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A series of fexaramine analogs were synthesized and evaluated to develop an intestine-selective/specific FXR partial agonist. Introduction of both a CN substituent at the C-2 in the biphenyl ring and a fluorine at the C-5 in the aniline ring in fexaramine markedly increased FXR agonistic activity.

Several lines of evidence indicated that generation of NADPH oxidase (Nox)-mediated reactive oxygen species are associated with neuronal inflammation, leading to Parkinson's disease (PD). Novel benzylidene-1-methyl-2-thioxoimidazolidin-one derivatives as Nox inhibitors were designed and synthesized in order to increase blood-brain barrier (BBB) permeability to target Nox in brain cells. In lucigenin chemiluminescence assay, eight compounds showed excellent inhibition activity against NADPH oxidases and parallel artificial membrane permeability assay (PAMPA) identified compound 11 with high passive permeability. To validate the effect of compound 11 on neuronal inflammation, we tested the regulatory activity of compound 11 in lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines in BV-2 microglial cells and LPS-mediated microglial migration. Treatment of BV2 cells with compound 11 resulted in suppressed production of pro-inflammatory cytokines and migration activity of BV2 cells in response to LPS. To evaluate the therapeutic efficacy of compound 11 in PD animal model, compound 11 was applied to MPTP-induced PD mouse model. Oral administration of compound 11 (30 mg/kg/daily, 4 weeks) into the mice resulted in suppression of dopaminergic neuronal death in substantia nigra (SN) and in striatum as well as inhibition of microglial migration into SN. These results implicate compound 11 as a novel therapeutic agent for the treatment of PD.

Cockroaches live in places where various pathogens exist and thus are more likely to use antimicrobial compounds to defend against pathogen intrusions. We previously performed an in silico analysis of the

A novel method for microwave-assisted digestion of milk samples using diluted HNO3 and H2O2 with a single reaction chamber was developed for elemental analysis by ICP-based techniques. The optimal conditions for digestion were 0.25 g of sample mass, 6 mL of 0.1 mol L−1 HNO3 and 2 mL of 30% H2O2 at 250 ℃ and 160 bar. The optimized procedure resulted in low residual carbon content and residual acidity of 260 mg L−1 and 0.06 mol L-1, respectively. The limits of detection ranged from 0.286 ոg g−1 (Ca) to 82.990 ոg g−1 (Fe). In addition, the proposed method was considered an excellent green analysis method with a final score of 87 based on the analytical Eco-Scale. Finally, the method was validated and applied to the determination of Al, As, Ba, Ca, Cd, Co, Cr, Cu, Fe, Mg, Mn, Ni, Pb, Sb, Se, and Zn in milk samples from South Korea.

Previously, we performed an

The field of gene therapy has evolved over the past two decades after the first introduction of nucleic acid drugs, such as plasmid DNA (pDNA). With the development of in vitro transcription (IVT) methods, synthetic mRNA has become an emerging class of gene therapy. IVT mRNA has several advantages over conventional pDNA for the expression of target proteins. mRNA does not require nuclear localization to mediate protein translation. The intracellular process for protein expression is much simpler and there is no potential risk of insertion mutagenesis. Having these advantages, the level of protein expression is far enhanced as comparable to that of viral expression systems. This makes IVT mRNA a powerful alternative gene expression system for various applications in regenerative medicine. In this review, we highlight the synthesis and preparation of IVT mRNA and its therapeutic applications. The article includes the design and preparation of IVT mRNA, chemical modification of IVT mRNA, and therapeutic applications of IVT mRNA in cellular reprogramming, stem cell engineering, and protein replacement therapy. Finally, future perspectives and challenges of IVT mRNA are discussed.

Objectives The inhibitors of P-glycoprotein (P-gp) which limits an access of exogenous compounds in the luminal membrane of the intestine have been studied to enhance the intestinal P-gp-mediated absorption of anticancer drugs. Methods Inhibition of the efflux pump by synthesized benzoxanthone derivatives was investigated in vitro and in vivo. MCF-7/ADR cell line was used for cytotoxicity assay and [3H]-daunomycin (DNM) accumulation/efflux study. Eight benzoxanthone analogues were tested for their effects on DNM cytotoxicity. Among them, three analogues were selected for the accumulation/efflux and P-gp ATPase studies. Paclitaxel (PTX), a P-gp substrate anticancer drug, was orally administered to rats with/without compound 1 (8,10-bis(thiiran-2-ylmethoxy)-7H-benzo[c]xanthen-7-one). The pharmacokinetic parameters of PTX in the presence/absence of compound 1 were evaluated from the plasma concentration-time profiles. Key-findings Compound 1 increased the DNA accumulation to 6.5-fold and decreased the DNM efflux to approximately 1/2 in the overexpressing P-gp cell line. Relative bioavailability (RB) of PTX in rats was significantly increased up to 3.2-fold by compound 1 (0.5 or 2 mg/kg). Conclusions Benzoxanthone analogue, compound 1 is strongly suggested to be a promising inhibitor of P-gp to improve an oral absorption of compounds for cancer therapy.

Given the challenges posed by antibiotic resistant microbes and the high mortality rate associated with sepsis, there is an urgent need to develop novel peptide antibiotics that exhibit both antimicrobial and anti-inflammatory activities. Herein, we evaluated antimicrobial activity and anti-inflammatory activity of psacotheasin 2, one of the antimicrobial peptide candidates identified previously using an in silico analysis on the transcriptome of Psacothea hilaris. In addition to exhibiting antimicrobial activities against microorganisms without inducing hemolysis, psacotheasin 2 also decreased the nitric oxide production in lipopolysaccharide (LPS)-induced Raw264.7 cells. Moreover, ELISA and western blot analysis revealed that psacotheasin 2 reduced the expression levels of pro-inflammatory enzymes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Further, we found that psacotheasin 2 markedly reduced the expression levels of pro-inflammatory cytokines (IL-6 and IL-1β) by regulating mitogen-activated protein kinases (MAPKs) and nuclear factor-kB (NF-kB) signaling in LPS-induced Raw264.7 cells. We also confirmed that the binding of psacotheasin 2 to bacterial cell membranes occurs via a specific interaction with LPS. In mouse models of LPS-induced shock, psacotheasin 2 significantly enhanced the survival rate and recovered weight by attenuating pro-inflammatory cytokines. Thus, psacotheasin 2 could be a promising candidate as a peptide antiseptic agent.

Yong Joo Jun,1,* Jung Hyun Park,2,* Prakash G Avaji,1 Kyung Su Park,3 Kyung Eun Lee,3 Hwa Jeong Lee,2 Youn Soo Sohn1 1C & Pharm, Ewha Womans University, Seodaemun-gu, Seoul, Republic of Korea; 2Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seodaemun-gu, Seoul, Republic of Korea; 3Advanced Analysis Center, Korea Institute of Science and Technology, Seongbuk-gu, Seoul, Republic of Korea *These authors contributed equally to this work Abstract: To prepare an efficient theranostic polyphosphazene–docetaxel (DTX) conjugate, a new drug delivery system was designed by grafting a multifunctional lysine ethylester (LysOEt) as a spacer group along with methoxy poly(ethylene glycol) (MPEG) to the polyphosphazene backbone ([NP]n), and then DTX was conjugated to the carrier polymer using acid-cleavable cis-aconitic acid (AA) as a linker. The resultant polyphosphazene–DTX conjugate, formulated as [NP(MPEG550)3(Lys-OEt)(AA)(DTX)]n and named “Polytaxel”, exhibited high water solubility and stability by forming stable polymeric micelles as shown in its transmission electron microscopy image and dynamic light scattering measurements. Another important aspect of Polytaxel is that it can easily be labeled with various imaging agents using the lysine amino group, enabling studies on various aspects, such as its organ distribution, tumor-targeting properties, pharmacokinetics, toxicity, and excretion. The pharmacokinetics of Polytaxel was remarkably improved, with prolonged elimination half-life and enhanced area under the curve. Ex vivo imaging study of cyanine dye-labeled Polytaxel showed that intravenously injected Polytaxel is long circulating in the blood stream and selectively accumulates in tumor tissues. Polytaxel distributed in other organs was cleared from all major organs at ~6weeks after injection. The invitro study of DTX release from the carrier polymer showed that >95% of conjugated DTX was released at pH 5.4 over a period of 7days. Xenograft trials of Polytaxel using nude mice against the human gastric tumor cell line MKN-28 showed complete tumor regression, with low systemic toxicity. Polytaxel is currently in preclinical study. Keywords: docetaxel, polyphosphazene, anticancer drug, nanomedicine, theranostics

PARK2 encodes for the E3 ubiquitin ligase parkin and is implicated in the development of Parkinson's disease (PD). Although the neuroprotective role of parkin is well known, the mechanism of PARK2's function in neural stem differentiation has not yet been thoroughly studied. Co-expressions network analysis showed that synaptosomal-associated protein 25 (SNAP-25) and brain-derived neurotrophic factor (BDNF) were positively correlated with parkin, but negatively correlated with p21 in human patient brain. We investigated a link between the ubiquitin E3 ligase parkin and proteasomal degradation of p21 for the control of neural stem cell differentiation. We found that the neurogenesis was lowered in PARK2 knockout (KO) mice compared with non-tg mice. Expression of the marker protein for neural cell differentiation such as class III beta tubulin (TUBBIII), glial fibrillary acidic protein (GFAP) and neurofilament, as well as SNAP25 and BDNF, was down regulated in PARK2 KO mice. Associated with the loss of differentiation function, p21 protein was highly accumulated in the neural stem cells of PARK2 KO mice. We discovered that p21 directly binds with parkin and is ubiquitinated by parkin which resulted in the loss of cell differentiation ability. Introduction of p21 shRNA in PARK2 KO mice significantly rescued the differentiation efficacy as well as SNAP25 and BDNF expression. c-Jun N-terminal kinase (JNK) pathway is implicated in neurogenesis and p21 degradation. We also defined the decreased p21 ubiquitination and differentiation ability were reversed after treatment with JNK inhibitor, SP600125 in PARK2 KO mice derived neural stem cells. Thus, the present study indicated that parkin knockout inhibits neural stem cell differentiation by JNK-dependent proteasomal degradation of p21.

Tetradecanol is a straight-chain saturated fatty alcohol purified from Dendropanax morbifera leaves. We found that tetradecanol (30μM) reduced specifically the growth of T cells such as EL-4 T cell and isolated murine CD4+ T cells. In this study, we investigated the effects of tetradecanol on the regulation of interlukin-2 (IL-2), a potent T cell growth factor. Tetradecanol significantly inhibited IL-2 secretion in EL-4 T cells activated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin (Io) and also in isolated murine CD4+ T cells activated with anti-CD3 and anti-CD28 antibodies. Next, we examined the effect of tetradecanol on the transcriptional activity related to IL-2 production in T cells. Tetradecanol decreased PMA/Io-induced promoter activity of NF-κB in EL-4 T cells, but did not show any significant effects on the promoters of activator protein 1 (AP-1) and nuclear factor of activated T cells (NF-AT). Tetradecanol inhibited IκBα degradation and nuclear translocation of NF-κB subunit, p65 in PMA/Io-activated EL-4 T cells. These results suggest that tetradecanol might have immunosuppressive effects on T cell mediated disorders. Using a chronic allergic contact dermatitis model induced by repeated application of oxazolone, we showed that tetradecanol reduced ear thickness induced by oxazolone.

Basavaraj R Patil,1 Su Yeon Kang,2 Da Hee Jung,2 Prakash G Avaji,1 Yong Joo Jun,1 Hwa Jeong Lee,2 Youn Soo Sohn1 1C & Pharm, Ewha Womans University, Seoul 03760, Republic of Korea; 2Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 03760, Republic of KoreaCorrespondence: Youn Soo SohnC & Pharm, Ewha Womans University, Room 304, University-Industry Cooperation Building, 52 Ewhayeodae-Gil, Seodaemun-Gu, Seoul 03760, Republic of KoreaTel +82 70 7008 2120Fax +82 2 362 8813Email yssohn@ewha.ac.krHwa Jeong LeeGraduate School of Pharmaceutical Sciences, Ewha Womans University, 52 Ewhayeodae-Gil, Seodaemun-Gu, Seoul 03760, Republic of KoreaTel +82 2 3277 3409Fax +82 2 3277 2851Email hwalee@ewha.ac.krPurpose: A new theranostic nanomedicine involving anticancer-active cisplatin moiety was designed to study its tumor-targeting properties as well as its drug efficacy and toxicity.Methods: A cisplatin carrier polymer was prepared by grafting equimolar polyethylene glycol of a molecular weight of 550 (PEG550) and aminoethanol to the poly(dichlorophosphazene) backbone. Cisplatin was conjugated to the carrier polymer using cis-aconitic acid as a linker.Results: The cisplatin-loaded polyphosphazene, named “Polycisplatin” was found to be amphiphilic in aqueous solution and self-assembled into nanoparticles with an average particle size of 18.6 nm in diameter. The time-dependent organ distribution study of Cy5.5-labeled Polycisplatin in the A549-tumor-bearing mice exhibited a high tumor selectivity of Polycisplatin by EPR effect despite the relatively small particle size. In order to compare the in vivo efficacy of Polycisplatin and cisplatin, their xenograft trials were performed using nude mice against the human gastric cell line MKN-28. Polycisplatin exhibited slightly less tumor suppression effect compared with cisplatin at the same dose of 1.95 mg Pt/kg, which is the maximum tolerate dose of cisplatin, but at the higher double dose of 3.9 mg Pt/kg, Polycisplatin exhibited a little better efficacy than cisplatin. Furthermore, mice treated with cisplatin at the dose of 1.95 mg Pt/kg exhibited severe body weight decrease by about 25%, while mice treated with Polycisplatin did not show serious body weight decrease even at its double dose of 3.9 mg Pt/kg. Furthermore, kidney indicators including kidney index, BUN, and creatinine values measured displayed that Polycisplatin is much less nephrotoxic than cisplatin.Conclusion: Nanoparticular Polycisplatin was successfully prepared by conjugating cisplatin to a hydrophilic polyphosphazene carrier polymer using the acid-cleavable cis-aconitic acid. Polycisplatin nanoparticles exhibit excellent tumor-targeting properties by EPR effect. The xenograft trials exhibited excellent antitumor efficacy and reduced systemic toxicity of Polycisplatin.Keywords: cisplatin, polyphosphazene, platinum drug, drug delivery, nanomedicine

In a previous work, we performed

Recent studies have shown that many types of cancer cells have increased levels of reactive oxygen species (ROS) and enhance antioxidant capacity as an adaptation to intrinsic oxidative stress, suggesting that cancer cells are more vulnerable to oxidative insults and are more dependent on antioxidant systems compared with normal cells. Thus, disruption of redox homeostasis caused by a decline in antioxidant capacity may provide a method for the selective death of cancer cells. Here we show that ROS-mediated selective death of tumor cells can be caused by inhibiting sulfiredoxin (Srx), which reduces hyperoxidized peroxiredoxins, leading to their reactivation. Srx inhibitor increased the accumulation of sulfinic peroxiredoxins and ROS, which led to oxidative mitochondrial damage and caspase activation, resulting in the death of A549 human lung adenocarcinoma cells. Srx depletion also inhibited the growth of A549 cells like Srx inhibition, and the cytotoxic effects of Srx inhibitor were considerably reversed by Srx overexpression or antioxidants such as N-acetyl cysteine and butylated hydroxyanisol. Moreover, Srx inhibitor rendered tumorigenic ovarian cells more susceptible to ROS-mediated death compared with nontumorigenic cells and significantly suppressed the growth of A549 xenografts without acute toxicity. Our results suggest that Srx might serve as a novel therapeutic target for cancer treatment based on ROS-mediated cell death.

Docetaxel formulated by micelle-encapsulation using a tripodal cyclotriphosphazene amphiphile [NP(MPEG750)(GlyPheLeu) 2 Et] 3 (CP750) was named “Phostaxel” and compared in efficacy and stability with Taxotere ® formulated using the surfactant polysorbate 80, which is currently in clinical use. Phostaxel has always shown better efficacy than Taxotere ® in various xenograft trials at the same dosage and administration schedule against the tumor cell lines tested. The better efficacy of Phostaxel could be explained based on the difference in pharmacokinetic and biodistribution profiles of Phostaxel and Taxotere ® . Phostaxel exhibited significantly slower clearance rate and larger AUC last value compared with Taxotere ® . Phostaxel has also shown higher DTX distribution in tumor than Taxotere ® . In addition, Phostaxel displayed better solution stability compared with Taxotere ® both in distilled water and in saline solution at room and refrigerator temperatures.

A series of 2-substituted-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)imidazoles was synthesized and evaluated to optimize a prototype inhibitor of TGF-β type I receptor kinase (ALK5), 6. Combination of replacement of a quinoxalin-6-yl moiety of 6 with a [1,2,4]triazolo[1,5-a]pyridin-6-yl moiety, insertion of a methyleneamino linker, and a o-F substituent in the phenyl ring markedly increased ALK5 inhibitory activity, kinase selectivity, and oral bioavailability. The 12b (EW-7197) inhibited ALK5 with IC50 value of 0.013 μM in a kinase assay and with IC50 values of 0.0165 and 0.0121 μM in HaCaT (3TP-luc) stable cells and 4T1 (3TP-luc) stable cells, respectively, in a luciferase assay. Selectivity profiling of 12b using a panel of 320 protein kinases revealed that it is a highly selective ALK5/ALK4 inhibitor. Pharmacokinetic study with 12b·HCl in rats showed an oral bioavailability of 51% with high systemic exposure (AUC) of 1426 ng × h/mL and maximum plasma concentration (Cmax) of 1620 ng/mL. Rational optimization of 6 has led to the identification of a highly potent, selective, and orally bioavailable ALK5 inhibitor 12b.

Na(+)/H(+) exchanger regulatory factor 3 (NHERF3) is a PSD-95/discs large/ZO-1 (PDZ)-based adaptor protein that regulates several membrane-transporting proteins in epithelia. However, the in vivo physiologic role of NHERF3 in transepithelial transport remains poorly understood. Multidrug resistance protein 4 (MRP4) is an ATP binding cassette transporter that mediates the efflux of organic molecules, such as nucleoside analogs, in the gastrointestinal and renal epithelia. Here, we report that Nherf3 knockout (Nherf3(-/-)) mice exhibit profound reductions in Mrp4 expression and Mrp4-mediated drug transport in the kidney. A search for the binding partners of the COOH-terminal PDZ binding motif of MRP4 among several epithelial PDZ proteins indicated that MRP4 associated most strongly with NHERF3. When expressed in HEK293 cells, NHERF3 increased membrane expression of MRP4 by reducing internalization of cell surface MRP4 and consequently, augmented MRP4-mediated efflux of adefovir, a nucleoside-based antiviral agent and well known substrate of MRP4. Examination of wild-type and Nherf3(-/-) mice revealed that Nherf3 is most abundantly expressed in the kidney and has a prominent role in modulating Mrp4 levels. Deletion of Nherf3 in mice caused a profound reduction in Mrp4 expression at the apical membrane of renal proximal tubules and evoked a significant increase in the plasma and kidney concentrations of adefovir, with a corresponding decrease in the systemic clearance of this drug. These results suggest that NHERF3 is a key regulator of organic transport in the kidney, particularly MRP4-mediated clearance of drug molecules.

Since P-glycoprotein (P-gp) acts as a barrier to intestinal absorption of various drugs, P-gp inhibitors have been studied as oral absorption enhancers of P-gp substrate drugs. Here, we investigated the in vitro and in vivo effects of a novel coumarin derivative (LL-348) for its P-gp inhibitory activity. With LL-348, accumulation of daunomycin (DNM) increased 270% and efflux of DNM decreased 63% compared to that of DNM alone. Paclitaxel (PTX, 25mg/kg) after oral administration with LL-348 (5mg/kg), the optimal dose of LL-348 as an oral absorption enhancer of PTX, improved the relative bioavailability (RB) of PTX to 961%. In a xenograft animal model, PTX (40mg/kg) treated with LL-348 (10mg/kg) significantly increased the efficacy of PTX. The results collectively demonstrate that LL-348 can provide a therapeutic benefit in the oral absorption of P-gp substrate anticancer drugs.

The expression of P-glycoprotein (P-gp), an ATP-dependent efflux transporter, is closely associated with the failure of chemotherapy and drug absorption. Two synthesized optically active phenylbutenoid dimers, 3S-(3,4-dimethoxyphenyl)-4R-{(E)-3,4-dimethoxystyryl}cyclohex-1-ene (1) and 3R-(3,4-dimethoxyphenyl)-4S-{(E)-3,4-dimethoxystyryl}cyclohex-1-ene (2), were tested for their P-gp inhibitory effects by measuring cellular accumulation and efflux of daunomycin in P-gp-overexpressed human breast cancer cells (MCF-7/ADR). Compound 2 significantly increased the accumulation of daunomycin (539%) and decreased the efflux of this compound (55.4%), and similar results were observed for 1. ATPase assays and Western blot analysis were performed to identify the mechanisms by which compounds 1 and 2 inhibit P-gp. In addition, changes in the pharmacokinetic profile of paclitaxel coadministered with 2 in rats were evaluated. Paclitaxel (25 mg/kg) when orally administered with 2 (5 mg/kg) improved its relative bioavailability by 185%. Compound 2 effectively improved cellular accumulation by reducing the efflux of daunomycin and significantly enhanced oral exposure to paclitaxel. Therefore, compound 2 may be useful for improving oral exposure and cellular availability of drugs that are also substrates of P-gp.

Nonsteroidal anti-inflammatory drugs (NSAIDs), which are globally prescribed, exhibit mainly anti-inflammatory and analgesic effects but also can cause adverse effects including gastrointestinal erosions, ulceration, bleeding, and perforation. The purpose of this study was to investigate surrogate biomarkers associated with the gastrointestinal (GI) damage caused by NSAID treatment using pattern recognition analysis of (1)H-nuclear magnetic resonance ((1)H NMR) spectra of rat urine. Urine was collected for 5h after oral administration of the following NSAIDs at low or high doses: acetylsalicylic acid (10 or 200mgkg(-1)), diclofenac (0.5 or 15mgkg(-1)), piroxicam (1 or 10mgkg(-1)), indomethacin (1 or 25mgkg(-1)), or ibuprofen (10, or 150mgkg(-1)) as nonselective COX inhibitors and celecoxib (10 or 100mgkg(-1)) as a COX-2 selective inhibitor. The urine was analyzed using 500MHz (1)H NMR for spectral binning and targeted profiling and the level of gastric damage was examined. The nonselective COX inhibitors caused severe gastric damage while no lesions were observed in the celecoxib-treated rats. The (1)H NMR urine spectra were divided into spectral bins (0.04ppm) for global profiling, and a total of 44 endogenous metabolites were assigned for targeted profiling. Multivariate data analyses were performed to recognize the spectral pattern of endogenous metabolites related to NSAIDs using partial least square-discrimination analysis (PLS-DA). The (1)H NMR spectra clustered differently according to gastric damage score in global profiling. In targeted profiling, the endogenous metabolites of citrate, allantoin, 2-oxoglutarate, acetate, benzoate, glycine, and trimethylamine N-oxide were selected as putative biomarkers for gastric damage caused by NSAIDs. These putative biomarkers might be useful for predicting the risk of adverse effects caused by NSAIDs in the early stage of drug development process.

Osteoclast cells (OCs) are differentiated from bone marrow-derived macrophages (BMMs) by activation of receptor activator of nuclear factor κB (NF-κB) ligand (RANKL). Activation of NADPH oxidase (Nox) isozymes is involved in RANKL-dependent OC differentiation, implicating Nox isozymes as therapeutic targets for treatment of osteoporosis. Here, we show that a novel pyrazole derivative, Ewha-18278 has high inhibitory potency on Nox isozymes. Blocking the activity of Nox with Ewha-18278 inhibited the responses of BMMs to RANKL, including reactive oxygen species (ROS) generation, activation of mitogen-activated protein (MAP) kinases and NF-κB, and OC differentiation. To evaluate the anti-osteoporotic function of Ewha-18278, the derivative was applied to estrogen-deficient ovariectomized (OVX) ddY mice. Oral administration of Ewha-18278 (10 mg/kg/daily, 4 weeks) into the mice recovered bone mineral density, trabecular bone volume, trabecular bone length, number and thickness, compared to control OVX ddY mice. Moreover, treatment of OVX ddY mice with Ewha-18278 increased bone strength by increasing cortical bone thickness. We provide that Ewha-18278 displayed Nox inhibition and blocked the RANKL-dependent cell signaling cascade leading to reduced differentiation of OCs. Our results implicate Ewha-18278 as a novel therapeutic agent for the treatment of osteoporosis.

Prakash G Avaji,1,2,* Jung Hyun Park,1,* Hyun Jeong Lee,1 Yong Joo Jun,2 Kyung Su Park,3 Kyung Eun Lee,3 Soo-Jin Choi,4 Hwa Jeong Lee,1 Youn Soo Sohn2 1Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul, Republic of Korea; 2C & Pharm, Ewha Womans University, Seoul, Republic of Korea; 3Advanced Analysis Center, Korea Institute of Science and Technology, Seoul, Republic of Korea; 4Department of Food Science and Technology, Seoul Women’s University, Seoul, Republic of Korea *These authors contributed equally tothis work Abstract: To develop a theranostic nanomedicine involving the antitumor-active moiety (dach)Pt(II) (dach: trans-(±)-1,2-diaminocyclohexane) of oxaliplatin (OX), a new biocompatible polyphosphazene carrier polymer was designed by grafting with a methoxy poly(ethylene glycol) (MPEG) to increase duration of circulation in the blood and with aminoethanol (AE) as a spacer group. The antitumor (dach)Pt moiety was conjugated to the carrier polymer using cis-aconitic acid (AA) as a linker, resulting in a polymer conjugate formulated as [NP(MPEG550)(AE-AA)Pt(dach)]n, named “Polyplatin” (PP). PP was found to self-assemble into very stable polymeric nanoparticles with a mean diameter of 55.1 nm and a critical aggregation concentration of 18.5 mg/L in saline. PP could easily be labeled with a fluorescence dye such as Cy5.5 for imaging studies. The time-dependent ex vivo image studies on organ distributions and clearance of Cy-labeled PP have shown that PP accumulated in the tumor with high selectivity by the enhanced permeability and retention effect but was cleared out from all the major organs including the liver in about 4 weeks postinjection. Another time-dependent bioimaging study on distribution and clearance of PP in mouse kidney using laser ablation inductively coupled plasma mass spectroscopy has shown that PP accumulates much less in kidney and is more rapidly excreted than monomeric OX, which is in accord with the very low acute toxicity of PP as shown by its high LD50 value of more than 2000 mg/kg. The pharmacokinetic study of PP has shown that it has a much longer half-life (t1/2β) of 13.3 hours compared with the 5.21 hours of OX and about a 20 times higher area under the curve value of 42,850.8 ng h/mL compared with the 2,320.4 ng h/mL of OX. The nude mouse xenograft trials of PP against the gastric MKN-28 tumor cell line exhibited remarkably better tumor efficacy compared with OX at the higher tolerated dose, with lower systemic toxicity. Keywords: polyphosphazene, anticancer agent, theranostics, nanomedicine, oxaliplatin

Cancer cells have been suggested to be more susceptible to oxidative damages and highly dependent on antioxidant capacity in comparison with normal cells, and thus targeting antioxidant enzymes has been a strategy for effective cancer treatment. Sulfiredoxin (Srx) is an enzyme that catalyzes the reduction of sulfinylated peroxiredoxins and thereby reactivates them. In this study we developed a Srx inhibitor, K27 (N-[7-chloro-2-(4-fluorophenyl)-4-quinazolinyl]-N-(2-phenylethyl)-β-alanine), and showed that it induces the accumulation of sulfinylated peroxiredoxins and oxidative stress, which leads to mitochondrial damage and apoptotic death of cancer cells. The effects of K27 were significantly reversed by ectopic expression of Srx or antioxidant N-acetyl cysteine. In addition, K27 led to preferential death of tumorigenic cells over non-tumorigenic cells, and suppressed the growth of xenograft tumor without acute toxicity. Our results suggest that targeting Srx might be an effective therapeutic strategy for cancer treatment through redox-mediated cell death.

Cancer gene therapy involves the replacement of missing or altered genes with healthy ones. In this paper, we have proposed tumor suppressor gene-carrying superparamagnetic iron oxide nanoparticles (SPIONs) for anti-cancer gene therapy. Thermally crosslinked SPIONs (TCL-SPIONs) were conjugated with branched polyethylenimine (PEI 1800 Da) by EDC-NHS chemistry for p53 plasmid DNA delivery. The morphology of the bPEI conjugated TCL-SPIONs (bPEI-TCL-SPION) and pDNA-loaded bPEI-TCL-SPION nanoparticles was measured using transmission electron microscopy (TEM). The particle sizes of the pDNA-loaded bPEI-TCL-SPION nanoparticles were also confirmed by dynamic light scattering, and ranged from 100 to 130 nm, depending on the molar charge ratio. The fluorescently labeled pDNA was complexed with bPEI-TCL-SPION and its intracellular internalization was investigated using confocal microscopy. The p53 plasmid-loaded bPEI-TCL-SPION nanoparticles achieved significantly higher p53 tumor suppressor gene expression and cellular viability compared to positive controls. The expressed wild-type p53 protein suppressed tumor cell proliferation as compared to the mutant control. When transgene expression of the p53 tumor suppressor gene was evaluated at the mRNA level and quantified using real-time PCR, the results were highly dependent on the molar charge ratio (N/P) as well as the cancer cell type. SPIONs internalized within cancer cells were tracked by magnetic resonance (MR) imaging. It was concluded that bPEI-TCL-SPION could be used as efficient gene delivery carriers that can be tracked by MR imaging.

Non-steroidal anti-inflammatory drugs (NSAIDs) have side effects including gastric erosions, ulceration and bleeding. In this study, pattern recognition analysis of the 1 H-nuclear magnetic resonance (NMR) spectra of urine was performed to develop surrogate biomarkers related to the gastrointestinal (GI) damage induced by indomethacin in rats. Urine was collected for 5 h after oral administration of indomethacin (25 mg kg −1 ) or co-administration with cimetidine (100 mg kg −1 ), which protects against GI damage. The 1 H-NMR urine spectra were divided into spectral bins (0.04 ppm) for global profiling, and 36 endogenous metabolites were assigned for targeted profiling. The level of gastric damage in each animal was also determined. Indomethacin caused severe gastric damage; however, indomethacin administered with cimetidine did not. Simultaneously, the patterns of changes in their endogenous metabolites were different. Multivariate data analyses were carried out to recognize the spectral pattern of endogenous metabolites related to indomethacin using partial least square-discrimination analysis. In targeted profiling, a few endogenous metabolites, 2-oxoglutarate, acetate, taurine and hippurate, were selected as putative biomarkers for the gastric damage induced by indomethacin. These metabolites changed depending on the degree of GI damage, although the same dose of indomethacin (10 mg kg −1 ) was administered to rats. The results of global and targeted profiling suggest that the gastric damage induced by NSAIDs can be screened in the preclinical stage of drug development using a NMR based metabolomics approach.

The aims of the present study were to investigate the effects of silymarin, an inhibitor of the P-glycoprotein efflux pump, on oral bioavailability of paclitaxel in rats, and to compare pharmacokinetic parameters of paclitaxel between a commercial formulation of paclitaxel (Taxol®) and a paclitaxel microemulsion. Oral bioavailability of paclitaxel in a Taxol® formulation was enhanced in the combination with silymarin (10 and 20mg/kg). In particular, the mean maximum plasma concentration (C(max)) and the mean area under the plasma concentration-time curve (AUC(0-)(t)) of paclitaxel in the Taxol® formulation were significantly increased 3-fold and 5-fold compared with control, respectively, following oral co-administration with 10mg/kg of silymarin (p

Daumone, 6-(3,5-dihydroxy-6-methyl-tetrahydro-pyran-2-yloxy)-heptanoic acid is a pheromone secreted by Caenorhabditis elegans, and has been known as a pivotal regulator of chemosensory processes in development and ageing. A quantification method using mass spectrometry was developed for the determination of daumone in rat plasma. After simple protein precipitation with acetonitrile including an internal standard, the analytes were chromatographed on a reversed-phase column and detected by liquid chromatography/tandem mass spectrometry with electrospray ionization. The accuracy and precision of the assay were in accordance with FDA regulations for validation of bioanalytical methods. This method was applied to measure the plasma daumone concentrations after a single intravenous administration of daumone in rats.

For the quantification of azasetron in rat plasma samples, a column-switching HPLC method was developed and validated. Following dilution of plasma samples with mobile phase A (17 mM potassium phosphate buffer (pH 3.0)) and simple protein precipitation by addition of perchloric acid (60%), the mixture was directly injected onto the pre-column. After endogenous plasma substances were eluted to waste, the analyte was transferred to the trap column by switching the system. Then, the analyte was back-flushed to the analytical column for separation with mobile phase B (a 22:78 v/v mixture of acetonitrile and 17 mM potassium phosphate buffer (pH 3.0)) and detected at 250 nm using a photodiode array detector. A linear standard curve was obtained in the concentration range of 10-800 ng/mL with the correlation coefficient (r) of 0.9998. The intra- and inter-day precision and accuracy values for azasetron were in the ranges of 0.3-12.9% and 89.7-101.4%, respectively. The method was valid in terms of specificity, precision, and accuracy. In addition, this efficient analytical method was successfully applied to determine plasma concentrations of azasetron following oral administration of azasetron at a dose of 4.0 mg/kg to rats.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used to treat rheumatoid arthritis, osteoarthritis, acute pain, and fever. However, NSAIDs have side effects that include gastric erosions, ulceration, bleeding, and perforation, etc. Selective cyclooxygenase (COX)-2 inhibitors have been developed to avoid the adverse drug reaction of traditional NSAIDs. The COX-2 inhibitors have a different mechanism of action from nonselective COX inhibitors. In this study, pattern recognition analysis of the (1)H nuclear magnetic resonance (NMR) spectra of urine was performed to develop surrogate biomarkers related to the gastrointestinal (GI) damage induced by NSAIDs in rats. Urine was collected for 5 h after administering the following NSAIDs at high doses: celecoxib (133 mg kg(-1), p.o.), a COX-2-selective inhibitor; and indomethacin (25 mg kg(-1), p.o.) or ibuprofen (800 mg kg(-1), p.o.), nonselective COX inhibitors. The urine was analyzed using 600 M (1)H NMR for spectral binning and targeted profiling. The level of gastric damage in each animal was also determined. Indomethacin and ibuprofen caused severe gastric damage, but no lesions were observed in the celecoxib-treated rats. The (1)H NMR urine spectra were divided into spectral bins (0.04 ppm) for global profiling, and 36 endogenous metabolites were assigned for targeted profiling. Multivariate data analyses were carried out to recognize the spectral pattern of endogenous metabolites related to NSAIDs using partial least-squares discrimination analysis (PLS-DA). There were different clusterings of (1)H NMR spectra according to the gastric damage scores in global profiling. In targeted profiling, a few endogenous metabolites of allantoin, taurine, and dimethylamine were selected as putative biomarkers for the gastric damage induced by NSAIDs. The results of global and targeted profilings suggest that the gastric damage induced by NSAIDs can be screened in the preclinical stage of drug development using a current metabolomics study. In addition, the putative biomarkers might also be useful for predicting the risk of adverse effects caused by NSAIDs.

Citral is a major compound of lemongrass (Cymbopogon citratus L.) that has many pharmacological activities such as anti-fungal and anti-bacterial effects. In this study, we investigated the anti-inflammatory effect of citral and defined its mechanism of action in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Citral (3-12 microg/mL) significantly inhibited LPS-induced nitric oxide (NO) production in a concentration-dependent manner (IC50: 6.5 microg/mL). Furthermore, it was found that citral effectively inhibited the transcriptional activity and expression of iNOS, and potently suppressed the DNA binding activity and nuclear translocation of NF-kappa B as well as I kappa B phosphorylation in a concentration dependent manner. These results suggest that citral is anti-inflammatory, and its effects may be due to the inhibition of NO production through the suppression of NF-kappa B activation.

Nanofibers are one-dimensional nanomaterial in fiber form with diameter less than 1 µm and an aspect ratio (length/diameter) larger than 100:1. Among the different types of nanoparticle-loaded nanofiber systems, nanofibers loaded with magnetic nanoparticles have gained much attention from biomedical scientists due to a synergistic effect obtained from the unique properties of both the nanofibers and magnetic nanoparticles. These magnetic nanoparticle-encapsulated or -embedded nanofiber systems can be used not only for imaging purposes but also for therapy. In this review, we focused on recent advances in nanofibers loaded with magnetic nanoparticles, their biomedical applications, and future trends in the application of these nanofibers.

Nitric oxide (NO) inactivates iron-sulfur enzymes in bacterial amino acid biosynthetic pathways, causing amino acid auxotrophy. We demonstrate that exogenous supplementation with branched-chain amino acids (BCAA) can restore the NO resistance of hmp mutant Salmonella Typhimurium lacking principal NO-metabolizing enzyme flavohemoglobin, and of mutants further lacking iron-sulfur enzymes dihydroxy-acid dehydratase (IlvD) and isopropylmalate isomerase (LeuCD) that are essential for BCAA biosynthesis, in an oxygen-dependent manner. BCAA supplementation did not affect the NO consumption rate of S. Typhimurium, suggesting the BCAA-promoted NO resistance independent of NO metabolism. BCAA supplementation also induced intracellular survival of ilvD and leuCD mutants at wild-type levels inside RAW 264.7 macrophages that produce constant amounts of NO regardless of varied supplemental BCAA concentrations. Our results suggest that the NO-induced BCAA auxotrophy of Salmonella, due to inactivation of iron-sulfur enzymes for BCAA biosynthesis, could be rescued by bacterial taking up exogenous BCAA available in oxic environments.

A column-switching high-performance liquid chromatographic (HPLC) method has been developed and validated for quantification of fluvastatin in rat plasma. Plasma samples were diluted with an equal volume of mobile phase, i.e. acetonitrile–5 mM potassium phosphate buffer (pH 6.8) (15:85, v/v), and the mixture was directly injected onto the HPLC system. The analyte was enriched in a pre-treatment column, while endogenous components were eluted to waste. The analyte was then back-flushed onto an analytical column and quantified with fluorescence detection (λex = 305 nm; λem = 390 nm). The standard curve for the drug was linear in the range 0.5–100 ng mL−1 in rat plasma. The limit of quantitation for plasma was found to be 0.5 ng mL−1. This method has been fully validated and shown to be specific, accurate and precise. The method is simple and rapid because of a minimized sample preparation and appears to be useful for the pharmacokinetic study of fluvastatin.

Gender differences have been well described in pharmacokinetics and contribute to the interindividual variation in drug disposition, therapeutic response, and drug toxicity. Sex-related differences in the membrane transport of endogenous substrates and xenobiotics have been reported in various organs of the body including kidney, liver, intestine, and brain. These gender-related differences in transport systems could also contribute to interindividual variability in pharmacokinetics and pharmacodynamics. This review will focus on current knowledge of gender-associated differences in the transport of endogenous and exogenous compounds in a variety of body organs and will discuss the implications and the clinical significance of these observations.

The 528 murine monoclonal antibody (MAb) to the human epidermal growth factor receptor (EGFR) was sequentially cationized with hexamethylenediamine and conjugated with diethylenetriaminepentaacetic acid (DTPA) as a potential antibody radiopharmaceutical for imaging EGFR-expressing cancer. The cationized 528 MAb was characterized with isoelectric focusing and electrophoresis, and an immunoradiometric assay, which showed the affinity of the 528 MAb for the human EGFR was retained following cationization. The native or cationized 528 MAb, labeled with (111)In, was injected intravenously in scid mice bearing human U87 flank tumors, which express the EGFR, and tumor imaging was performed with both external detection in live animals and with whole body autoradiography. However, the tumor signal was not increased with the cationized MAb, relative to the native MAb, and this was due to a serum inhibition phenomenon that was confirmed by a pharmacokinetics analysis in control mice. In an attempt to block the serum inhibition, the cationized 528 MAb was pegylated with 2000 Da poly(ethylene glycol), and the cationized/pegylated MAb was conjugated with DTPA and labeled with (111)In. However, a pharmacokinetics analysis showed the pegylation did not reverse the serum inhibition of the cationic charge on the MAb. These studies describe methods for reformulating monoclonal antibodies to develop improved radiopharmaceuticals, but show that radiolabeling a cationized MAb with DTPA produces a serum neutralization of the initial cationization modification.

Aβ1–40is a potential peptide radiopharmaceutical that could be used to image the brain Aβ amyloid of Alzheimer disease in vivo, should this peptide be made transportable through the blood–brain barrier in vivo. The blood–brain barrier transport of [125I]-Aβ1–40in a transgenic mouse model was enabled by conjugation to the rat 8D3 monoclonal antibody to the mouse transferrin receptor. The Aβ1–40–8D3 conjugate is a bifunctional molecule that binds the blood–brain barrier TfR and undergoes transport into brain and binds the Aβ amyloid plaques of Alzheimer disease. AppSW/ Psen1 double-transgenic and littermate control mice were administered either unconjugated Aβ1–40or the Aβ1–40–8D3 conjugate intravenously, and brain scans were obtained 6 hours later. Immunocytochemical analysis showed abundant Aβ immunoreactive plaques in the brains of the AppSW/ Psen1 transgenic mice and there was a selective retention of radioactivity in the brains of these mice at 6 hours after intravenous administration of the conjugate. In contrast, there was no selective sequestration either of the conjugate in control littermate mouse brain or of unconjugated Aβ1–40in transgenic mouse brain. In conclusion, the results show that it is possible to image the Aβ amyloid burden in the brain in vivo with an amyloid imaging agent, provided the molecule is conjugated to a blood–brain barrier drug-targeting system.

Non ionic, amphipathic molecules form vesicles and this property correlates with the disruption of membranes. In the present studies, high mM concentrations of aliphatic alcohols, 1-O-hexyldiglycerol (HDG) and 1-O-heptyltriglycerol (HTG), are shown to cause enhanced drug transport into brain via disruption of the blood-brain barrier (BBB) in vivo, as determined with an internal carotid artery perfusion method. The intravenous administration of comparable concentrations of HDG or HTG caused no increase in BBB transport of drug. The enhanced transport of drug showed a dependency on molecular weight as 45 mM HTG increased the transport of sucrose, 360 Da, but did not increase the transport of arginine vasopressin (AVP), 1084 Da, although AVP transport across the BBB was increased by 80 mM HDG or HTG. Quasielastic light scattering measurements provided evidence for the formation of vesicular structures in aqueous solutions containing high mM concentrations of the HDG or HTG. In summary, these studies demonstrate BBB disruption following the internal carotid arterial infusion of high mM concentrations of membrane active alkyl glycerols.

MicroRNAs (miRNA) are short oligonucleotides of endogenous origin involved in post-transcriptional regulation and are altered in disease, making them potential therapeutic targets. miRNA replacement is necessary in cells with downregulated miRNAs levels in response to disease. miRNA 145 is a novel tumor suppressor gene involved in cell suppression, invasion and migration of cancer cells; it is downregulated in most cancers. Delivery of therapeutic miRNA using nanoparticles enhances the chances of successful delivery and expression of genes at the target site. We evaluated polysorbitol-mediated transporter (PSMT) in the cellular delivery of miRNA 145. The polysorbitol backbone possesses osmotic properties and leads to enhanced cellular uptake. PSMT delivers genes into cells by a caveolae-mediated endocytic pathway. Caveolae expression is usually altered in transformed cancer cells. Physicochemical characterization, and the transfection efficiency and transgene expression capability of PSMT/reporter plasmid DNA nanoparticles, were determined. GFP-tagged miRNA 145 delivery with PSMT was confirmed by confocal microscopy and Western blotting. The functional effects of miRNA 145 delivered with PSMT were analyzed by confocal microscopy, as well as in apoptosis, proliferation and wound healing assays. Finally, the expression of an miRNA 145 target protein, c-myc, was determined by Western blotting after intracellular delivery of PSMT/miRNA 145 nanoparticle (NP).

Intestinal P-glycoprotein (P-gp) is a limiting step for oral absorption of drugs. Therefore, P-gp inhibitors have been studied as enhancers of oral absorption of drugs that are P-gp substrates. We investigated the in vitro and in vivo P-gp inhibitory activity of synthesized xanthone analogues. With 3-(3-chloro-2-hydroxypropoxy)-1-hydroxy-9H-thioxanthen-9-one, compound 13, accumulation of daunomycin (DNM) increased 707% and efflux of DNM decreased 66% compared to DNM alone. Relative bioavailability (RB) of paclitaxel (PTX, 25 mg/kg) increased 2.5-fold after oral administration with 13 (5 mg/kg). In a xenograft animal model, oral administration of PTX (40 mg/kg) with 13 (10 mg/kg) significantly inhibited tumour growth and was more effective than intravenously administered PTX (10 mg/kg) alone. Therefore, the synthesized xanthone analogue 13 might have therapeutic benefits for oral absorption of P-gp substrate anticancer drugs.

Aiming at tumor targeting delivery of oxaliplatin using polymer therapy, a new monomeric platinum(II) complex (dach)Pt[HEDM] (dach: trans-(±)-1,2-diaminocyclohexane; HEDM: 2-hydroxyethoxydiethylmalate) was designed to include the antitumor moiety (dach)Pt and HEDM as a linker to the polyphosphazene backbone. This monomeric Pt-complex could easily be grafted to the PEGylated polyphosphazene backbone to prepare a novel polyphosphazene-Pt conjugate, [NP(MPEG550)(dach)Pt(EM)]n [MPEG550: methoxy poly(ethylene glycol) with an average molecular weight of 550; EM: ethoxymalate]. This amphiphilic polyphosphazene-Pt conjugate was found to self-assemble into stable polymeric micelles of a mean diameter of 130nm, which is suitable for passive tumor targeting by enhanced permeability and retention (EPR) effect. Pharmacokinetic study of this polymer conjugate exhibited long blood circulation as expected and longer half-life (t1/2β=9.52h) compared with oxaliplatin (3.47h), and much larger AUC (area under the curve) value (25,831ng·h/mL) compared with oxaliplatin (1194ng·h/mL). Biodistribution study of the polymer conjugate has shown excellent tumor selectivity with the tumor to tissue ratio of 3.84 at 2h post injection and 11.7 at 24h post injection probably due to the EPR effect of the polymer conjugate while no tumor selectivity was observed for monomeric oxaliplatin. Furthermore, accumulation of this polymer conjugate in kidney was much lower compared with oxaliplatin. Also the nude mouse xenograft trial of the polymer conjugate has shown higher antitumor efficacy compared with oxaliplatin.

A beta1-40 peptide radiopharmaceuticals could be used to image A beta brain amyloid in transgenic mouse models of Alzheimer's disease should the A beta peptide radiopharmaceutical be made transportable through the blood-brain barrier (BBB) in vivo. The present studies used the RI7-217 rat monoclonal antibody to the mouse transferrin receptor as a BBB drug targeting vector for the delivery to brain of A beta1-40 radiolabeled with either 125-Iodine or 111-Indium. The A beta peptide radiopharmaceutical is conjugated to the RI7 MAb using avidin biotin technology, wherein the A beta1-40 peptide radiopharmaceutical is monobiotinylated (bio) and bound to a conjugate of the RI7 MAb and streptavidin (SA). The [125 I]-bio-A beta1-40 or the [111 In]-bio-A beta1-40 either free or bound to the RI7/SA conjugate was injected intravenously into anesthetized adult mice and plasma pharmacokinetics and organ uptake were measured over the next 60 minutes. The A beta1-40 peptide radiopharmaceutical radiolabeled with 111-Indium was the preferred formulation, compared to peptide labeled with 125-Iodine, because there was a greater metabolic stability and reduced artifactual organ uptake of metabolites associated with the use of the 111-Indium nuclide. However, biotinylated A beta1-40 peptide radiopharmaceuticals conjugated to the RI7/SA brain drug targeting system were metabolically unstable in mice in vivo owing to active biotinidase activity. Future work involving brain drug targeting in mice that utilizes avidin biotin technology will need to incorporate biotin analogues that are resistant to biotinidase.

Changes in membrane fluidity have been shown to alter the sodium-dependent renal transport of glucose and phosphate; however, this has not been examined for sodium/sulfate cotransport in the renal proximal tubule. Sodium/sulfate cotransport regulates the homeostasis of sulfate in mammals. The objective of this study was to investigate the influence of alterations of membrane fluidity on sodium-coupled sulfate transport in the Madin-Darby canine kidney cells, which have been stably transfected with sodium/sulfate cotransporter (NaSi-1) cDNA (MDCK-Si). Preincubation of cells with 0. 2 mM cholesterol significantly decreased the V(max) for sodium/sulfate cotransport (13.69 +/- 1.11 vs 10.15 +/- 1.17 nmol/mg protein/5 min, mean +/- SD, n = 4, p0.01) with no significant alteration in K(m). The addition of benzyl alcohol (20 mM) to cells increased the V(max) of sulfate uptake by 20% (11.97 +/- 0.91 vs 14. 35 +/- 0.56 nmol/mg protein/5 min, mean +/- SD, n = 3, p0.05) with no significant change in K(m). Membrane fluidity, as measured by the fluorescence polarization of 1,6-diphenyl 1,3,5-hexatriene (DPH), was significantly increased in MDCK-Si cells treated with 20 mM benzyl alcohol and decreased in the cells preincubated with 0.2 mM cholesterol, compared with control cells. Our results suggest that alterations in membrane fluidity that may occur as a result of disease states, aging, and pregnancy may play an important role in the modulation of renal sodium/sulfate cotransport.

We recently reported that overexpression of thymosin beta-4 (Tβ4) in transgenic mice promotes abnormal hair growth and tooth development, but the role of Tβ4 in dental pulp regeneration was not completely understood. The aim of this study was to investigate the role of Tβ4 on odontoblastic differentiation and the underlying mechanism regulating pulp regeneration in human dental pulp cells (HDPCs). Our results demonstrate that mRNA and protein expression of Tβ4 is upregulated during odontogenic differentiation in HDPCs. Transfection with Tβ4 siRNA decreases OM-induced odontoblastic differentiation by decreasing alkaline phosphatase (ALP) activity, mRNA expression of differentiation markers, and calcium nodule formation. In contrast, Tβ4 activation with a Tβ4 peptide promotes these processes by enhancing the phosphorylation of p38, JNK, and ERK mitogen-activated protein kinases (MAPKs), bone morphogenetic protein (BMP) 2, BMP4, phosphorylation of Smad1/5/8 and Smad2/3, and expression of transcriptional factors such as Runx2 and Osterix, which were blocked by the BMP inhibitor noggin. The expression of integrin receptors α1, α2, α3, and β1 and downstream signaling molecules including phosphorylated focal adhesion kinase (p-FAK), p-paxillin, and integrin-linked kinase (ILK) were increased by Tβ4 peptide in HDPCs. ILK siRNA blocked Tβ4-induced odontoblastic differentiation and activation of the BMP and MAPK transcription factor pathways in HDPCs. In conclusion, this study demonstrates for the first time that Tβ4 plays a key role in odontoblastic differentiation of HDPCs and activation of Tβ4 could provide a novel mechanism for regenerative endodontics.

1. The interactions between herbal dietary supplements and therapeutic drugs have emerged as an important issue and P-glycoprotein (P-gp) has been reported as one of the significant factors of these interactions. 2. The objective of this article is to examine the effects of single and repeated administrations of silymarin on pharmacokinetics of a P-gp substrate, risperidone, and its major metabolite, 9-hydroxyrisperidone, in rats. 3. To determine the plasma levels of risperidone and 9-hydroxyrisperidone in rats, a HPLC method was developed using a liquid-liquid acid back extraction. When risperidone (6 mg/kg) was co-administered with silymarin (40 mg/kg) to rats orally, the C(max) of 9-hydroxyrisperidone was significantly increased to1.3-fold (p0.05), while the other pharmacokinetic parameters did not show any significant differences. Expanding the experiment where rats were repeatedly administered with silymarin for 5 days prior to giving risperidone, the C(max) of risperidone and 9-hydroxyrisperidone were significantly increased to 2.4-fold (p0.001) and 1.7-fold (p0.001), respectively, and the AUC(0-t), as well to 1.7-fold (p0.05) and 2.1-fold (p0.01), respectively. 4. The repeated exposures of silymarin, compared to single administration of silymarin, increased oral bioavailability and affected the pharmacokinetics of risperidone and 9-hydroxyrisperidone, by inhibiting P-gp.

Poor aqueous solubility and the unpleasant taste of aripiprazole (APZ) have been recurring problems, owing to its low bioavailability and low patient tolerance, respectively. Herein, we prepared a nanohybrid system that was based on a bentonite clay material, montmorillonite (MMT), which could both mask the taste and enhance the solubility of APZ (i.e., APZ-MMT). To further improve the efficacy of this taste masking and drug solubility, APZ-MMT was also coated with a cationic polymer, polyvinylacetal diethylamino acetate (AEA). In vitro dissolution tests at neutral pH showed that the amount of drug that was released from the AEA-coated APZ-MMT was greatly suppressed (

Ji-Hee Lee1,*, Goeun Choi1,*, Yeon-Ji Oh1, Je Won Park1, Young Bin Choy3, Mung Chul Park1, Yeo Joon Yoon1, Hwa Jeong Lee2, Hee Chul Chang4, Jin-Ho Choy1 1Center for Intelligent Nano-Bio Materials (CINBM), Department of Bioinspired Science and Department of Chemistry and Nano Science, 2Division of Life and Pharmaceutical Sciences and College of Pharmacy, Ewha Womans University, Seoul, Korea; 3Department of Biomedical Engineering, College of Medicine and Institute of Medical and Biological Engineering, Medical Research Center, Seoul National University, Seoul, Korea; 4Global Strategy Center and Pharmaceutical Research Institute, Daewoong Pharmaceutical Co., Ltd., Seoul, Korea*These authors contributed equally to this workAbstract: A nanohybrid was prepared with an inorganic clay material, montmorillonite (MMT), for taste masking of sildenafil (SDN). To further improve the taste-masking efficiency and enhance the drug-release rate, we coated the nanohybrid of SDN–MMT with a basic polymer, polyvinylacetal diethylaminoacetate (AEA). Powder X-ray diffraction and Fourier transform infrared experiments showed that SDN was successfully intercalated into the interlayer space of MMT. The AEA-coated SDN–MMT nanohybrid showed drug release was much suppressed at neutral pH (release rate, 4.70 ± 0.53%), suggesting a potential for drug taste masking at the buccal cavity. We also performed in vitro drug release experiments in a simulated gastric fluid (pH = 1.2) and compared the drug-release profiles of AEA-coated SDN–MMT and Viagra®, an approved dosage form of SDN. As a result, about 90% of SDN was released from the AEA-coated SDN–MMT during the first 2 hours while almost 100% of drug was released from Viagra®. However, an in vivo experiment showed that the AEA-coated SDN–MMT exhibited higher drug exposure than Viagra®. For the AEA-coated SDN–MMT, the area under the plasma concentration–time curve from 0 hours to infinity (AUC0-∞) and maximum concentration (Cmax) were 78.8 ± 2.32 µg • hour/mL and 12.4 ± 0.673 µg/mL, respectively, both of which were larger than those obtained with Viagra® (AUC0-∞ = 69.2 ± 3.19 µg • hour/mL; Cmax = 10.5 ± 0.641 µg/mL). Therefore, we concluded that the MMT-based nanohybrid is a promising delivery system for taste masking of SDN with possibly improved drug exposure.Keywords: montmorillonite, nanohybrids, polyvinylacetal diethylaminoacetate, sildenafil citrate, taste masking

To evaluate the metabolic interaction between host and gut microflora on drug metabolism, pseudo germ-free rats were prepared with an antibiotics cocktail to change their gut conditions. The usefulness of the pseudo germ-free model was evaluated for observing the DMPK of acetaminophen (APAP). Pseudo germ-free rats were prepared by orally administering antibiotic cocktails consisting of bacitracin, streptomycin and neomycin, and then APAP was orally administered to control and pseudo germ-free rats. The plasma concentration of APAP and its six metabolites were quantified using a validated LC-MS/MS method. A non-compartment model estimated the pharmacokinetic parameters of APAP and its metabolites, and the ratios of the area under curve (AUC; AUC(metabolite) /AUC(APAP) ) were also observed to evaluate the change of APAP metabolism. The AUCs of APAP and APAP-Glth (glutathione) were higher and the AUC(APAP-Sul) /AUC(APAP) (metabolic efficiency of sulfate conjugation) was lower in pseudo germ-free rats than those in the control rats. The decrease in metabolic efficiency of sulphate conjugation could result from the reduction of the sulphate supply, causing an increase of the AUC of APAP and APAP-Glth. The activities of gut microflora can affect the state of hepatic sulphate for drug conjugation, indirectly leading to characteristic APAP metabolism. These results indicate that gut microflora may play an important role in the pharmacokinetics and metabolism of APAP. Thus, the metabolic interaction between host and gut microflora should be considered upon drug administration and pseudo germ-free rats prepared in the present study can be competent for investigating the metabolic interaction between host and gut microflora on drug metabolism.

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug. It is mainly metabolized by phase 1 and 2 reactions in the liver, and thus it could be involved in many drug-drug interactions. Therefore, the study of APAP metabolism is important in toxicological and pharmacokinetic studies. The objective of this study was to develop a rapid and sensitive method for the determination of APAP and its six metabolites in rat plasma for the pharmacokinetic studies. APAP and its metabolites were separated through a Capcell Pak MGII C(18) column and quantitated with a 16 min run in a triple-quadruple mass spectrometer. The mobile phases were composed of 0.1% formic acid in either 95% water or 95% acetonitrile and analysis was performed twice in positive and negative modes. Validations such as accuracy, precision, recovery, matrix effect and stability were found to be within acceptance criteria of validation guidelines, indicating that the assay was applicable to the determination of the plasma concentrations of drug and its six metabolites. In conclusion, we developed an LC-MS/MS method for the quantitative analysis of APAP and its six metabolites in rat plasma, and this method appears to be useful for pharmacokinetic/toxicokinetic studies of APAP and its metabolites in rats.