Pharmacokinetics of the novel 5-HT4 receptor agonist, DA-6886, in dogs.

The pharmacokinetics of a new 5-hydroxytryptamine receptor 4 agonist, DA-6886, intended for the treatment of constipation-predominant irritable bowel syndrome, were evaluated in beagle dogs following both intravenous and oral administration of DA-6886 (1-10 mg/kg). The study also examined the effects of gender and food on the pharmacokinetics of DA-6886 in dogs.DA-6886 demonstrated dose-proportional area under the plasma concentration-time curve (AUC) values and dose-independent clearance (21.0-24.6 mL/min/kg) after administration via both routes. The steady-state volume of distribution (Vss) for DA-6886 was dose-independent and relatively large (6.76-8.57 L/kg), aligning with its observed high distribution in rat tissues.No significant differences were observed in the pharmacokinetics of DA-6886 between male and female dogs. Post oral administration, extent of absolute oral bioavailability (BA) was relatively high (48.2-96.1%) in contrast to the rates observed in rats (18.9-55.0%).Dogs that were fed exhibited a significantly lower Cmax and a delayed Tmax in comparison to those that were fasted. However, the AUC values were similar between the two groups. The extended stomach transit time in the fed state may account for this delayed absorption of DA-6886 without substantial changes in AUC.

Pharmacokinetic Comparison of Chitosan-Derived and Biofermentation-Derived Glucosamine in Nutritional Supplement for Bone Health.

Glucosamine and chondroitin sulfate have been used as nutritional supplementation for joint tissues and osteoarthritis (OA). Biofermented glucosamine is of great interest in the supplement industry as an alternative source of glucosamine. The purpose of this study is to compare the pharmacokinetics of chitosan-derived glucosamine and biofermentation-derived glucosamine as nutritional supplementation. In a randomized, double-blind and cross-over study design, we recruited subjects of healthy men and women. The pharmacokinetics of glucosamine were examined after a single dose of glucosamine sulfate 2KCl (1500 mg) with two different sources of glucosamine (chitosan-derived glucosamine and biofermentation-derived glucosamine) to male and female subjects fitted with intravenous (iv) catheters for repeated blood sampling up to 8 h. According to plasma concentration-time curve of glucosamine after an oral administration of 1500 mg of glucosamine sulfate 2KCl, AUC0-8h and AUC0-∞ values of glucosamine following oral administration of chitosan-derived and biofermentation-derived glucosamine formulations were within the bioequivalence criteria (90% CI of ratios are within 0.8-1.25). The mean Cmax ratios for these two formulations (90% CI of 0.892-1.342) did not meet bioequivalence criteria due to high within-subject variability. There were no statistically significant effects of sequence, period, origin of glucosamine on pharmacokinetic parameters of glucosamine such as AUC0-8h, AUC0-∞, Cmax. Our findings suggest that biofermentation-derived glucosamine could be a sustainable source of raw materials for glucosamine supplement.

Pharmacokinetics of DA-6886, A New 5-HT4 Receptor Agonist, in Rats.

DA-6886 is a novel serotonin (5-hydroxytrypamine [5-HT]) receptor 4 agonist for the potential treatment of constipation-predominant irritable bowel syndrome. The purpose of this study was to validate the quantitative assay of DA-6886 in rat plasma and to evaluate the pharmacokinetics and tissue distribution of DA-6886 in rats. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the robust quantification of DA-6886 in rat plasma was successfully validated and applied to the pharmacokinetic studies in rats. The pharmacokinetic parameters of DA-6886 in rats were evaluated following single intravenous or oral administration at three dose levels (2, 10, and 20 mg/kg). DA-6886 exhibited a smaller dose-normalized area under the plasma concentration-time curve (AUC) values and faster clearances in the low-dose group than in the high-dose group following both intravenous and oral administration. The steady-state volume of distribution (Vss) of DA-6886 was relatively large (4.91-7.84 L/kg), which was consistent with its high distribution to the liver, kidney, lung, and digestive tract, and was dose-independent. After oral administration, the extent of absolute bioavailability (F) tended to increase (18.9-55.0%) with an increasing dose. The slope of the log-transformed AUC and/or Cmax values versus log dose was greater than unity and greater for oral administration (~1.9) than for intravenous administration (~1.1). Because the nonlinear pharmacokinetics of DA-6886 was more obviously observed after oral administration, it appears that the saturation of pre-systemic intestinal and/or hepatic first-pass extraction of DA-6886 at high doses occurred.

Metformin and Niclosamide Synergistically Suppress Wnt and YAP in APC-Mutated Colorectal Cancer.

The Wnt and Hippo pathways are tightly coordinated and understanding their reciprocal regulation may provide a novel therapeutic strategy for cancer. Anti-helminthic niclosamide is an effective inhibitor of Wnt and is now in a phase II trial for advanced colorectal cancer (CRC) patients. We found that Axin2, an authentic target gene of canonical Wnt, acts as aYAP phosphorylation activator in APC-mutated CRC. While niclosamide effectively suppresses Wnt, it also inhibits Hippo, limiting its therapeutic potential for CRC. To overcome this limitation, we utilized metformin, a clinically available AMPK activator. This combinatory approach not only suppresses canonical Wnt activity, but also inhibits YAP activity in CRC cancer cells and in patient-derived cancer organoid through the suppression of cancer stemness. Further, combinatory oral administration suppressed in vivo tumorigenesis and the cancer progression of APC-MIN mice models. Our observations provide not only a reciprocal link between Wnt and Hippo, but also clinically available novel therapeutics that are able to target Wnt and YAP in APC-mutated CRC.

Changes in digoxin pharmacokinetics associated with hepatic P-glycoprotein upregulation in rats with non-alcoholic fatty liver disease.

BACKGROUND & OBJECTIVES: Upregulation of hepatic P-glycoprotein (P-gp) expression has been reported in patients with non-alcoholic fatty liver disease (NAFLD) and rodent models thereof. Here, we explored the changes hepatic P-gp expression and activity in a NAFLD rat model and the effects thereof on the pharmacokinetics of digoxin (a probe substrate of P-gp). METHODS: Rats were fed a 1% (w/w) orotic acid-containing diet for 20 days to induce NAFLD; control rats received a normal diet. P-gp expression and biliary digoxin excretion were examined. The pharmacokinetics of digoxin were evaluated after it had been administered intravenously (10 µg·kg-1 ) and orally (200 µg·kg-1 ) to control and NAFLD rats. RESULTS: The total areas under the plasma concentration-time curves (AUCs) of digoxin after intravenous and oral administration were significantly smaller (by 39.1% and 73.0%, respectively) in NAFLD rats because of faster biliary digoxin excretion, reflecting elevations of hepatic P-gp expression and activity. Notably, the steady-state volume of distribution rose by 98.2%, while extent of oral bioavailability fell by 55.5% in NAFLD rats. CONCLUSION: This is the first study to report digoxin pharmacokinetic changes caused by hepatic P-gp upregulation in NAFLD. Further studies are needed to explore the clinical impact of enhanced P-gp-mediated biliary excretion on pharmacotherapies using P-gp substrates in patients with NAFLD.

Development and Validation of an LC-MS/MS Method for Quantification of the Novel Antibacterial Candidate DA-7010 in Plasma and Application to a Preclinical Pharmacokinetic Study.

DA-7010 is a new candidate for an antibacterial agent that targets Gram-negative pathogens by acting as a leucyl-tRNA synthetase inhibitor. In this study, a simple and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed to determine DA-7010 levels in the plasma from mice, rats, and dogs. Plasma samples were mixed with methanol for protein precipitation. Chromatographic separation was carried out using a reversed-phase C18 column (Agilent Poroshell 120, 50 × 3.0 mm, 2.7 µm). An isocratic elution of the mobile phase consisting of 5 mM formic acid in water and acetonitrile at a ratio of 84:16 (v/v) was applied at a flow rate of 0.3 mL/min. The total chromatographic run time was 3.5 min. Multiple reaction monitoring (MRM) mode was used for mass spectrometric detection using an Agilent 6460 triple quadrupole coupled with an electrospray ionization (ESI) source operated in positive-ion mode. The MRM transitions analyzed were m/z 220.1→162.1 for DA-7010 and m/z 206.1→170.1 for the internal standard (structural analogue of DA-7010). Calibration curves were constructed in the range of 10-10,000 ng/mL. The intra- and interday precision and accuracy were within 11.3%, excluding those for the lower limit of quantification (LLOQ) samples, which were within 17.1%. The developed LC-MS/MS method was successfully validated and applied in preclinical pharmacokinetic studies of DA-7010 in mice, rats, and dogs following single oral administrations. The oral absorption of DA-7010 was rapid, and the systemic exposure was approximately four times higher in the dogs than in the mice or rats.

Pharmacokinetic changes of clozapine and norclozapine in a rat model of non-alcoholic fatty liver disease induced by orotic acid.

Impaired in vitro oxidation of clozapine has been reported in steatotic rat liver due to downregulation of cytochrome P450 (CYP) 1A. Pharmacokinetic changes of clozapine and its major metabolite, norclozapine, were evaluated in a rat model of non-alcoholic fatty liver disease (NAFLD) induced by orotic acid. Significantly slower in vitro CLint for formation of norclozapine from clozapine was observed in NAFLD rats than in control rats as a result of the reduced protein expression and metabolic activity of CYP1A1/2. However, systemic exposures to clozapine in NAFLD rats were comparable to those in controls after intravenous (4 mg/kg) and oral (10 mg/kg) administration of clozapine. Of note, the AUC of the norclozapine and AUCnorclozapine/AUCclozapine ratio following intravenous and oral administration of clozapine rather increased significantly in NAFLD rats, as a result of the slowed subsequent metabolism of norclozapine via CYP1A1/2. Steady-state brain concentrations of both clozapine and norclozapine were significantly higher in NAFLD rats than those in control rats following intravenous infusion of clozapine. Increased systemic exposure to norclozapine and elevated brain concentrations of clozapine and norclozapine observed in NAFLD rats imply that further studies are warranted on the pharmacotherapy of clozapine in patients with pre-existing or drug-induced hepatic steatosis.

Transcriptional Expression in Human Periodontal Ligament Cells Subjected to Orthodontic Force: An RNA-Sequencing Study.

: This study was performed to investigate the changes in gene expression in periodontal ligament (PDL) cells following mechanical stimulus through RNA sequencing. In this study, premolars extracted for orthodontic treatment were used. To stimulate the PDL cells, an orthodontic force of 100× g was applied to the premolar (experimental group; n = 11), whereas the tooth on the other side was left untreated (control group; n = 11). After the PDL cells were isolated from the extracted teeth, gene set enrichment analysis (GSEA), differentially expressed gene (DEG) analysis, and real-time PCR were performed to compare the two groups. GSEA demonstrated that gene sets related to the cell cycle pathway were upregulated in PDL. Thirteen upregulated and twenty downregulated genes were found through DEG analysis. Real-time PCR results confirmed that five upregulated genes (CC2D1B, CPNE3, OPHN1, TANGO2, and UAP-1) and six downregulated genes (MYOM2, PPM1F, PCDP1, ATP2A1, GPR171, and RP1-34H18.1-1) were consistent with RNA sequencing results. We suggest that, from among these eleven genes, two upregulated genes, CPNE3 and OPHN1, and one downregulated gene, PPM1F, play an important role in PDL regeneration in humans when orthodontic force is applied.

The pharmacokinetics of mycophenolic acid in rats with orotic acid induced nonalcoholic fatty liver disease.

Post-transplantation nonalcoholic fatty liver disease (NAFLD) is common in liver transplant recipients. Changes in the expression levels and activities of drug-metabolizing enzymes and drug transporters have been reported in patients with NAFLD and relevant rodent models. Here, we evaluated whether the pharmacokinetics of mycophenolic acid (MPA), an immunosuppressant, would be altered in rats with NAFLD. NAFLD was induced by feeding a diet containing 1% (w/w) orotic acid for 20 days. The extent of hepatic glucuronidation of MPA to a major metabolite, mycophenolic acid-7-O-glucuronide (MPAG), did not differ between rats with NAFLD and controls. The expression levels of hepatic multidrug resistance-associated protein 2, responsible for biliary excretion of MPAG, were comparable in rats with NAFLD and controls; the biliary excretion of MPAG was also similar in the two groups. Compared with control rats, rats with NAFLD did not exhibit significant changes in the areas under the plasma concentration - time curves of MPA or MPAG after intravenous (5 mg/kg) or oral (10 mg/kg) administration of MPA. However, delayed oral absorption of MPA was observed in rats with NAFLD compared with controls; the MPA and MPAG peak plasma concentrations fell significantly and the times to achieve them were prolonged following oral administration of MPA.

Optimization of Canalicular ABC Transporter Function in HuH-7 Cells by Modification of Culture Conditions.

Human hepatoma cell lines are useful for evaluation of drug-induced hepatotoxicity, hepatic drug disposition, and drug-drug interactions. However, their applicability is compromised by aberrant expression of hepatobiliary transporters. This study was designed to evaluate whether extracellular matrix (Matrigel) overlay and dexamethasone (DEX) treatment would support cellular maturation of long-term HuH-7 hepatoma cell cultures and improve the expression, localization, and activity of canalicular ATP-binding cassette (ABC) transporters, multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1), multidrug resistance-associated protein 2 (MRP2/ABCC2), and bile salt export pump (BSEP/ABCB11). Matrigel overlay promoted the maturation of HuH-7 cells toward cuboidal, hepatocyte-like cells displaying bile canaliculi-like structures visualized by staining for filamentous actin (F-actin), colocalization of MRP2 with F-actin, and by accumulation of the MRP2 substrate 5(6)-carboxy-2',7'-dichlorofluorescein (CDF) within the tubular canaliculi. The cellular phenotype was rather homogenous in the Matrigel-overlaid cultures, whereas the standard HuH-7 cultures contained both hepatocyte-like cells and flat epithelium-like cells. Only Matrigel-overlaid HuH-7 cells expressed MDR1 at the canaliculi and excreted the MDR1 probe substrate digoxin into biliary compartments. DEX treatment resulted in more elongated and branched canaliculi and restored canalicular expression and function of BSEP. These findings suggest that hepatocyte polarity, elongated canalicular structures, and proper localization and function of canalicular ABC transporters can be recovered, at least in part, in human hepatoma HuH-7 cells by applying the modified culture conditions. SIGNIFICANCE STATEMENT: We report the first demonstration that proper localization and function of canalicular ABC transporters can be recovered in human hepatoma HuH-7 cells by modification of cell culture conditions. Matrigel overlay and dexamethasone supplementation increased the proportion of hepatocyte-like cells, strongly augmented the canalicular structures between the cells, and restored the localization and function of key canalicular ABC transporters. These results will facilitate the development of reproducible, economical, and easily achievable liver cell models for drug development.

Effects of Orotic Acid-Induced Non-Alcoholic Fatty Liver on the Pharmacokinetics of Metoprolol and its Metabolites in Rats.

PURPOSE: Preliminary study results have shown that rats with non-alcoholic fatty liver disease (NAFLD) induced by 1% orotic acid-containing diet have decreased hepatic CYP2D activity. This study aims to evaluate the possible pharmacokinetic changes in NAFLD as a result of reduced metabolic activity of CYP2D. METHODS: The pharmacokinetics of metoprolol and its metabolites, O-desmethyl metoprolol (DMM) and α-hydroxy metoprolol (HM), was investigated in NAFLD and control rats following intravenous (1 mg/kg) and oral (2 mg/kg) administration of metoprolol. The hepatic CYP2D expression was also investigated. RESULTS: NAFLD rats had lower CYP2D expression (by 36.6%) and slower intrinsic clearance (CLint) of metoprolol and formation of HM (by 40.1% and 37.2%, respectively). There were no significant changes in the pharmacokinetics of metoprolol and its metabolites following intravenous administration. In contrast, oral administration of metoprolol resulted in significantly increased total area under plasma concentration-time curve (AUC) of metoprolol (by 127%) and decreased metabolite formation ratios (AUCDMM/AUCMetoprolol [by 42.8%], AUCHM/AUCMetoprolol [by 35.0%]) in NAFLD rats. Moreover, these changes were well correlated with severity of steatosis as quantified by hepatic triglyceride contents. CONCLUSIONS: NALFD can lead to a reduction in the hepatic CLint of a drug if it is a substrate of the CYP2D subfamily. The decreased clearance may result in elevated drug concentrations and increased exposure.

Protein expression and function of organic anion transporters in short-term and long-term cultures of Huh7 human hepatoma cells.

Human-derived hepatic cell lines are a valuable alternative to primary hepatocytes for drug metabolism, transport and toxicity studies. However, their relevance for investigations of drug-drug and drug-organic anion (e.g., bile acid, steroid hormone) interactions at the transporter level remains to be established. The aim of the present study was to determine the suitability of the Huh7 cell line for transporter-dependent experiments. Huh7 cells were cultured for 1 to 4 weeks and subsequently were analyzed for protein expression, localization and activity of solute carrier (SLC) and ATP-binding cassette (ABC) transporters involved in organic anion transport using liquid chromatography-tandem mass spectroscopy, immunocytochemistry, and model substrates [3H]taurocholate (TCA), [3H]dehydroepiandrosterone sulfate (DHEAS) and 5(6)-carboxy-2',7'-dichlorofluorescein (CDF) diacetate. The extended 4-week culture resulted in a phenotype resembling primary hepatocytes and differentiated HepaRG cells: cuboidal hepatocyte-like cells with elongated bile canaliculi-like structures were surrounded by epithelium-like cells. Protein expression of OSTα, OSTß and OATP1B3 increased over time. Moreover, the uptake of the SLC probe substrate DHEAS was higher in 4-week than in 1-week Huh7 cultures. NTCP, OATP1B1, BSEP and MRP3 were barely or not detectable in Huh7 cells. OATP2B1, MRP2 and MRP4 protein expression remained at similar levels over the four weeks of culture. The activity of MRP2 and the formation of bile canaliculi-like structures were confirmed by accumulation of CDF in the intercellular compartments. Results indicate that along with morphological maturation, transporters responsible for alternative bile acid secretion pathways are expressed and active in long-term cultures of Huh7 cells, suggesting that differentiated Huh7 cells may be suitable for studying the function and regulation of these organic anion transporters.

Therapeutic implications of cancer epithelial-mesenchymal transition (EMT).

The epithelial-mesenchymal transition (EMT) comprises an essential biological process involving cancer progression as well as initiation. While the EMT has been regarded as a phenotypic conversion from epithelial to mesenchymal cells, recent evidence indicates that it plays a critical role in stemness, metabolic reprogramming, immune evasion and therapeutic resistance of cancer cells. Interestingly, several transcriptional repressors including Snail (SNAI1), Slug (SNAI2) and the ZEB family constitute key players for EMT in cancer as well as in the developmental process. Note that the dynamic conversion between EMT and epithelial reversion (mesenchymal-epithelial transition, MET) occurs through variable intermediate-hybrid states rather than being a binary process. Given the close connection between oncogenic signaling and EMT repressors, the EMT has emerged as a therapeutic target or goal (in terms of MET reversion) in cancer therapy. Here we review the critical role of EMT in therapeutic resistance and the importance of EMT as a therapeutic target for human cancer.

Pharmacokinetic alterations in poloxamer 407-induced hyperlipidemic rats.

1. Plasma lipid profile abnormalities in hyperlipidemia can potentially alter the pharmacokinetics of a drug in a complex manner. To evaluate these pharmacokinetic alterations in hyperlipidemia and to determine the underlying mechanism(s), poloxamer 407-induced hyperlipidemic rats (HL rats), a well-established animal model of hyperlipidemia have been used. 2. In this review, we summarize findings on the pathophysiological and gene expression changes in drug-metabolizing enzymes and transporters in HL rats. We discuss pharmacokinetic changes in drugs metabolized primarily via hepatic cytochrome P450 (CYPs) in terms of alterations in hepatic intrinsic clearance (CL'int), free fraction in plasma (fu) and hepatic blood flow rate (QH), depending on the hepatic excretion ratio, as well as drugs eliminated primarily by mechanisms other than hepatic CYPs. 3. For lipoprotein-bound drugs, increased binding to lipoproteins resulted in lower fu values and volumes of distribution, with some exceptions. Generally, slower non-renal clearance (or total body clearance) of drugs that are substrates of hepatic CYP3A and CYP2C is well explained by the following factors: alterations in CL'int (due to down-regulation of hepatic CYPs), decreased fu and/or possible decreased QH. 4. These consistent findings across studies in HL rats suggest more studies are needed at the clinical level for optimal pharmacotherapies for hyperlipidemia.

Pharmacokinetics and Brain Distribution of the Active Components of DA-9805, Saikosaponin A, Paeonol, and Imperatorin in Rats.

DA-9805 is a botanical anti-Parkinson's drug candidate formulated from ethanol extracts of the root of Bupleurum falcatum, the root cortex of Paeonia suffruticosa, and the root of Angelica dahurica. The pharmacokinetics (PKs) and brain distribution of active/representative ingredients of DA-9805, Saikosaponin a (SSa; 1.1⁻4.6 mg/kg), Paeonol (PA; 14.8⁻59.2 mg/kg), and Imperatorin (IMP; 1.4⁻11.5 mg/kg) were evaluated following the intravenous or oral administration of each pure component and the equivalent dose of DA-9805 in rats. All three components had greater dose-normalized areas under the plasma concentration-time curve (AUC) and slower clearance with higher doses, following intravenous administration. By contrast, dose-proportional AUC values of SSa, PA, and IMP were observed following the oral administration of each pure component (with the exception of IMP at the highest dose) or DA-9805. Compared to oral administration of each pure compound, DA-9805 administration showed an increase in the AUC of SSa (by 96.1⁻163%) and PA (by 155⁻164%), possibly due to inhibition of their metabolism by IMP or other component(s) in DA-9805. A delay in the absorption of PA and IMP was observed when they were administered as DA-9805. All three components of DA-9805 showed greater binding values in brain homogenates than in plasma, possibly explaining why the brain-to-plasma ratios were greater than unity following multiple oral administrations of DA-9805. By contrast, their levels in cerebrospinal fluid were negligible. Our results further our understanding of the comprehensive PK characteristics of SSa, PA, and IMP in rats and the comparative PKs between each pure component and DA-9805.

Dishevelled has a YAP nuclear export function in a tumor suppressor context-dependent manner.

Phosphorylation-dependent YAP translocation is a well-known intracellular mechanism of the Hippo pathway; however, the molecular effectors governing YAP cytoplasmic translocation remains undefined. Recent findings indicate that oncogenic YAP paradoxically suppresses Wnt activity. Here, we show that Wnt scaffolding protein Dishevelled (DVL) is responsible for cytosolic translocation of phosphorylated YAP. Mutational inactivation of the nuclear export signal embedded in DVL leads to nuclear YAP retention, with an increase in TEAD transcriptional activity. DVL is also required for YAP subcellular localization induced by E-cadherin, α-catenin, or AMPK activation. Importantly, the nuclear-cytoplasmic trafficking is dependent on the p53-Lats2 or LKB1-AMPK tumor suppressor axes, which determine YAP phosphorylation status. In vivo and clinical data support that the loss of p53 or LKB1 relieves DVL-linked reciprocal inhibition between the Wnt and nuclear YAP activity. Our observations provide mechanistic insights into controlled proliferation coupled with epithelial polarity during development and human cancer.

Frequent oncogenic BRAF V600E mutation in odontogenic keratocyst.

OBJECTIVES: Odontogenic keratocyst (OKC), also known as keratocystic odontogenic tumor (KCOT), has clinical significance due to its high incidence as well as high recurrence rate after surgical enucleation. Current clinical management for OCK is entirely dependent on surgical approach. While various genetic alterations, such as PTCH1 mutation and loss of heterozygosity in tumor suppressor genes, have been reported, the molecular background of OKC is not well-understood. Although recent identification of BRAF V600E mutation and subsequent activation of mitogen-activated protein kinase (MAPK) pathway in ameloblastoma and odontogenic tumors provide additional options with targeted therapeutics, the molecular background of OKC is not well understood. MATERIALS AND METHODS: In this study, we examined BRAF V600E mutation from paraffin embedded OKC samples by tumor cell enriched microdissection and TA cloning of amplified DNA. We further examined the relationship between BRAF V600E mutation and clinical parameters. RESULTS: We found frequent BRAF V600E mutation in OKC (24 of 38 samples, 63.2%). However, BRAF V600E mutational status is not related with clinical indexes such as size, location, and recurrence. In orthokeratinized odontogenic cyst, there is one case of BRAF 600E mutation from 11 samples (9.1%). CONCLUSION: These results indicate that BRAF V600E mutation occurs in OKCs at a high rate and plays an important role in the pathogenesis of OKCs.

Simultaneous determination of saikosaponin a, paeonol, and imperatorin, components of DA-9805, in rat plasma by LC-MS/MS and application to a pharmacokinetic study.

DA-9805 is a new botanical antiparkinson drug candidate formulated using an ethanolic extract of the root of Bupleurum falcatum, the root cortex of Paeonia suffruticosa, and the root of Angelica dahurica. In this study, a sensitive and rapid LC-MS/MS method was developed to simultaneously determine, saikosaponin a, paeonol, and imperatorin, three active/representative ingredients of DA-9805, in rat plasma. Plasma was extracted by mixture of ethyl acetate and methyl tertiary butyl ether. Chromatographic separation was carried out using a C18 column and a gradient elution of mobile phases consisting of 5mM formic acid in water and acetonitrile. Total chromatographic run time was 10.5min. Multiple reaction monitoring mode was used for mass spectrometry; the transitions were m/z 779.5→617.2 for saikosaponin a in negative-ion mode, m/z 167→149 for paeonol and m/z 271.1→203 for imperatorin in positive-ion mode. Calibration curves were constructed in the range of 0.5-1000ng/mL for saikosaponin a, 20-10000ng/mL for paeonol, and 0.2-1000ng/mL for imperatorin. All the validation data, including the selectivity, linearity, precision, accuracy, recovery, matrix effect, and stability satisfied the acceptance requirements. The method was successfully applied in a pharmacokinetic study of saikosaponin a, paeonol, and imperatorin following oral administration of DA-9805.

Anti-helminthic niclosamide inhibits Ras-driven oncogenic transformation via activation of GSK-3.

Despite the importance of Ras oncogenes as a therapeutic target in human cancer, their 'undruggable' tertiary structures limit the effectiveness of anti-Ras drugs. Canonical Wnt signaling contributes to Ras activity by glycogen synthase kinase 3 (GSK-3)-dependent phosphorylation at the C-terminus and subsequent degradation. In the accompanying report, we show that the anti-helminthic niclosamide directly binds to GSK-3 and inhibits Axin functions in colon cancer cells, with reversion of Snail-mediated epithelial-mesenchymal transition. In this study, we report that niclosamide effectively suppresses Ras and nuclear NFAT activities regardless of the mutational status of Ras at nM levels. Mechanistically, niclosamide increased endogenous GSK-3 activity, shortening the half-life of mutant Ras. Further, niclosamide activates Raf-1 kinase inhibitory protein, a downstream target of Snail repressor. Niclosamide treatment attenuates Ras-induced oncogenic potential in vitro and in vivo. These findings provide a clinically available repositioned Ras inhibitor as well as a novel strategy for inhibiting the Ras via GSK-3.

Effects of poloxamer 407-induced hyperlipidemia on hepatic multidrug resistance protein 2 (Mrp2/Abcc2) and the pharmacokinetics of mycophenolic acid in rats.

Hepatic multidrug resistance-associated protein 2 (Mrp2) is responsible for the majority of the biliary elimination of endogenous and exogenous substances, therefore it is important to evaluate possible functional changes in Mrp2 activity under conditions of hyperlipidemia (HL). Thus, the present study assessed the protein expression and transporting activity of hepatic Mrp2 based on the in vivo biliary excretion of phenolsulfonphthalein (PSP) as a model anionic substrate for Mrp2 in poloxamer 407-induced hyperlipidemic rats (HL rats) and compared these values with those for control rats. The pharmacokinetics of mycophenolic acid (MPA) and mycophenolic acid-7-O-glucuronide (MPAG) were evaluated after the intravenous (5 mg/kg) and oral (10 mg/kg) administration of MPA to control and HL rats. In HL rats, the protein expression of hepatic Mrp2 and its biliary transporting activity exhibited significant reductions (by 24.3% and 24.6%, respectively) in the absence of a change in bile flow rate. Unexpectedly, HL and control rats showed comparable biliary excretion rates of MPAG due to the counter effects of the reduced expression and activity of Mrp2 and a 484% increase in the free fraction of MPAG in HL rats. The estimated biliary clearance value of free MPAG in HL rats was considerably slower (by 77.1%) than that in control rats. Although significant pharmacokinetic changes in total MPA and MPAG levels were not observed in HL rats, there was a marked increase in free MPA and MPAG levels. Clinically relevant pharmacokinetic changes in subjects with HL that are related to MRP2 could not be ruled out. Copyright © 2016 John Wiley & Sons, Ltd.