetaminophen; drug metabolism; drug transporters; hepatotoxicity; nuclear receptors

Hepatocyte Nuclear Factor 4 Alpha and Farnesoid X Receptor Co-regulates Gene Transcription in Mouse Livers on a Genome-Wide Scale by Ann M. Thomas; Steve N. Hart; Guodong Li; Hong Lu; Yaping Fang; Jianwen Fang; Xiao-bo Zhong; Grace L. Guo (2188-2198).
Farnesoid X receptor (Fxr) is a ligand-activated nuclear receptor critical for liver function. Reports indicate that the functions of Fxr in the liver may overlap with those of hepatocyte nuclear factor 4α (Hnf4α), but studies of their precise genome-wide interaction to regulate gene transcription in the liver are lacking. Thus, we compared the genome-wide binding of Fxr and Hnf4α in the liver of mice and characterized their cooperative activity on binding to and activating target gene transcription.Genome-wide ChIP-Seq data of Fxr and Hnf4α in mouse liver were analyzed by MACS, BEDTools, and DAVID. Co-immunoprecipitation, ChIP-qPCR, and luciferase assays were done to test for protein-protein interaction and cooperative binding.ChIP-seq analysis showed nearly 50% binding sites of Fxr and Hnf4α in mouse liver overlap and Hnf4α bound to shared target sites upstream and in close proximity to Fxr. Moreover, genes co-bound by Fxr and Hnf4α are enriched in complement and coagulation cascades and drug metabolism. A direct Fxr-Hnf4α protein interaction dependent on Fxr activity was detected and transcriptional assays suggest that Hnf4α can increase Fxr transcriptional activity. Conversely, binding assays showed Hnf4α can be either Fxr-dependent or -independent at different shared binding sites.Our results showed that Fxr cooperates with Hnf4α in the liver to modulate gene transcription. This study provides the first evidence on a genome-wide scale of both cooperative and independent interactions between Fxr and Hnf4α in regulating gene transcription in the liver.
Keywords: ChIP-Seq; co-regulation; Fxr; Hnf4α; nuclear receptor interaction

Identification of Three Novel Natural Product Compounds that Activate PXR and CAR and Inhibit Inflammation by Suticha Kittayaruksakul; Wenchen Zhao; Meishu Xu; Songrong Ren; Jing Lu; Ju Wang; Michael Downes; Ronald M. Evans; Raman Venkataramanan; Varanuj Chatsudthipong; Wen Xie (2199-2208).
To investigate the effects of three natural product compounds, carapin, santonin and isokobusone, on the activity of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in induction of drug-metabolizing enzymes and inhibition of inflammation.The monkey kidney-derived fibroblast (CV-1) cells and human embryonic kidney HEK293 cells were used for transient transfection and luciferase reporter gene assays. Human primary hepatocytes and primary hepatocytes from wild type, PXR−/−, and hPXR transgenic mice were used to study the induction of drug-metabolizing enzymes and the implication of these compounds in inflammation.Carapin, santonin and isokobusone activated both PXR and CAR in transient transfection and luciferase reporter gene assays. Mutagenesis studies showed that two amino acid residues, Phe305 of the rodent PXR and Leu308 of the human PXR, are critical for the recognition of these compounds by PXR. Importantly, the activation of PXR and CAR by these compounds induced the expression of drug-metabolizing enzymes in primary human and mouse hepatocytes. Furthermore, activation of PXR by these compounds inhibited the expression of inflammatory mediators in response to lipopolysaccharide (LPS). The effects of these natural compounds on drug metabolism and inflammation were abolished in PXR−/− hepatocytes.Our results show that carapin, santonin and isokobusone activate PXR and CAR and induce drug-metabolizing enzymes. In addition, these compounds inhibited the expression of inflammatory mediators in response to LPS through the activation of PXR.
Keywords: drug metabolism; gene regulation; natural products; nuclear receptor

Pregnancy Represses Induction of Efflux Transporters in Livers of Type I Diabetic Mice by Lauren M. Aleksunes; Jialin Xu; Eugenia Lin; Xia Wen; Michael J. Goedken; Angela L. Slitt (2209-2220).
To determine whether down-regulation of transcription factor signaling during pregnancy disrupts the induction of efflux transporters in type I diabetic mice.Type I diabetes was induced in female C57BL/6 mice with multiple low dose intraperitoneal injections of streptozotocin (STZ) at least 2 weeks prior to mating with normoglycemic male mice. On gestation day 14, livers were collected from vehicle- and STZ-treated non-pregnant and pregnant mice for quantification of efflux transporter and transcription factor signaling.STZ treatment up-regulated expression of Mrp1-5, Mdr1, Abcg5, Abcg8, Bcrp, and Bsep mRNA and/or protein in the livers of non-pregnant mice. Interestingly, little to no change in transporter expression was observed in STZ-treated pregnant mice compared to vehicle- and STZ-treated non-pregnant mice.This study demonstrates the opposing regulation of hepatobiliary efflux transporters in response to diabetes and pregnancy and points to PPARγ, Nrf2, and FXR as candidate pathways underlying the differential expression of transporters.
Keywords: ABC transporter; diabetes; efflux transporters; liver; pregnancy

CR increases fatty acid oxidation to decrease tissue lipid content. The Nuclear factor E2-related factor 2 (Nrf2)-Kelch like ECH associated Protein 1 (Keap1) pathway is an antioxidant gene regulatory pathway that has been previously investigated in weight gain. However, limited interaction of Nrf2/Keap1 and CR exists. The purpose of this study was to determine how Keap1 knockdown (Keap1-KD), which is known to increase Nrf2 activity, affects the CR response, such as weight loss, hepatic lipid decrease, and induction of fatty acid oxidation gene expression.C57BL/6 and Keap1-KD mice were maintained on 40% CR or fed ad libitum for 6 weeks. Hepatic lipid content, lipid metabolic gene, and miRNA expression was quantified.CR lowered hepatic lipid content, and induced fatty acid oxidation gene expression to a greater degree in Keap1-KD compared to C57BL/6 mice. CR differentially altered miRNA 34a, 370, let-7b* in livers of Keap1-KD compared to C57BL/6 mice.CR induced induction of fatty acid oxidation gene expression was augmented with Keap1 knockdown, which was associated with differential expression of several miRNAs implicated in fatty acid oxidation and lipid accumulation.
Keywords: caloric restriction; gene expression; liver; Nfe2l2; nuclear receptor

Fatty liver alters liver transporter expression. Caloric restriction (CR), the recommended therapy to reverse fatty liver, increases Sirtuin1 deacetylase activity in liver. This study evaluated whether CR and CR mimetics reversed obesity-induced transporter expression in liver and hepatocytes.mRNA and protein expression was determined in adult lean (lean) and leptin-deficient obese (OB) mice fed ad libitum or placed on 40% (kCal) reduced diet. Hepatocytes were isolated from lean and OB mice, treated with AMP Kinase activators, and gene expression was determined.CR decreased Oatp1a1, Oatp1b2, and Abcb11 mRNA expression in lean, but not OB mice. CR increased Abcc2 mRNA OB livers, whereas protein expression increased in both genotypes. CR increased Abcc3 protein expression increased in OB livers. CR did not alter Abcc1, 4 and 5 mRNA expression in lean mice but decreased expression in livers of OB mice. CR increased Abcc4 protein in lean, but not OB mice.CR restriction reversed the expression of some, but not all transporters in livers of OB mice. Overall, these data indicate a potential for CR to restore some hepatic transporter changes in OB mice, but suggest a functional leptin axis is needed for reversal of expression for some transporters.
Keywords: gene expression; liver; nuclear receptor; steatosis; transport

Divergent Effects of Sulforaphane on Basal and Glucose-Stimulated Insulin Secretion in β-Cells: Role of Reactive Oxygen Species and Induction of Endogenous Antioxidants by Jingqi Fu; Qiang Zhang; Courtney G. Woods; Hongzhi Zheng; Bei Yang; Weidong Qu; Melvin E. Andersen; Jingbo Pi (2248-2259).
Oxidative stress is implicated in pancreatic β-cell dysfunction, yet clinical outcomes of antioxidant therapies on diabetes are inconclusive. Since reactive oxygen species (ROS) can function as signaling intermediates for glucose-stimulated insulin secretion (GSIS), we hypothesize that exogenously boosting cellular antioxidant capacity dampens signaling ROS and GSIS.To test the hypothesis, we formulated a mathematical model of redox homeostatic control circuit comprising known feedback and feedforward loops and validated model predictions with plant-derived antioxidant sulforaphane (SFN).SFN acutely (30-min treatment) stimulated basal insulin secretion in INS-1(832/13) cells and cultured mouse islets, which could be attributed to SFN-elicited ROS as N-acetylcysteine or glutathione ethyl ester suppressed SFN-stimulated insulin secretion. The mathematical model predicted an adapted redox state characteristic of strong induction of endogenous antioxidants but marginally increased ROS under prolonged SFN exposure, a state that attenuates rather than facilitates glucose-stimulated ROS and GSIS. We validated the prediction by demonstrating that although 24-h treatment of INS-1(832/13) cells with low, non-cytotoxic concentrations of SFN (2–10 μM) protected the cells from cytotoxicity by oxidative insult, it markedly suppressed insulin secretion stimulated by 20 mM glucose.Our study indicates that adaptive induction of endogenous antioxidants by exogenous antioxidants, albeit cytoprotective, inhibits GSIS in β-cells.
Keywords: antioxidant; computational model; insulin secretion; Nrf2; ROS; sulforaphane

Organic Anion Transporting Polypeptides (OATPs) are expressed in various epithelial tissues in the body. Because they can be expressed in cancers and because they can transport anticancer drugs, OATPs could be potential targets for cancer therapy. Therefore we examined their expression in human pancreatic ductal adenocarcinomas.Expression of all 11 human OATPs was measured at the mRNA level and OATPs with highest expression were characterized at the protein level.Transcripts of SLCO1B3, SLCO2A1, SLCO3A1 and SLCO4A1 were detected in all the tested pancreatic tissues. OATP1B3, OATP2A1, OATP3A1 and OATP4A1 protein expression was confirmed in these tissues and expression of all four transporters increased in pancreatic adenocarcinoma compared to normal pancreas. OATP1B3 expression was highest in pancreatic hyperplasia and stage one adenocarcinomas compared to stage two and three adenocarcinomas.OATP1B3, OATP2A1, OATP3A1 and OATP4A1 are up-regulated in pancreatic adenocarcinoma and could potentially be used to target anticancer drugs to pancreatic cancer. Additionally, because expression of OATP1B3 is highest in pancreatitis and stage one adenocarcinoma, which leads to pancreatic cancer, OATP1B3 is a potential marker to diagnose patients with early stage pancreatic adenocarcinomas.
Keywords: cancer biomarker; OATP; pancreatic ductal adenocarcinoma; SLCO; uptake transporters

Extra-Hepatic Cancer Represses Hepatic Drug Metabolism Via Interleukin (IL)-6 Signalling by Marina Kacevska; Andre Mahns; Rohini Sharma; Stephen J. Clarke; Graham R. Robertson; Christopher Liddle (2270-2278).
In many cancer patients, the malignancy causes reduced hepatic drug clearance leading to potentially serious complications from the use of anticancer drugs. The mechanisms underlying this phenomenon are poorly understood. We aimed to identify tumor-associated inflammatory pathways that alter drug response and enhance chemotherapy-associated toxicity.We studied inflammatory pathways involved in extra-hepatic tumor mediated repression of CYP3A, a major hepatic drug metabolizing cytochrome P450 subfamily, using a murine Engelbreth-Holm-Swarm sarcoma model. Studies in IL-6 knockout mice determined the source of elevated IL-6 in tumor-bearing animals and monoclonal antibodies against IL-6 were used to intervene in this inflammatory pathway.Our studies confirm elevated plasma IL-6 levels and reveal activation of Jak/Stat and Mapk signalling pathways and acute phase proteins in livers of tumor-bearing mice. Circulating IL-6 was predominantly produced by the tumor xenograft, rather than being host derived. Anti IL-6 antibody intervention partially reversed tumor-mediated inflammation and Cyp3a gene repression.IL-6 is an important player in cancer-related repression of CYP3A-mediated drug metabolism and activation of the acute phase response. Targeting IL-6 in cancer patients may prove an effective approach to alleviating cancer-related phenomena, such as adverse drug-related outcomes commonly associated with cancer chemotherapy.
Keywords: cancer; cancer chemotherapy; CYP3A; drug metabolism; inflammation; interleukin-6

Role of Intracellular Calcium in Proteasome Inhibitor-Induced Endoplasmic Reticulum Stress, Autophagy, and Cell Death by Jessica A. Williams; Yifeng Hou; Hong-Min Ni; Wen-Xing Ding (2279-2289).
Proteasome inhibition induces endoplasmic reticulum (ER) stress and compensatory autophagy to relieve ER stress. Disturbance of intracellular calcium homeostasis can lead to ER stress and alter the autophagy process. It has been suggested that inhibition of the proteasome disrupts intracellular calcium homeostasis. However, it is unknown if intracellular calcium affects proteasome inhibitor-induced ER stress and autophagy.Human colon cancer HCT116 Bax positive and negative cell lines were treated with MG132, a proteasome inhibitor. BAPTA-AM, a cell permeable free calcium chelator, was used to modulate intracellular calcium levels. Autophagy and cell death were determined by fluorescence microscopy and immunoblot analysis.MG132 increased intracellular calcium levels in HCT116 cells, which was suppressed by BAPTA-AM. MG132 suppressed proteasome activity independent of Bax and intracellular calcium levels in HCT116 cells. BAPTA-AM inhibited MG132-induced cellular vacuolization and ER stress, but not apoptosis. MG132 induced autophagy with normal autophagosome-lysosome fusion. BAPTA-AM seemed not to affect autophagosome-lysosome fusion in MG132-treated cells but further enhanced MG132-induced LC3-II levels and GFP-LC3 puncta formation, which was likely via impaired lysosome function.Blocking intracellular calcium by BAPTA-AM relieved MG132-induced ER stress, but it was unable to rescue MG132-induced apoptosis, which was likely due to impaired autophagic degradation.
Keywords: autophagy; cell death; ER stress; intracellular calcium; proteasome inhibitor

Benzylmorpholine Analogs as Selective Inhibitors of Lung Cytochrome P450 2A13 for the Chemoprevention of Lung Cancer in Tobacco Users by Linda C. Blake; Anuradha Roy; David Neul; Frank J. Schoenen; Jeffrey Aubé; Emily E. Scott (2290-2302).
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), one of the most prevalent and procarcinogenic compounds in tobacco, is bioactivated by respiratory cytochrome P450 (CYP) 2A13, forming DNA adducts and initiating lung cancer. CYP2A13 inhibition offers a novel strategy for chemoprevention of tobacco-associated lung cancer.Twenty-four analogs of a 4-benzylmorpholine scaffold identified by high throughput screening were evaluated for binding and inhibition of both functional human CYP2A enzymes, CYP2A13 and the 94%-identical hepatic CYP2A6, whose inhibition is undesirable. Thus, selectivity is a major challenge in compound design.A key feature resulting in CYP2A13-selective binding and inhibition was substitution at the benzyl ortho position, with three analogs being >25-fold selective for CYP2A13 over CYP2A6.Two such analogs were negative for genetic and hERG toxicities and metabolically stable in human lung microsomes, but displayed rapid metabolism in human liver and in mouse and rat lung and liver microsomes, likely due to CYP2B-mediated degradation. A specialized knockout mouse mimicking the human lung demonstrates compound persistence in lung and provides an appropriate test model. Compound delivered by inhalation may be effective in the lung but rapidly cleared otherwise, limiting systemic exposure.
Keywords: chemopreventative; cytochrome P450 2A13; cytochrome P450 2A6; lung cancer; tobacco

Image Analysis Quantification of Sticking and Picking Events of Pharmaceutical Powders Compressed on a Rotary Tablet Press Simulator by Germinal Mollereau; Vincent Mazel; Virginie Busignies; Pierre Tchoreloff; Fabrice Mouveaux; Philippe Rivière (2303-2314).
The aim of this work was to develop a quantification method based on image analysis, able to characterize sticking during pharmaceutical tableting. Relationship between image analysis features and relevant mechanical parameters recorded on an instrumented tablet press simulator were investigated.Image analysis, based on gray levels co-occurrence matrices (GLCM), generated textural features of the tablet surface. The tableting simulator (Stylcam® 200R, Medelpharm), instrumented with force and displacement transducers, provided accurate records. The tablet defects and compaction process parameters were studied using three pharmaceutical powders (Fast-Flo® lactose, anhydrous Emcompress® and Avicel® PH200 microcrystalline cellulose), five compression pressures (60 to 250 MPa), five lubricating levels, and three types of punches (standard steel, amorphous hard carbon and anti-sticking punches).Texture parameters made it possible to quantify with precision tablets’ aspect. The selected parameter IC2 (Information on Correlation 2) plotted versus the ratio between the ejection shear stress (Esh) and the compression pressure (Cp) let appear a relevant knowledge space where it was possible to identify a normal and a degraded tableting mode. A positive link between those two parameters was shown.Since the Esh/Cp ratio was related to image analysis results, it proved to be an interesting defect tag.
Keywords: coated punches; image analysis; lubrication; sticking; tablet press simulator

A Solid-State NMR Study of Amorphous Ezetimibe Dispersions in Mesoporous Silica by Frederick G. Vogt; Karen Roberts-Skilton; Sonya A. Kennedy-Gabb (2315-2331).
The purpose of this work is to examine the ability of methods based on multinuclear and multidimensional solid-state NMR (SSNMR) to perform detailed characterization of amorphous dispersions of ezetimibe adsorbed on mesoporous silica.Ezetimibe was loaded into two types of mesoporous silica with average pore sizes of 2.5 and 21 nm. The mesoporous materials were characterized by powder X-ray diffraction (PXRD), vibrational spectroscopy, differential scanning calorimetry, and 1H, 13C, 19F, and 29Si SSNMR analysis including relaxation time measurements. Interactions between the drug and silica were investigated using 1D and 2D SSNMR methods based on dipolar correlation using cross-polarization (CP) and spin diffusion.PXRD was used to show the absence of crystalline ezetimibe in the mesoporous materials, and 19F SSNMR was used to assess drug physical state and study mobility. 19F-29Si CP methods were used to directly detect adsorbed ezetimibe. 1H-13C, 1H-19F, and 1H-29Si, and heteronuclear correlation and 1H homonuclear correlation experiments were used to investigate interactions between the drug and silica through 1H environments.SSNMR methods were able to detect interactions between the drug and the silica substrate. Differences between the drug loaded onto silica with two different pore sizes were observed, including differences in hydrogen bonding environment and molecular mobility. These methods should be useful for characterization of similar systems.
Keywords: amorphous dispersion; dipolar correlation; mesoporous silica; powder X-ray diffraction; solid-state NMR

Bone Morphogenetic Protein-2 Release from Composite Hydrogels of Oligo(poly(ethylene glycol) fumarate) and Gelatin by Lucas A. Kinard; Chia-Ye Chu; Yasuhiko Tabata; F. Kurtis Kasper; Antonios G. Mikos (2332-2343).
Hydrogel composites of oligo(poly(ethylene glycol) fumarate) (OPF) and gelatin microparticles (GMs) were investigated as carriers of bone morphogenetic protein-2 (BMP-2) for bone tissue engineering applications.Hydrogel composites with different physical characteristics were prepared by changing the amount and type (acidic vs. basic) of gelatin incorporated in the OPF bulk phase. Composites with differing physical properties (degradation, swelling, and mechanical properties) and differing BMP-2 loading phase were investigated to determine the effect of these factors on BMP-2 release profiles over 28 days.Overall, higher gelatin amount increased the degradation and swelling of composites, and acidic GMs further increased the degradation and swelling and reduced the compressive modulus of the composites. The most significant factor affecting the release of BMP-2 from composites was the loading phase of the growth factor: GM loading reduced the burst release, increased BMP-2 release during the later phases of the experiment, and increased the cumulative release in faster degrading samples.The results indicate that the physical properties and the BMP-2 release kinetics of hydrogel composites can be controlled by adjusting multiple parameters at the time of the hydrogel composite fabrication.
Keywords: bone morphogenetic protein-2; controlled release; gelatin microparticles; hydrogel composites; oligo(poly(ethylene glycol) fumarate)

The Co-Delivery of Oxaliplatin Abrogates the Immunogenic Response to PEGylated siRNA-Lipoplex by Eman Alaaeldin; Amr S. Abu Lila; Naoto Moriyoshi; Hatem A. Sarhan; Tatsuhiro Ishida; Khaled A. Khaled; Hiroshi Kiwada (2344-2354).
In vivo application of siRNA/PEGylated cationic liposome complex (lipoplex) is impeded by two main obstacles: cytokine responses and anti-PEG IgM responses to PEGylated siRNA-lipoplex. Here, we investigated whether co-administration of oxaliplatin (l-OHP) abrogates the cytokine release and anti-PEG IgM production by PEGylated siRNA-lipoplex.Free l-OHP was administered either simultaneously or 30 min prior to PEGylated siRNA-lipoplex administration, and cytokine response and anti-PEG IgM production were evaluated. In addition, the effect of the liposomal encapsulation of l-OHP on the immunogenic response of PEGylated siRNA-lipoplex was investigated.Simultaneous co-administration of free l-OHP with PEGylated siRNA-lipoplex caused a significant reduction in anti-PEG IgM production, along with an increase in the cytokine response. Free l-OHP injected prior to the lipoplex injection, however, successfully reduced cytokine release and anti-PEG IgM response. Platination of siRNA by simultaneously administered free l-OHP might facilitate the dissociation of double-stranded siRNA to single-stranded siRNA, resulting in the inducement of a potent immuno-stimulation of siRNA via endosomal toll-like receptors (TLRs). On the other hand, encapsulation of l-OHP into the siRNA-lipoplex resulted in a reduction of both anti-PEG IgM production and cytokine responses.Our results suggest that, besides the expected therapeutic efficacy of co-administration, encapsulation of l-OHP into the PEGylated siRNA-lipoplex has great potential for minimizing the immunostimulation of PEGylated siRNA-lipoplex, resulting in a safe, applicable, and compliant treatment regimen for sequential clinical administration.
Keywords: anti-PEG IgM; cytokines; PEGylated siRNA cationic liposome (lipoplex); short interfering RNA (siRNA); β-galctosidase

When information is sparse, individual parameters derived from a non-linear mixed effects model analysis can shrink to the mean. The objective of this work was to predict individual parameter shrinkage from the Bayesian information matrix (M BF ). We 1) Propose and evaluate an approximation of M BF by First-Order linearization (FO), 2) Explore by simulations the relationship between shrinkage and precision of estimates and 3) Evaluate prediction of shrinkage and individual parameter precision.We approximated M BF using FO. From the shrinkage formula in linear mixed effects models, we derived the predicted shrinkage from M BF . Shrinkage values were generated for parameters of two pharmacokinetic models by varying the structure and the magnitude of the random effect and residual error models as well as the design. We then evaluated the approximation of M BF FO and compared it to Monte-Carlo (MC) simulations. We finally compared expected and observed shrinkage as well as the predicted and estimated Standard Errors (SE) of individual parameters. M BF FO was similar to M BF MC. Predicted and observed shrinkages were close . Predicted and estimated SE were similar. M BF FO enables prediction of shrinkage and SE of individual parameters. It can be used for design optimization.
Keywords: bayesian fisher information matrix; non-linear mixed effects model; optimal design; pharmacokinetics

To elucidate the role of metabolism in the pharmacokinetics and pharmacodynamics of intranasal loxapine in conscious animals.At pre-determined time points after intranasal or oral loxapine administration, levels of loxapine, loxapine metabolites, and neurotransmitters in rat brain were quantified after catalepsy assessments (block test and paw test). Cataleptogenicity of loxapine was also compared with its metabolites.Intranasally administered loxapine was efficiently absorbed into systemic circulation followed by entering brain, with tmax ≤15 min in all brain regions. Oral route delivered minimal amounts of loxapine to plasma and brain. Brain AUC0–240min values of 7-hydroxy-loxapine were similar after intranasal and oral administration. Intranasal loxapine tended to induce less catalepsy than oral loxapine, although statistical significance was not reached. The catalepsy score was positively and significantly correlated with the striatal concentration of 7-hydroxy-loxapine, but not with loxapine. 7-hydroxy-loxapine was more cataleptogenic than loxapine, while the presence of loxapine tended to reduce rather than intensify 7-hydroxy-loxapine-induced catalepsy. The increases in striatal dopamine turnover were comparable after intranasal and oral loxapine administration.The metabolite 7-hydroxy-loxapine, but not loxapine, was the main contributor to the catalepsy observed after intranasal and oral loxapine treatment. Intranasal route could effectively deliver loxapine to brain.
Keywords: antipsychotics; catalepsy; central nervous system; intranasal administration; metabolism

The Shape/Morphology Balance: A Study of Stealth Liposomes via Fractal Analysis and Drug Encapsulation by Natassa Pippa; Faidra Psarommati; Stergios Pispas; Costas Demetzos (2385-2395).
Fractal analysis was used as a tool in order to study the morphological characteristics of PEGylated liposomes. We report on the morphological characteristics of stealth liposomes composed of DPPC and DPPE-PEG 3000 in two dispersion media using fractal analysis.Light scattering techniques were used in order to elucidate the size, the morphology and the surface charge of PEGylated liposomes as a function of PEGylated lipid concentration and temperature. Fluorescence spectroscopy studies revealed a microenvironment of low polarity inside the liposomal membranes.All formulations were found to retain their physicochemical characteristics for at least 3 weeks. The hydrodynamic radii (Rh) of stealth liposomes were stable in the process of heating up to 50°C; while the fractal dimension values (df) which correspond to their morphology, have been changed during heating. Hence, these results are a first indication of the presence of a heterogeneous microdomain structure of the stealth liposomal system. The amphiphilic drug indomethacin (IND) was successfully encapsulated within the liposomes and led to an increased size of stealth liposomes, while the morphology of liposomal vectors changed significantly at the highest molar ratio of PEGylated lipid.We can state that this approach can promote a new analytical concept based on the morphological characteristics and quantify the shape of drug carriers complementary to that of the conventional analytical techniques.
Keywords: fractal dimension; indomethacin; morphology; pegylated nanoparticles; shape

Preparation, optimization and in vitro evaluation of core-shell nanoparticles comprising of a hydrophilic core of BSA surrounded by a hydrophobic shell of PLGA for loading water-soluble drugs.A double emulsion method was optimized for preparation of BSA-PLGA based core-shell nanoparticles. Proof of concept for core-shell type structure was established by visual techniques like confocal microscopy and TEM. Characterization was done for particle size, encapsulation efficiency, drug loading and in vitro drug release. Cellular uptake was assessed using confocal microscopy, bio-TEM and HPLC assay, and cytotoxic activity was tested by MTT assay in MG-63 osteosarcoma cells.The optimized core-shell nanoparticles showed a particle size of 243 nm (PDI-0.13) and encapsulation efficiency of 40.5% with a drug loading of 8.5% w/w. In vitro drug release studies showed a sustained release for 12 h. Cellular uptake studies indicated a rapid and efficient uptake within 2 h. TEM studies indicated that the core-shell nanoparticles were localized in cytoplasm region of the cells. Gemcitabine loaded core-shell nanoparticles showed enhanced cytotoxicity against MG-63 cells as compared to marketed formulation of gemcitabine (GEMCITE®).These results indicate that core-shell nanoparticles can be a good carrier system for delivering hydrophilic drugs like gemcitabine successfully to the cells with enhanced efficacy. Figure Core-Shell Nanoparticles with a hydrophilic BSA core and hydrophobic PLGA shell for carrier system of hydrophilic drugs
Keywords: BSA; core-shell nanoparticles; gemcitabine; water-soluble drugs

Contribution of Protein Binding, Lipid Partitioning, and Asymmetrical Transport to Drug Transfer into Milk in Mouse Versus Human by Naoki Ito; Kousei Ito; Hiroki Koshimichi; Akihiro Hisaka; Masashi Honma; Takashi Igarashi; Hiroshi Suzuki (2410-2422).
Drug transfer into milk is a general concern during lactation. Because data are limited in human subjects, particularly for new drugs, experimental animal models of lactational drug transfer are critical. This study analyzed drug transfer into milk in a mouse model, as well as the contribution of similar and dissimilar host factors.Milk/plasma drug concentration ratios (M/P) in humans were obtained from the literature, while those in mice were determined experimentally after intraperitoneal implantation of osmotic pumps containing drugs of interest. Unbound drug fractions in plasma and milk were determined in vitro for both species.M/P values were determined for 27 drugs in mice and compared with those in human. These values were increased in mice for 21 drugs; the geometric mean ratio of M/P between mice and humans was 2.03 (95% CI, 1.42–2.89) for all 27 drugs. These results were reasonably explained by the relatively high protein and lipid content in mouse milk. Moreover, species-specific asymmetrical transport systems were suggested for 9 drugs.In addition to species-specific differences in milk protein and lipid content, variances in asymmetrical drug transport across the mammary epithelium may yield discordant M/P values in humans and mice.
Keywords: breastfeeding; lactation; mammary epithelium; species-specific differences; transporter

AAPS Connection (2423-2425).