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Abstract Although, steroid profiling is being applied in clinical and biochemical studies, improvement of the technique is still needed for accurate quantification of steroids in case of limited biological sample volumes. To improve analytical sensitivity and selectivity in comparison to that of conventional methods, a method that comprises supported liquid extraction (SLE) and gas chromatography-mass spectrometry with a combination of selected-reaction and selected-ion monitoring modes (GC-SRM/SIM-MS) was developed. Here, this combination of SLE purification with GC-MS method was optimized with 37 different types of steroids and the results were compared to a solid-phase extraction (SPE) method. The devised assay led to an increase in extraction efficiency with the good chromatographic selectivity through a single extraction step. The limits of quantification of the serum steroids, ranged from 0.2 to 5 ng mL−1, except for cholesterol (0.2 μg mL−1), and the correlation coefficients for calibration curves were higher than 0.99. The precision and accuracy were 1.4%–10.5% and 82.7%–115.3%, respectively. The overall recoveries of 30 steroids ranged from 62.1% to 104.3%, while that of 7 sterols was 44.7%–75.7%. Then, this validated method was applied to monitor the serum steroid levels of mice, which showed significant sex and age dependent metabolic patterns. This technique can be used to evaluate the metabolic changes occurring in animal models as well as in clinical patients. Graphical abstract Image 1 Highlights • GC-MS-based analysis for different types of serum steroids was evaluated. • All 37 steroids were measured in SIM and SRM simultaneously. • Supported liquid extraction (SLE) ensured a good chromatographic selectivity. • The validated method showed sex and age dependent changes of mouse serum steroids. [ABSTRACT FROM AUTHOR]

Objective: The aim of this study was to investigate the prevalence and causes of early discontinuation and non-adherence to upfront and extended adjuvant letrozole therapy in breast cancer patients. Methods: Adherence was assessed using medical charts and longitudinal pharmacy records of 609 patients who initiated adjuvant letrozole between January 2002 and April 2011. A Cox proportional hazards regression model was adopted to identify potential predictors of non-adherence. Results: The overall adherence rate after 1 year of therapy was 79.5%, with cumulative rates declining to 63.7% after 3 years and 57.1% after 5 years. A significantly lower rate of adherence in the extended adjuvant group was observed compared with the upfront adjuvant group (49.0 vs. 72.5%, p < 0.001). Adverse events (50.4%) were the major cause of early discontinuation, with musculoskeletal pain (73.2%) being the single most cited reason. Additional factors correlating with non-adherence in the upfront adjuvant group included a delay in initiation of adjuvant hormone therapy, breast-conserving surgery, calcium supplements, bisphosphonate therapy and concomitant medication for co-morbidity. Conclusions: We observed that approximately 57% of patients fully adhered to letrozole therapy over a 5-year treatment period, and that the adherence to extended letrozole was meaningfully lower than the upfront adjuvant letrozole in a clinical practice setting. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Targeting of magnolin, a natural compound found in the Magnolia species, on ERKs active pocket inhibits EGF-induced cell proliferation and transformation. Thus, magnolin might be a naturally occurring chemoprevention and therapeutic agent by targeting ERK1 and ERK2.Mitogen-activated protein kinases play a key role in cell proliferation, cell cycle progression and cell transformation, and activated Ras/extracellular signal-regulated kinases (ERKs)/ribosomal S6 kinase 2 (RSK2) signaling pathways have been widely identified in many solid tumors. In this study, we found that magnolin, a compound found in the Magnolia species, directly targeted and inhibited ERK1 and ERK2 kinase activities with IC50 values of 87 and 16.5 nM by competing with adenosine triphosphate in an active pocket. Further, we demonstrated that magnolin inhibited epidermal growth factor (EGF)-induced p90RSKs phosphorylation at Thr359/Ser363, but not ERKs phosphorylation at Thr202/Tyr204, and this resulted in inhibition of cell proliferation by suppression of the G1/S cell cycle transition. Additionally, p38 kinases, Jun N-terminal kinases and Akts were not involved in the magnolin-mediated inhibitory signaling. Magnolin targeting of ERK1 and 2 activities suppressed the phosphorylation of RSK2 and downstream target proteins including ATF1 and c-Jun and AP-1, a dimer of Jun/Fos, and the transactivation activities of ATF1 and AP-1. Notably, ERKs inhibition by magnolin suppressed EGF-induced anchorage-independent cell transformation and colony growth of RasG12V-harboring A549 human lung cancer cells and NIH3T3 cells stably expressing RasG12V in soft agar. Taken together, these results demonstrated that magnolin might be a naturally occurring chemoprevention and therapeutic agent capable of inhibiting cell proliferation and transformation by targeting ERK1 and ERK2. [ABSTRACT FROM AUTHOR]

The aim of this work was to identify the form(s) of human liver cytochrome P450 (CYP) involved in the hepatic transformation of myristicin to its major metabolite, 5-allyl-1-methoxy-2,3-dihydroxybenzene. When microsomes prepared from different human liver samples were compared, the activity of 5-allyl-1-methoxy-2,3-dihydroxybenzene formation was well correlated (r2=0.87) with nifedipine oxidation (a marker of CYP3A4). With a microsomal sample having high CYP3A4 activity, microsomal oxidation of myristicin to the major metabolite (5-allyl-1-methoxy-2,3-dihydroxybenzene) was markedly inhibited by gestodene and ketoconazole, selective inhibitors of CYP3A enzymes, but not by any of several other P450 inhibitors. Antibodies raised against CYPs 3A4 and 1A2 could also inhibit the oxidation of myristicin, but antibodies recognizing other CYPs had no effect. The oxidation of myristicin to 5-allyl-1-methoxy-2,3-dihydroxybenzene was catalyzed by purified bacterial recombinant CYPs 3A4 and 1A2. These results provide evidence that CYP3A4 (and possibly other CYP3A enzymes) and CYP1A2 play roles in the formation of the major metabolite, 5-allyl-1-methoxy-2,3-dihydroxybenzene. [Copyright &y& Elsevier]

1. The effects of flupyrazofos on liver microsomal cytochrome P450 were investigated in the male Fischer 344 rat. When rats were treated intraperitoneally with flupyrazofos for 3 consecutive days, the activities of ethoxyresorufin O-deethylase and testosterone 2 β-hydroxylase were significantly reduced, whereas the activities of pentoxyresorufin O-depentylase and testosterone 6 β- and 7 α-hydroxylases were induced in liver microsomes. 2. Within 24 h after treatment with 50 m kg[sup -1] flupyrazofos, most enzyme activities were decreased, indicating the interaction of flupyrazofos with cytochrome P450. 3. In Western immunoblotting, cytochrome P4502B1/2 proteins were clearly induced by treatment with flupyrazofos, whereas P4501A1/2 and 2C6 proteins were reduced in liver microsomes. 4. The present results indicate that flupyrazofos modulates the expression of cytochrome P450 in rat. [ABSTRACT FROM AUTHOR]

Zastaprazan (JP-1366), a novel potassium-competitive acid blocker, is a new drug for the treatment of erosive esophagitis. JP-1366 is highly metabolized in human, mouse, and dog hepatocytes but moderately metabolized in rat and monkey hepatocytes when estimated from the metabolic stability of this compound in hepatocyte suspension and when 18 phase I metabolites and 5 phase II metabolites [i.e., N-dearylation (M6), hydroxylation (M1, M19, M21), dihydroxylation (M7, M8, M14, M22), trihydroxylation (M13, M18), hydroxylation and reduction (M20), dihydroxylation and reduction (M9, M16), hydrolysis (M23), hydroxylation and glucuronidation (M11, M15), hydroxylation and sulfation (M17), dihydroxylation and sulfation (M10, M12), N-dearylation and hydroxylation (M3, M4), N-dearylation and dihydroxylation (M5), and N-dearylation and trihydroxylation (M2)] were identified from JP-1366 incubation with the hepatocytes from humans, mice, rats, dogs, and monkeys. Based on the cytochrome P450 (CYP) screening test and immune-inhibition analysis with CYP antibodies, CYP3A4 and CYP3A5 played major roles in the metabolism of JP-1366 to M1, M3, M4, M6, M8, M9, M13, M14, M16, M18, M19, M21, and M22. CYP1A2, 2C8, 2C9, 2C19, and 2D6 played minor roles in the metabolism of JP-1366. UDP-glucuronosyltransferase (UGT) 2B7 and UGT2B17 were responsible for the glucuronidation of M1 to M15. However, JP-1366 and active metabolite M1 were not substrates for drug transporters such as organic cation transporter (OCT) 1/2, organic anion transporter (OAT) 1/3, organic anion transporting polypeptide (OATP)1B1/1B3, multidrug and toxic compound extrusion (MATE)1/2K, P-glycoprotein (P-gp), and breast cancer-resistant protein (BCRP). Only M1 showed substrate specificity for P-gp. The findings indicated that drug-metabolizing enzymes, particularly CYP3A4/3A5, may have a significant role in determining the pharmacokinetics of zastaprazan while drug transporters may only have a small impact on the absorption, distribution, and excretion of this compound. [ABSTRACT FROM AUTHOR]

Although previous studies have examined the signaling pathway involved in melanogenesis through which ultraviolet (UV) or α-melanocyte-stimulating hormones (α-MSH) stimuli act as key inducers to produce melanin at the stratum basal layer of the epidermis, the signaling pathway regulating melanogenesis is still controversial. This study reports that α-MSH, not UVA and UVB, acted as a major stimulus of melanogenesis in B16F10 melanoma cells. Signaling pathway analysis using gene knockdown technology and chemical inhibitors, the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)/p90 ribosomal S6 kinase 2 (RSK2) played an important role in melanogenesis. Unexpectedly, LY294002, a PI3K inhibitor, increased melanogenesis without UV or α-MSH stimulation, suggesting that the PI3K/AKT signaling pathway may not be a major signaling pathway for melanogenesis. Chemical inhibition of the MEKs/ERKs/RSK2 signaling pathway using U0126 or BI-D1870 suppressed melanogenesis by stimulation of UVA or α-MSH stimulation, or both. In particular, the genetic depletion of RSK2 or constitutive active (CA)-RSK2 overexpression showed that RSK2 plays a key role in melanogenesis. Interestingly, forkhead box protein O4 (FOXO4) was phosphorylated by RSK2, resulting in the increase of FOXO4’s transactivation activity. Notably, the FOXO4 mutant harboring serine-to-alanine replacement at the phosphorylation sites totally abrogated the transactivation activity and reduced melanin production, indicating that RSK2-mediated FOXO4 activity plays a key role in melanogenesis. Furthermore, kaempferol, a flavonoid inhibiting the RSK2 activity, suppressed melanogenesis. In addition, FOXO4-wt overexpression showed that FOXO4 enhance melanin synthesis. Overall, the RSK2-FOXO4 signaling pathway plays a key role in modulating melanogenesis. [ABSTRACT FROM AUTHOR]

Licoricidin is a major prenylated isoflavone of Glycyrrhiza uralensis Fisch. (Leguminosae), and its pharmacological effects have been reported frequently. Typically, flavonoids having multiple hydroxyl groups are unambiguous substrates for glucuronyl conjugation by UDP‐glucuronosyltransferases (UGTs). The pharmacological effects of flavonoids are derived from the conjugation of glucuronide to yield the bioactive metabolite. Here, the metabolism of licoricidin in pooled human liver microsomes (HLMs) was investigated using high‐resolution quadrupole‐orbitrap mass spectrometry. One metabolite (M1) was identified in HLMs after incubation with licoricidin in the presence of uridine 5′‐diphosphoglucuronic acid (UDPGA) and NADPH. The structure of M1 was determined as a monoglucuronyl licoricidin, which was selectively produced by UGT1A9. Licoricidin showed a higher metabolic ratio and rapid metabolism with the recombinant human UGT1A9 than mycophenolic acid, a well‐known UGT1A9 substrate. In conclusion, the selective formation of 7‐glucuronyl licoricidin by UGT1A9 in HLMs could serve as a new selective substrate to determine the activity of UGT1A9 in vitro. [ABSTRACT FROM AUTHOR]

Bakuchicin is a furanocoumarin isolated from the seeds of Psoralea corylifolia , which is used in oriental medicine. However, limited information on the pharmacokinetics of bakuchicin is available and in addition, no determined method has been devised to quantify bakuchicin levels in the plasma. In the present study, we developed and validated a quantification method using liquid chromatography (LC) coupled with tandem mass spectrometry (LC-MS/MS), which was applied to a pharmacokinetic investigation in mouse plasma. LC was performed using an ACE 5 C18 column, and a mixture of acetonitrile and water containing 0.1% formic acid was used as the mobile phase at a flow rate of 220 μL/min. Bakuchicin transition ions in multiple reaction-monitoring modes using positive ionization were observed at m / z 187.0 to m / z 131.0. Bakuchicin and the internal standard (reserpine) had retention times of 4.5 and 4.3 min, respectively. Acceptable linearity (r 2 = 0.996) was observed over the concentration range of 20–1000 ng/mL, with a lower quantification limit of 20 ng/mL in mouse plasma. This method was successfully applied to determine the pharmacokinetic parameters of bakuchicin in mouse plasma and showed that the bioavailability of bakuchicin was 58.3% at 5 mg/kg oral administration. [ABSTRACT FROM AUTHOR]

Simultaneous multiresidual pesticide analysis of saliva samples was performed using scaled-down QuEChERS extraction with LC-MS/MS and GC-MS/MS. The optimum extraction procedure using acidified acetonitrile was applicable to 336 pesticides (287 for LC-MS/MS and 49 for GC-MS/MS). To determine pesticide multiresidues in saliva, 100 μL of the sample was extracted with 200 μL of 0.1% formic acid in acetonitrile, and the initial extract was partitioned with 40 mg of MgSO 4 and 10 mg of NaCl. The organic supernatants (120 μL) were then mixed with acetonitrile (30 μL) for matrix-matching (4:1, v/v), and the final extract solution was injected into the LC-MS/MS (4 μL) and GC-MS/MS (2 μL) systems. The established analytical method showed a good LOQs between 5 and 25 ng/mL with reliable accuracy/precision values and recovery results (50–140%) for the target pesticides. Under the two different storage conditions, most of the analytes did not undergo chemical changes in the saliva samples, whereas some pesticides were more stable in freeze-thaw processes than those left at room temperature. Biomonitoring of farmers (ten mixers and ten sprayers) was successfully applied using the validated method, and two carbamates (fenobucarb and propamocarb) were determined at trace concentrations (12.5–675.0 ng/mL from 11 positively detected samples). [Display omitted] • A modified QuEChERS was developed for multiresidual pesticide analysis in saliva. • Acidified acetonitrile exhibited excellent extraction efficiency for the target pesticides. • Chemical stability of each pesticide was evaluated under two different conditions. • It can be used for agricultural, forensic, and clinical purposes. [ABSTRACT FROM AUTHOR]

The well-known hepatotoxicity mechanism resulting from alpha-amanitin (α-AMA) exposure arises from RNA polymerase II (RNAP II) inhibition. RNAP Ⅱ inhibition occurs through the dysregulation of mRNA synthesis. However, the signaling pathways in hepatocytes that arise from α-AMA have not yet been fully elucidated. Here, we identified that the RAS/RAF/ERK signaling pathway was activated through quantitative phosphoproteomic and molecular biological analyses in Huh-7 cells. Bioinformatics analysis showed that α-AMA exposure increased protein phosphorylation in a time-dependent α-AMA exposure. In addition, phosphorylation increased not only the components of the ERK signaling pathway but also U2AF65 and SPF45, known splicing factors. Therefore, we propose a novel mechanism of α-AMA as follows. The RAS/RAF/ERK signaling pathway involved in aberrant splicing events is activated by α-AMA exposure followed by aberrant splicing events leading to cell death in Huh-7 cells. [ABSTRACT FROM AUTHOR]

The toxicokinetics of β-amanitin, a toxic bicyclic octapeptide present abundantly in Amanitaceae mushrooms, was evaluated in mice after intravenous (iv) and oral administration. The area under plasma concentration curves (AUC) following iv injection increased in proportion to doses of 0.2, 0.4, and 0.8 mg/kg. β-amanitin disappeared rapidly from plasma with a half-life of 18.3–33.6 min, and 52.3% of the iv dose was recovered as a parent form. After oral administration, the AUC again increased in proportion with doses of 2, 5, and 10 mg/kg. Absolute bioavailability was 7.3–9.4%, which resulted in 72.4% of fecal recovery from orally administered β-amanitin. Tissue-to-plasma AUC ratios of orally administered β-amanitin were the highest in the intestine and stomach. It also readily distributed to kidney > spleen > lung > liver ≈ heart. Distribution to intestines, kidneys, and the liver is in agreement with previously reported target organs after acute amatoxin poisoning. In addition, β-amanitin weakly or negligibly inhibited major cytochrome P450 and 5′-diphospho-glucuronosyltransferase activities in human liver microsomes and suppressed drug transport functions in mammalian cells that overexpress transporters, suggesting the remote drug interaction potentials caused by β-amanitin exposure. [ABSTRACT FROM AUTHOR]

Atopic dermatitis is a chronic inflammatory skin disease, of which incidence is closely related to exposure to environmental pollutants and allergens. Thymic stromal lymphopoietin (TSLP) plays an important role in the early stages of atopic dermatitis development by inducing Th2 immune responses. In addition, TSLP regulates activation of group 2 innate lymphoid cells (ILC2), promoting the pathogenesis of atopic dermatitis. The aim of this study was to investigate whether celastrol alleviated atopic dermatitis symptoms by regulating TSLP expression and ILC2 stimulation. Celastrol suppressed TSLP production in mouse keratinocyte cells by inhibiting NF-ĸB activation. Topical application of celastrol significantly improved atopic dermatitis symptoms induced by house dust mite (HDM) in NC/Nga mice as determined by dermatitis score and histological assessment. Celastrol decreased the levels of TSLP in atopic dermatitis skin lesions of HDM-stimulated NC/Nga mice. Celastrol reduced levels of Th2 cytokines including IL-4, IL-5, and IL-13 in atopic dermatitis skin lesions of NC/Nga mice. Further, celastrol significantly reduced ILC2 population in atopic dermatitis skin lesions of NC/Nga mice. These results indicate that topical application of celastrol improved atopic dermatitis symptoms by lowering TSLP levels and concomitant immune responses. Data demonstrated that reduced TSLP levels and associated lower number of ILC2 cells alleviate atopic dermatitis symptoms induced by house dust mite. [ABSTRACT FROM AUTHOR]

Autophagy is a critical cellular process required for maintaining cellular homeostasis in health and disease states, but the molecular mechanisms and impact of autophagy on cancer is not fully understood. Here, we found that Sox2, a key transcription factor in the regulation of the “stemness” of embryonic stem cells and induced-pluripotent stem cells, strongly induced autophagic phenomena, including intracellular vacuole formation and lysosomal activation in colon cancer cells. The activation occurred through Sox2-mediated ATG10 gene expression and resulted in the inhibition of cell proliferation and anchorage-independent colony growth ex vivo and tumor growth in vivo. Further, we found that Sox2-induced-autophagy enhanced cellular senescence by up-regulating tumor suppressors or senescence factors, including p16INK4a, p21 and phosphorylated p53 (Ser15). Notably, knockdown of ATG10 in Sox2-expressing colon cancer cells restored cancer cell properties. Taken together, our results demonstrated that regulation of autophagy mediated by Sox2 is a mechanism-driven novel strategy to treat human colon cancers. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine pharmacokinetics of α-amanitin, a toxic bicyclic octapeptide isolated from the poisonous mushrooms, following intravenous (iv) or oral (po) administration in mice using a newly developed and validated liquid chromatography-high resolution mass spectrometry. The iv injected α-amanitin disappeared rapidly from the plasma with high a clearance rate (26.9–30.4 ml/min/kg) at 0.1, 0.2, or 0.4 mg/kg doses, which was consistent with a rapid and a major excretion of α-amanitin via the renal route (32.6%). After the po administration of α-amanitin at doses of 2, 5, or 10 mg/kg to mice, the absolute bioavailability of α-amanitin was 3.5–4.8%. Due to this low bioavailability, 72.5% of the po administered α-amanitin was recovered from the feces. When α-amanitin is administered po, the tissue to plasma area under the curve ratio was higher in stomach > large intestine > small intestine > lung ~ kidneys > liver but not detected in brain, heart, and spleen. The high distribution of α-amanitin to intestine, kidneys, and liver is in agreement with the previously reported major intoxicated organs following acute α-amanitin exposure. In addition, α-amanitin weakly or negligibly inhibited cytochrome P450 and 5′-diphospho-glucuronosyltransferase enzymes activity in human liver microsomes as well as major drug transport functions in mammalian cells overexpressing transporters. Data suggested remote drug interaction potential may be associated with α-amanitin exposure. [ABSTRACT FROM AUTHOR]

Effective on‐demand release of therapeutics at an intracellular drug supply hub, the cytosol, is among the important steps for successful drug delivery. To improve cytosolic drug release, this study selects diselenide because the bond is cleaved by both glutathione (GSH) and reactive oxygen species (ROS) in the cytosol. Specifically, upon diselenide cleavage, the levels of GSH or ROS are reduced, resulting in decreased or increased cell viability and either the synergistic or antagonistic death of cancer cells with an anticancer drug, respectively, because GSH and ROS trigger two conflicting functions (i.e., antioxidant vs prooxidant activity). Thus, this study designs a diselenide‐based drug carrier to determine which trigger is the major cause of diselenide degradation, how the disrupted balance between GSH and ROS levels influences cell viability and drug efficacy, and whether the combined use of a diselenide drug carrier and a drug has a synergistic or antagonistic effect. Using a multiple diselenide‐containing nanoparticle (MSePCL‐NP), the study shows that diselenide is cleaved to a greater extent by GSH than by ROS; MSePCL‐NP induces a greater decrease in the viability of cancer cells, but not normal cells; a combination of DOX@MSePCL‐NP synergistically kills cancer cells and inhibits tumor growth in vivo. [ABSTRACT FROM AUTHOR]

Dronedarone and ticagrelor have high co-administration potential in patients with both acute coronary syndrome and atrial fibrillation. The objective of the present in vivo study was to investigate the potential interaction between dronedarone (5 and 10 mg/kg) and ticagrelor (5 and 10 mg/kg) when administered orally to rats. Forty Sprague-Dawley rats were randomly distributed into eight groups; consisting of a dronedarone only group, a ticagrelor only group, a dronedarone with ticagrelor-pretreatment group, and a ticagrelor with dronedarone-pretreatment group. Pharmacokinetic exposure (AUCinf = 1472 ng·h/mL) associated with administration of 10 mg/kg of dronedarone increased significantly, with delayed Tmax in the group that received ticagrelor-pretreatment when compared to the dronedarone only group (AUCinf = 723 ng·h/mL). In addition, pharmacokinetic exposure (AUCinf = 2391 ng·h/mL) associated with administration of 10 mg/kg of ticagrelor increased significantly, with increased Kel (0.31 h–1) and decreased Vz/F (14.6 L/kg) in the dronedarone-pretreatment group when compared to the ticagrelor only group (AUCinf = 1616 ng·h/mL; Kel = 0.21 h–1; Vz/F = 31.3 L/kg). Results of our study suggest that further investigation of a potential interaction between dronedarone and ticagrelor in humans is justified and that caution may need to be exercised when dronedarone and ticagrelor pharmacotherapies concomitantly. [ABSTRACT FROM AUTHOR]

The misuse of poisonous mushrooms containing amatoxins causes acute liver failure (ALF) in patients and is a cause of significant mortality. Although the toxic mechanisms of α-amanitin (α-AMA) and its interactions with RNA polymerase II (RNAP II) have been studied, α-AMA effector proteins that can interact with α-AMA in hepatocytes have not been systematically studied. Limited proteolysis-coupled mass spectrometry (LiP-MS) is an advanced technology that can quickly identify protein-ligand interactions based on global comparative proteomics. This study identified the α-AMA effector proteins found in human hepatocytes, following the detection of conformotypic peptides using LiP-MS coupled with tandem mass tag (TMT) technology. Proteins that are classified into protein processing in the endoplasmic reticulum and the ribosome during the KEGG pathway can be identified through affinity evaluation, according to α-AMA concentration-dependent LiP-MS and LiP-MS in hepatocytes derived from humans and mice, respectively. The possibility of interaction between α-AMA and proteins containing conformotypic peptides was evaluated through molecular docking studies. The results of this study suggest a novel path for α-AMA to induce hepatotoxicity through interactions with various proteins involved in protein synthesis, as well as with RNAP II. • α-Amanitin effector proteins were newly identified by Limited Proteolysis-Coupled Mass Spectrometry. • The conformotypic peptides of α-amanitin were highly detected in the non-cytoplasmic fraction. • The effectors were validated in a concentration-dependent affinity evaluation and prediction using 3D docking model. • It was found that proteins in the endoplasmic reticulum and the ribosome interact with α-amanitin. [ABSTRACT FROM AUTHOR]

The absorption, metabolism, and excretion (AME) profiles of KD101, currently under clinical development to treat obesity, were assessed in humans using accelerator mass spectrometry (AMS) after a single oral administration of KD101 at 400 mg and a microdose of 14C‐KD101 at ~ 35.2 μg with a total radioactivity of 6.81 kBq. The mean total recovery of administered radioactivity was 85.2% with predominant excretion in the urine (78.0%). The radio‐chromatographic metabolite profiling showed that most of the total radioactivity in the plasma and the urine was ascribable to metabolites. The UDP‐glucuronosyltransferase (UGT), including UGT1A1, UGT1A3, and UGT2B7, might have contributed to the interindividual variability in the metabolism and excretion of KD101. The microtracing approach using AMS is a useful tool to evaluate the AME of a drug under development without risk for high radiation exposure to humans. [ABSTRACT FROM AUTHOR]

Lipid metabolism is associated with colon cancer prognosis and incidence. Stearoyl-CoA desaturase 1 (SCD1), which converts fully saturated fatty acids (SFAs) to monounsaturated fatty acids (MUFAs), has been suggested as a vulnerable target for selective elimination of cancer stem cells (CSCs). However, the clinical significance and physiological role of SCD1 in CSCs has not been well demonstrated. Here, we showed the clinical and biochemical relevance of blocking SCD1 to target CSCs by analyzing human colon cancer data from TCGA and through lipidomic profiling of CSCs with or without SCD1 inhibition using mass spectrometry. Positive associations between SCD1 expression and colorectal cancer patient clinical status and the expression of CSC-related genes (WNT and NOTCH signaling) were found based on TCGA data analysis. Lipidomic profiling of CSCs and bulk cancer cells (BCCs) using mass spectrometry revealed that colon CSCs contained a distinctive lipid profile, with higher free MUFA and lower free SFA levels than in BCCs, suggesting that enhanced SCD1 activity generates MUFAs that may support WNT signaling in CSCs. In addition, all identified phosphatidyl-ethanolamine-containing MUFAs were found at higher levels in CSCs. Interestingly, we observed lower phosphatidyl-serine (18:1/18:0), phosphatidyl-choline (PC; p-18:0/18:1)), and sphingomyelin (SM; d18:1/20:0 or d16:1/22:0) levels in CSCs than in BCCs. Of those, SCD1 inhibition, which efficiently diminished free MUFA levels, increased those specific PC and SM and MUFAs in CSCs promptly. These results suggest that these specific lipid composition is critical for CSC stem cell maintenance. In addition, not only free MUFAs, which are known to be required for WNT signaling, but also other phospholipids, such as SM, which are important for lipid raft formation, may mediate other cell signaling pathways that support CSC maintenance. Comparison of the lipidomic profiles of colon cancer cells with those of previously reported for glioma cells further demonstrated the tissue specific characteristics of lipid metabolism in CSCs. • Expression of the fatty acid desaturase SCD1 is associated with colon cancer progression and stemness. • Colon CSCs have a unique lipidomic profile that contains more MUFAs and fewer SFAs than that of bulk culture cells. • Nine phospholipid components with levels that differed between CSCs and bulk cancer cells were identified. • A sphingomyelin may be a possible lipid component that mediates different signaling pathways in CSCs and bulk cancer cells. [ABSTRACT FROM AUTHOR]

Phalloidin, a bicyclic heptapeptide found in Amanita mushroom, specifically binds to F-actin in the liver causing cholestatic hepatotoxicity. However, the toxicokinetics and tissue distribution properties of phalloidin as well as their underlying mechanisms have to be studied further. The area under the plasma concentration curve (AUC) of phalloidin increased in proportion to the doses (0.2, 0.4, and 0.8 mg/kg for intravenous injection and 2, 5, and 10 mg/kg for oral administration). Phalloidin exhibited dose-independent low volume of distribution (395.6–456.9 mL/kg) and clearance (21.4–25.5 mL/min/kg) and low oral bioavailability (2.4%–3.3%). This could be supported with its low absorptive permeability (0.23 ± 0.05 × 10−6 cm/s) in Caco-2 cells. The tissue-to-plasma AUC ratios of intravenously injected and orally administered phalloidin were the highest in the liver and intestines, respectively, and also high in the kidneys, suggesting that the liver, kidneys, and intestines could be susceptible to phalloidin exposure and that active transport via the hepatic and renal organic anion transporters (OATP1B1, OATP1B3, and OAT3) may contribute to the higher distribution of phalloidin in the liver and kidneys. • LC-HRMS method was validated for quantification of phalloidin in mouse plasma. • Toxicokinetics and tissue distribution of phalloidin were characterized in mice. • Phalloidin is a substrate for OAT3, OATP1B1, and OATP1B3. • Phalloidin showed no inhibitory effects on human major CYP, UGT, and transporters. [ABSTRACT FROM AUTHOR]

Abstract: DNA nanostructures have been widely studied in biomedical research contributing to targeted treatment of chronic diseases. The immunostimulatory XL‐DNA nanostructures of X‐shaped oligodeoxynucleotides complex are previously reported, activating toll‐like receptor9 in dendritic cells. This study examines whether the XL‐DNA could be therapeutically applied to treat immune diseases such as atopic dermatitis. To optimize topical delivery, liposome‐encapsulated XL‐DNA (Lipo‐XL‐DNA) is generated using emulsion transfer method with lipid layers composed of 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine, 1,2‐dioleoyl‐sn‐glycero‐3‐phospho‐(1′‐rac‐glycerol), and cholesterol. Size distribution of Lipo‐XL‐DNA ranges around 90–160 nm with mean diameter of 115.44 ± 18.72 nm. The morphology is confirmed by transmission electron microscope. Zeta potential is −28.59 mV. Confocal microscopy shows that Lipo‐XL‐DNA is efficiently delivered into epidermis and dermis. Topical application of Lipo‐XL‐DNA effectively alleviates atopic dermatitis symptoms in mice, as shown by dermatitis score, histological evaluation, and serum immunoglobulin E levels. RNA‐seq analysis confirms that Lipo‐XL‐DNA reduces pro‐inflammatory products, but increases epidermal barrier homeostasis factors in atopic dermatitis lesions. Lipo‐XL‐DNA orchestrates immune balance by downregulating Th2 immunity, but upregulating Th1 immunity. Collectively, liposome encapsulation enables efficient transdermal delivery of XL‐DNA, for an effective treatment of atopic dermatitis in mice. The results provide a promising therapeutic strategy using XL‐DNA nanostructures to treat immune‐compromised diseases. [ABSTRACT FROM AUTHOR]

The profiling of fatty acids (FAs) or sterols has been applied in clinical studies, but still needs to be improved to enable their simultaneous quantification. Moreover, little progress has been made in determining the levels of FAs and sterols in human saliva in a single run. In this study, gas chromatography-tandem mass spectrometry (GC–MS/MS) using one-step tert -butyldimethylsilyl (TBDMS) derivatization was developed for comprehensive profiling of 18 FAs (eight saturated, five monounsaturated, and five polyunsaturated FAs) and 7 sterols (cholesterol and its precursors). The TBDMS derivatization process was also optimized in terms of reaction solvent, catalyst, temperature, and reaction time. The optimized conditions resulted in better derivatization efficiency with good chromatographic separation through a high-temperature column within 23 min. The present method provided good linearity ( r  > 0.993), precision (coefficient of variation, 2.7% to 10.4%), and accuracy (91.5% to 103.4%). The overall recovery ranged from 73.8% to 114.3% for the 18 FAs, and from 68.9% to 79.8% for the 7 sterols. The validated method was applied to characterize FAs and sterols in human saliva samples. This is the first report of a GC–MS/MS method for the simultaneous determination of various FAs and sterols from a small volume (100 μL) of saliva. This approach can be used as a primary screening tool to examine the levels of both FAs and sterols in saliva, providing detailed information about their homeostasis for diagnostic and prognostic purposes. [ABSTRACT FROM AUTHOR]

Abstract Purpose: Red ginseng is one of the world's most popular herbal medicines; it exhibits a wide range of pharmacologic activities and is often co-ingested with other herbal and conventional medicines. This open-label, randomized, 3-period study investigated the in vivo herb–drug interaction potential for red ginseng extract with cytochrome P-450 (CYP) enzymes and organic anion-transporting polypeptide (OATP) 1B1. Methods: Fifteen healthy male volunteers (22-28 years; 57.1-80.8 kg) were administered a single dose of cocktail probe substrates (caffeine 100 mg, losartan 50 mg, omeprazole 20 mg, dextromethorphan 30 mg, midazolam 2 mg, and pitavastatin 2 mg) and single or multiple doses of red ginseng extract for 15 days. Findings: The pharmacokinetic profiles of the probe substrates and metabolites after single- or multiple-dose administration of red ginseng extracts were comparable to the corresponding profiles of the control group. The geometric mean ratio of AUC 0–t and 90% CIs for the probe substrate drugs between the control and multiple doses of red ginseng for 15 days were within 0.8 to 1.25 (CYP2C9, CYP3A4, and OATP1B1 probe substrates) or slightly higher (CYP1A2, CYP2C19, and CYP2D6 probe substrates). Additional assessments of the in vitro drug interaction potential of red ginseng extracts and the ginsenoside Rb1 on drug-metabolizing enzymes and transporters using human liver microsomes, cryopreserved human hepatocytes, and transporter-overexpressed cells were negative. Implications: Red ginseng poses minimal risks for clinically relevant CYP- or OATP-mediated drug interactions and is well tolerated. Clinical Research Information Service registry no.: [ABSTRACT FROM AUTHOR]

Regnase-1 is an endoribonuclease that regulates the stability of target genes. Here, we investigated whether Regnase-1 plays a regulatory role in the pathophysiology of atopic dermatitis, a chronic inflammatory skin disease. Regnase-1 levels were decreased in skin and serum of atopic dermatitis patients and mice. Regnase-1 +/- mice exhibited more severe atopic dermatitis symptoms than wild-type mice in a house dust mite allergen-induced atopic dermatitis model. Regnase-1 deficiency led to the global changes in gene expression related with innate immune and inflammatory responses, in particular chemokines. The skin Regnase-1 level had an inverse relationship with chemokine expression when we analyzed samples of atopic dermatitis patients and Regnase-1-deficient mice, suggesting that potentiated chemokine production contributes to the augmented inflammation at lesion sites. Subcutaneous administration of recombinant Regnase-1 to mice significantly ameliorated atopic dermatitis-like skin inflammation with reduced chemokine production in a house dust mite-induced atopic dermatitis NC/Nga mouse model. These results indicate that Regnase-1 plays an essential role in maintaining skin immune homeostasis as a regulator of chemokine expression. Modulating Regnase-1 activity may be an efficient therapeutic strategy for treating chronic inflammatory diseases, including atopic dermatitis. [Display omitted] • Regnase-1 expression is decreased in skin and serum of atopic dermatitis patients. • Regnase-1 deficiency is linked to aggravated inflammation in atopic dermatitis. • Regnase-1 deficiency exacerbates chemokine expression in the lesional skin. • Recombinant Regnase-1 ameliorates atopic dermatitis symptoms and chemokine production. • Regnase-1 is an efficient therapeutic target for maintaining skin immune homeostasis. [ABSTRACT FROM AUTHOR]

The objective of this study was to determine the absorption, excretion, and metabolism of a novel, oral antihyperglycemic drug, evogliptin, in male rats and dogs. Plasma, urine, feces, and expired air samples were collected after a single oral dose administration of [14C]evogliptin, samples were analyzed by measuring overall radioactivity levels using high-performance liquid chromatography (HPLC), and radioactivity levels were measured by utilizing LC-tandem mass spectrometry (LC-MS/MS). The total amounts of radioactivity excreted in urine, feces, and expired air up to 168 h after administration of [14C]evogliptin tartrate to rats (30 mg evogliptin/kg) and dogs (10 mg evogliptin/kg) were 96.7% and 96.8% of initial doses administered, respectively. The extent of urinary and fecal excretion in the rat up to 168 h constituted 29.7% and 66.5% of the given dose, respectively; and in dog was 43.3% and 53.5%, respectively. A total of 23 possible metabolites were detected with radiochromatograms of plasma, urinary, and fecal samples, but only the structures of 12 metabolites were identified via LC-MS/MS analysis. Evogliptin was the major component. Regarding the total radiochromatographic peak areas, peaks 9 (evogliptin acid) and 11 (hydroxyevogliptin) were the major metabolites in rats, and peaks 8 [4(S)-hydroxyevogliptin glucuronide], 15 [4(S)-hydroxyevogliptin], and 17 [4(R)-hydroxyevogliptin] were the predominant metabolites in dogs. Data demonstrated that evogliptin was the major component excreted in urine and feces of rats and dogs, but the metabolite profiles varied between species. [ABSTRACT FROM AUTHOR]

Itraconazole (ITZ) is a first-generation triazole-containing antifungal agent that effectively treats various fungal infections. As ITZ has a better safety profile than that of ketoconazole (KCZ), ITZ has been used worldwide for over 25 years. However, few reports have explored the metabolic profile of ITZ, and the underlying mechanism of ITZ-induced liver injury is not clearly understood. In the present study, we revisited ITZ metabolism in humans, using a non-targeted metabolomics approach, and identified several novel metabolic pathways including O -dearylation, piperazine oxidation, and piperazine- N , N ′-deethylation. Furthermore, we explored the formation of reactive ITZ metabolites using trapping agents as surrogates, to assess the possibility of metabolism-mediated toxicity. We found that ITZ and its metabolites did not form any adducts with nucleophiles including glutathione, potassium cyanide, and semicarbazide. The present study expands our knowledge of ITZ metabolism and supports the suggestion that ITZ has a better safety profile than that of KCZ in terms of metabolism-mediated toxicity. [ABSTRACT FROM AUTHOR]

Objective. The aetiology of gout is closely linked to the deposition of monosodium uric acid (MSU) crystals and the consequent activation of the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. In this study, we investigated whether oral administration of an NLRP3 inhibitor would be effective to attenuate the symptoms of gout. Methods. The effects of oral administration with sulforaphane (SFN) were examined in two mouse models of acute gout induced by injection of MSU crystals into footpads or air pouch. The production of caspase- 1 (p10) and IL-1β was examined by immunoblotting and ELISA as hallmarks of NLRP3 inflammasome activation. Results. Oral administration of SFN attenuated MSU crystal-induced swelling and neutrophil recruitment in a mouse foot acute gout model, correlating with the suppression of the NLRP3 inflammasome activation in foot tissues. Consistently, oral administration of SFN blocked MSU-crystal-induced activation of the NLRP3 inflammasome in a mouse air pouch gout model. SFN suppressed NLRP3 inflammasome activation induced by MSU crystals, adenosine triphosphate and nigericin but not by poly(dA:dT) in primary mouse macrophages, independent of the reactive oxygen species pathway. SFN inhibited ligand-independent activation of the NLRP3 inflammasome, suggesting that SFN may act directly on the NLRP3 inflammasome complex. Conclusion. Oral administration of SFN effectively alleviated acute gouty inflammation by suppression of the NLRP3 inflammasome. Our results provide a novel strategy in which oral treatment with SFN may be beneficial in preventing acute attacks of gout. [ABSTRACT FROM AUTHOR]

Metabolomics combined with high-resolution mass spectrometry (HR-MS) and multivariate data analysis has broad applications in the study of xenobiotic metabolism. Although information about xenobiotic metabolism is essential to understand toxic mechanisms, pharmacokinetic parameters and excretion pathways, it is limited to predict all generated metabolites in biological fluids. Here, we revisited sildenafil metabolism in human liver microsomes using a metabolomics approach to achieve a global picture of sildenafil phase 1 metabolism. Finally, 12 phase 1 metabolites were identified in human liver microsomes; M1-M5 were previously known metabolites. The chemical structures of the novel metabolites were elucidated by MS 2 fragmentation using an HR-MS system as follows: M6, reduced sildenafil; M7, N , N -deethylation and mono-oxidation; M8, demethanamine, N , N -deethylation and mono-hydroxylation; M9, demethanamine and N , N -deethylation; M10 and M11, mono-oxidation in the piperazine ring after N -demethylation; and M12, mono-oxidation. All metabolites, except M1, were formed by CYP3A4 and CYP3A5. In conclusion, we successfully updated the metabolic pathway of sildenafil in human liver, including 7 novel metabolites using metabolomics combined with HR-MS and multivariate data analysis. [ABSTRACT FROM AUTHOR]

Hydrophobicity modulates both the nucleus-to-mitochondria targeting switch and slow disulfide cleavage, resulting in mitochondria-targeted delivery of DOX@REPL-NG and release of DOX from DOX@REPL-NG. [Display omitted] • A new nanogel delivers and releases hydrophobic payloads to mitochondria targets. • Hydrophobicity modulates both the switching of organelle targeting and slow disulfide cleavage. • Hydrophobic lysines preferentially select mitochondria. • Hydrophobic neighbors cause glutathione to slowly cleave the disulfide for slow drug release. Despite the significance of organelle-targeted delivery and the subsequent release of therapeutics, the therapeutic outcome of this technology is limited due to undesired organelle targeting and unwanted cytosolic drug release. In this study, an ε-poly(l -lysine) (EPL)-based bioreducible nanogel (REPL-NG) was designed to deliver and release hydrophobic payloads to mitochondria targets, and the NG was synthesized via a sequential thiolation and oxidation reaction. REPL-NG contains a hydrophilic NG surface, which provides colloidal stability in aqueous environments due to the ionizable amine in lysine. Hydrophobic chemicals were encapsulated as a result of the multiple disulfide-mediated and crosslinked inner compartments. The capability of the ionizable amines to buffer protons helped the NG escape endolysosomes. The neighboring hydrophobic lysine preferentially drove the NG into mitochondria rather than the nucleus, and the neighboring hydrophobic disulfide bonds were slowly cleaved by the intracellularly reduced glutathione (GSH) in mitochondria. In an HCT116 xenograft tumor-bearing mouse model, doxorubicin (DOX)-loaded REPL-NG (DOX@REPL-NG) resulted in better blood circulation, better tumor accumulation and retention, and ultimately more effective inhibition of tumor growth than free drugs. In conclusion, hydrophobicity could modulate both the nucleus-to-mitochondria targeting switch and slow disulfide cleavage, resulting in mitochondria-targeted delivery of DOX@REPL-NG and release of DOX from DOX@REPL-NG. [ABSTRACT FROM AUTHOR]

In vitro metabolism of a new synthetic cannabinoid, EAM-2201, has been investigated with human liver microsomes and major cDNA-expressed cytochrome P450 (CYP) isozymes using liquid chromatography–high resolution mass spectrometry (LC–HRMS). Incubation of EAM-2201 with human liver microsomes in the presence of NADPH resulted in the formation of 37 metabolites, including nine hydroxy-EAM-2201 (M1-M9), five dihydroxy-EAM-2201 (M10-M14), dihydrodiol-EAM-2201 (M15), oxidative defluorinated EAM-2201 (M16), two hydroxy-M16 (M17 and M18), three dihydroxy-M16 (M19-M21), N- dealkyl-EAM-2201 (M22), two hydroxy-M22 (M23 and M24), dihydroxy-M22 (M25), EAM-2201 N- pentanoic acid (M26), hydroxy-M26 (M27), dehydro-EAM-2201 (M28), hydroxy-M28 (M29), seven dihydroxy-M28 (M30-M36), and oxidative defluorinated hydroxy-M28 (M37). Multiple CYPs, including CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2J2, 3A4, and 3A5, were involved in the metabolism of EAM-2201. In conclusion, EAM-2201 is extensively metabolized by CYPs and its metabolites can be used as an indicator of EAM-2201 abuse. [ABSTRACT FROM AUTHOR]

Cedrol, β-cedrene, and thujopsene are bioactive sesquiterpenes found in cedar essential oil and exert antiseptic, anti-inflammatory, antispasmodic, tonic, astringent, diuretic, sedative, insecticidal, and antifungal activities. These compounds are used globally in traditional medicine and cosmetics. The aim of this study was to investigate the inhibitory effects of cedrol, β-cedrene, and thujopsene on the activities of eight major human cytochrome P-450 (CYP) enzymes using human liver microsomes to assess potential β-cedrene-, cedrol-, and thujopsene–drug interactions. Cedrol, β-cedrene, and thujopsene were found to be potent competitive inhibitors of CYP2B6-mediated bupropion hydroxylase with inhibition constant (Ki) values of 0.9, 1.6, and 0.8 μM, respectively, comparable with that of a selective CYP2B6 inhibitor, thioTEPA (Ki, 2.9 μM). Cedrol also markedly inhibited CYP3A4-mediated midazolam hydroxylation with aKivalue of 3.4 μM, whereas β-cedrene and thujopsene moderately blocked CYP3A4. Cedrol, β-cedrene, and thujopsene at 100 μMnegligibly inhibited CYP1A2, CYP2A6, and CYP2D6 activities. Only thujopsene was found to be a mechanism-based inhibitor of CYP2C8, CYP2C9, and CYP2C19. Cedrol and thujopsene weakly inhibited CYP2C8, CYP2C9, and CYP2C19 activities, but β-cedrene did not. These in vitro results indicate that cedrol, β-cedrene, and thujopsene need to be examined for potential pharmacokinetic drug interactions in vivo due to their potent inhibition of CYP2B6 and CYP3A4. [ABSTRACT FROM PUBLISHER]

The accurate, precise, and robust quantification of endogenous biomarkers is a challenging task because of the presence of significantly low levels of endogenous compounds in biological samples, the absence of analyte-free matrix-matched calibrators, and sample instability due to in-vitro production or degradation of the analytes. Gamma-hydroxybutyric acid (GHB), a compound often used in drug-facilitated crimes, is a human neurotransmitter produced during both the biosynthesis and metabolism of gamma-aminobutyric acid (GABA). Evidently, proving GHB intoxication through the quantification of GHB and its metabolites in biological samples is not straightforward. This study aimed to develop a sensitive and accurate quantitative method for the simultaneous determination of endogenous GHB and its metabolic precursors and products (glutamic acid, GABA, succinic acid, 2,4-dihydroxybutyric acid, 3,4-dihydroxybutyric acid, glycolic acid, and succinylcarnitine) in human urine using LC-MS/MS. For this purpose, chemical derivatization with benzoyl chloride was employed to improve the sensitivity to glutamic acid and GABA. Synthetic urine was used to prepare calibrators, and the validity of this approach was fully demonstrated, particularly focusing on the instability issues. The validation results proved the method to be selective, sensitive, accurate, and precise, with acceptable linearity within calibration ranges. Moreover, our results regarding the in-vitro production or degradation of metabolites highlight the effects of handling and storage conditions of urine samples. Finally, this effective analytical method is expected to be useful in studying the relationship between GHB intoxication and metabolic alterations and, thus, discovering practical biomarkers for GHB ingestion. [Display omitted] - Accurate and reproducible quantification of endogenous biomarkers is challenging - A quantitative method for endogenous GHB and biomarker metabolites was developed - Chemical derivatization-assisted LC-MS/MS improved sensitivity - The use of a surrogate matrix and the instability issue were fully investigated [ABSTRACT FROM AUTHOR]

1. Few studies describing the pharmacokinetic properties of chlorogenic acid (CA) and corydaline (CRD) which are marker compounds of a new prokinetic botanical agent, DA-9701, have been reported. The aim of the present study is to evaluate the pharmacokinetic properties CA and CRD following intravenous and oral administration of pure CA (1-8 mg/kg) or CRD (1.1-4.5 mg/kg) and their equivalent dose of DA-9701 to rats. 2. Dose-proportional AUC and dose-independent clearance (10.3-12.1 ml/min/kg) of CA were observed following its administration. Oral administration of CA as DA-9701 did not influence the oral pharmacokinetic parameters of CA. Incomplete absorption of CA, its decomposition in the gastrointestinal tract, and/or pre-systemic metabolism resulted in extremely low oral bioavailability ( F) of CA (0.478-0.899%). 3. CRD showed greater dose-normalized AUC in the higher dose group than that in lower dose group(s) after its administration due to saturation of its metabolism via decreased non-renal clearance (by 51.3%) and first-pass extraction. As a result, the F of CRD following 4.5 mg/kg oral CRD (21.1%) was considerably greater than those of the lower dose groups (9.10 and 13.8%). However, oral administration of CRD as DA-9701 showed linear pharmacokinetics as a result of increased AUC and F in lower-dose groups (by 182% and 78.5%, respectively) compared to those of pure CRD. The greater oral AUC of CRD for DA-9701 than for pure CRD could be due to decreased hepatic and/or GI first-pass extraction of CRD by other components in DA-9701. [ABSTRACT FROM AUTHOR]

1. Hederacoside C (HDC) is one of the active ingredients in Hedera helix leaf extract (Ivy Ex.) and AG NPP709, a new botanical drug to treat acute respiratory infection and chronic inflammatory bronchitis. However, information regarding its pharmacokinetic properties remains limited. 2. Here, we report the pharmacokinetics of HDC in rats after intravenous administration of HDC (3, 12.5, and 25 mg/kg) and after oral administration of HDC, Ivy Ex., and AG NPP709 (equivalent to 12.5, 25, and 50 mg/kg HDC). 3. Linear pharmacokinetics of HDC were identified upon its intravenous administration at doses of 3-25 mg/kg. Intravenous administration of HDC results in relatively slow clearance (1.46-2.08 mL/min/kg) and a small volume of distribution at steady state (138-222 mL/kg), while oral administration results in a low absolute oral bioavailability ( F) of 0.118-0.250%. The extremely low F of HDC may be due to poor absorption of HDC from the gastrointestinal (GI) tract and/or its decomposition therein. 4. The oral pharmacokinetics of HDC did not differ significantly among pure HDC, Ivy Ex., and AG NPP709. [ABSTRACT FROM AUTHOR]

α-Cedrene is a pharmacologically active ingredient isolated from the essential oil of cedar. A selective and sensitive GC-MS/MS method was developed for the quantification of α-cedrene in rat plasma for the first time. α-Cedrene was extracted from rat plasma using ethyl acetate at neutral pH. The analytes were determined in selective reaction monitoring mode using MS/MS: m/z 204.3→119.0 for α-cedrene and m/z 146.0→111.0 for 1,4-dichlorobenzene (internal standard). The standard curve was linear ( r2 ≥ 0.995) over the concentration ranges of 5-800 ng/mL. The lower limit of quantification was 5 ng/mL using 50 μL of rat plasma. The coefficient of variation and relative error for intra- and interassays at four quality control levels were 3.1-13.9% and −4.0-2.6%, respectively. The stability of processing (freeze-thaw, long-term storage at −80°C, and short-term storage at room temperature) and chromatography (reinjection) was shown to be of insignificant effect. The present method was applied successfully to the pharmacokinetic study of α-cedrene after its intravenous (10 mg/kg) and oral (25 mg/kg) administration in male Sprague-Dawley rats. [ABSTRACT FROM AUTHOR]

Highlights: [•] We demonstrated a novel function of RSK2 in stress tolerance. [•] RSK2 deficiency enhanced apoptosis by calcium stress. [•] RSK2-mediated GSK3β phosphorylation at serine 9 increased calcium-induced stress tolerance. [•] Calcium stress-induced apoptosis inhibited by adding back of RSK2 into RSK2−/− MEFs. [Copyright &y& Elsevier]

Due to increasing safety and intracellular delivery concerns about hydrophilic polymers in amphiphilic polymer-based nanoparticles (NPs), this study investigates small hydrophilic molecule-stabilized NPs for effective intracellular delivery with multiorganelle targetability and dual responsiveness to acidic pH/glutathione (GSH). In the construction of small hydrophilic molecule-stabilized NP (MSPCL-NP), the A-B-A-type amphiphilic polymer (MSPCL-P) is composed of two short hydrophilic carboxylate-capped disulfide derivatives (A) that replace hydrophilic polymers and assist in providing colloidal stability and preventing antibody (e.g., at least anti-PEG antibody)-mediated specific interactions and complement activation in the plasma and a hydrophobic multiple disulfide-containing poly(ε-caprolactone) block (B) that carries hydrophobic drugs. The carboxylates on the surface of MSPCL-NP target the acidic extratumoral/endolysosomal milieu by sensing and buffering acidic pH values, and the hydrophobic carboxylic acids improve adsorptive endocytosis and effective endosomal escape. Multiple disulfide linkages selectively target cytosolic GSH, resulting in rapid drug release from the destroyed MSPCL-NP via the cleavage of disulfide bonds in MSPCL-P. Doxorubicin (DOX)-loaded NP (DOX@MSPCL-NP) exerts strong effects on killing cells in vitro and inhibits tumor growth in HCT116 xenograft tumor-bearing mice. In conclusion, the multifunctionality and multispatial targetability of MSPCL-NP might effectively overcome various sequential drug delivery hurdles, ranging from blood circulation to drug release. Furthermore, the introduction of small hydrophilic molecules represents a potential strategy to make self-assembled NPs without the use of hydrophilic polymers. [ABSTRACT FROM AUTHOR]

Inflammation and immunity are linked to the onset and development of obesity and metabolic disorders. Pattern recognition receptors (PRRs) are key regulators of inflammation and immunity in response to infection and stress, and they have critical roles in metainflammation. In this study, we investigated whether RIG-I (retinoic acid-inducible gene I)-like receptors were involved in the regulation of obesity-induced metabolic stress in RIG-I knockout (KO) mice fed a high-fat diet (HFD). RIG-I KO mice fed an HFD for 12 weeks showed greater body weight gain, higher fat composition, lower lean body mass, and higher epididymal white adipose tissue (eWAT) weight than WT mice fed HFD. In contrast, body weight gain, fat, and lean mass compositions, and eWAT weight of MDA5 (melanoma differentiation-associated protein 5) KO mice fed HFD were similar to those of WT mice fed a normal diet. RIG-I KO mice fed HFD exhibited more severely impaired glucose tolerance and higher HOMA-IR values than WT mice fed HFD. IFN-β expression induced by ER stress inducers, tunicamycin and thapsigargin, was abolished in RIG-I-deficient hepatocytes and macrophages, showing that RIG-I is required for ER stress-induced IFN-β expression. Our results show that RIG-I deficiency promotes obesity and insulin resistance induced by a high-fat diet, presenting a novel role of RIG-I in the development of obesity and metabolic disorders. [ABSTRACT FROM AUTHOR]

ABSTRACT Obovatol, a major constituent of the leaves of Magnolia obovata Thunb, is known to inhibit nuclear factor-κB activity and arachidonic acid-induced platelet aggregation. This study was performed to identify the metabolites of obovatol in human liver microsomes. Human liver microsomes incubated with obovatol in the presence of NADPH and/or UDPGA resulted in the formation of six metabolites, M1-M6. M1 and M2 were identified as hydroxyobovatol, on the basis of liquid chromatography/tandem mass spectrometric (LC-MS/MS) analysis. M1, M2 and obovatol were further metabolized to their glucuronide conjugates, obovatol-glucuronide (M3), obovatol-diglucuronide (M4) and hydroxyobovatol-glucuronide (M5 and M6). The inhibitory potency of obovatol on eight major human P450s was also investigated in human liver microsomes. In these experiments, obovatol strongly inhibited CYP2C19-mediated S-mephenytoin hydroxylase activity with an IC50 value of 0.8 µ m, which could have implications for drug-drug interactions. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Our previous report demonstrated that RSK2 plays an important role in cell proliferation and transformation induced by tumor promoters such as epidermal growth factor mediated through the N-terminal kinase domain of RSK2 in JB6 Cl41 mouse skin epidermal cells in vitro. However, no direct evidence has been reported regarding the relationship of RSK2 activity and human skin cancer. To elucidate the relationship of RSK2 activity and human skin cancer, we examined the effect of knocking down RSK2 expression on epidermal growth factor-induced anchorage-independent transformation in the premalignant HaCaT human skin keratinocyte cell line and on soft agar colony growth of SK-MEL-28 malignant melanoma cells. We found that the phosphorylated protein levels of RSK2 were enhanced in cancer tissues compared with normal tissues in a human skin cancer tissue array. We found that UVB stimulation induced increased in not only the total and phosphorylated protein levels of ERKs and RSK2 but also the nuclear localization and gene expression of RSK2. RSK2 knockdown inhibited proliferation and anchorage-independent transformation of HaCaT cells and soft agar colony growth of malignant melanoma cells. Moreover, RSK2–/– mouse embryonic fibroblast (MEF) showed enhanced sub-G1 accumulation induced by UVB stimulation compared with RSK2+/+ MEFs, indicating that RSK2 might play an important role in tolerance against stress associated with ultraviolet. Importantly, activated RSK2 protein levels were highly abundant in human skin cancer tissues compared with matched skin normal tissues. Taken together, our results demonstrated that RSK2 plays a key role in neoplastic transformation of human skin cells and in skin cancer growth. [ABSTRACT FROM PUBLISHER]

Abstract: The objective of this study was to develop a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to determine the stability of collagen pentapeptide (KTTKS), which is a subfragment of collagen and has been proved to promote the extracellular release of collagen in skin fibroblast, in rat skin. The chromatographic condition was optimized on an Acclaim C-18 column (2.1mm×150mm, 3μm) under isocratic elution using a mobile phase consisting of deionized water and acetonitrile (87:13, v/v) mixture containing 5mM pentafluoropropionic acid as an ion-pairing reagent. The quantitation of KTTKS was performed on a triple quadrupole mass spectrometer in multiple reaction monitoring mode. The calibration curve showed good linearity in the concentration range of 0.05–10.0μg/mL (r 2 >0.999). The intra- and inter-day precisions were 0.8–6.5% and 2.4–5.8%, respectively, and the intra- and inter-day accuracies were 96.3–102.7% and 92.8–98.5%, respectively. The developed LC–MS/MS method was successfully applied to investigate the degradation rate and sites of KTTKS in rat skin homogenate. KTTKS was found to be very susceptible to the peptide bond cleavage by aminopeptidases present in the skin. [Copyright &y& Elsevier]

Background/objectives: Preoperative nutritional status is associated with postoperative complications. Prealbumin, a visceral protein, is sensitive to protein malnutrition. The objective of this study is to evaluate the role of preoperative prealbumin levels as a marker for predicting complications after gastric surgery.Methods: An observational study was performed on 183 patients who underwent gastric surgery due to benign or malignant gastric disease at Seoul National University Hospital (SNUH) between August 2009 and October 2010. Preoperative prealbumin levels were also measured. Nutritional variables such as prealbumin (cutoff value, 18 mg/dL), albumin, body mass index (BMI), and clinicopathologic data were collected. Postoperative hospital stay, 30-day complications and mortality rate were obtained to investigate outcomes.Results: The complication rate was 52% in the abnormal prealbumin group (n = 23) and 24% in the normal prealbumin group (n = 160; p = 0.005). The complication rate was higher in patients with low preoperative albumin levels (<3.5 g/dL) and abnormal BMI (<18.5 kg/m(2)), but the differences were not statistically significant. Comorbidity of diabetes mellitus (DM), resection extent, combined resection, TNM stage and prealbumin levels were associated with complications. In multivariate analysis, DM and combined resection were significantly correlated with complications (p = 0.001 for each). In subgroup analysis, resection extent, approach, combined resection, TNM stage, and prealbumin levels were significantly associated with infectious complications. Multivariate analysis identified combined resection (p = 0.001) and prealbumin levels (p = 0.032) as independent variables.Conclusions: Preoperative prealbumin levels could be a useful marker for predicting complications, especially infectious complications, after gastric surgery. [ABSTRACT FROM AUTHOR]

Magnolin is a major bioactive component found in Shin-i, the dried flower buds of Magnolia fargesii; it has anti-inflammatory and anti-histaminic activities. Incubation of magnolin in human liver microsomes with an nicotinamide adenine dinucleotide phosphate-generating system resulted in the formation of five metabolites, namely, O-desmethyl magnolin (M1 and M2), didesmethylmagnolin (M3), and hydroxymagnolin (M4 and M5). In this study, we characterized the human liver cytochrome P450 (CYP) enzymes responsible for the biotransformation of three major metabolites—M1, M2, and M4—of magnolin. CYP2C8, CYP2C9, CYP2C19, and CYP3A4 were identified as the major enzymes responsible for the formation of the two O-desmethyl magnolins (M1 and M2), on the basis of a combination of correlation analysis and experiments, including immunoinhibition of magnolin in human liver microsomes and metabolism of magnolin by human cDNA-expressed CYP enzymes. CYP2C8 played a predominant role in the formation of hydroxymagnolin (M4). These results suggest that the pharmacokinetics of magnolin may not be affected by CYP2C8, CYP2C9, CYP2C19, and CYP3A4 responsible for the metabolism of magnolin or by the co-administration of appropriate CYP2C8, CYP2C9, CYP2C19, and CYP3A4 inhibitors or inducers due to the involvement of multiple CYP enzymes in the metabolism of magnolin. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has received considerable attention as a potential anticancer agent. However, recombinant Apo2L/TRAIL has several limitations, which include a weak pharmacokinetic profile, namely, a short biological half-life and rapid renal clearance, and an inability to form a homotrimeric structure. In this research, we attempted to develop a sustained release nanoparticle (NP) formulation that stabilizes Apo2L/TRAIL and preserves its antitumor activity. Apo2L/TRAIL-loaded human serum albumin (HSA) NPs were prepared using a desolvation technique optimized by particle size, zeta-potential, and entrapment efficiency. Apo2L/TRAIL in HSA-NPs continuously released over 24 h at 37°C in phosphate buffered saline and rat plasma condition, and the biological activity of Apo2L/TRAIL-HSA-NPs was preserved (IC = 67.2 ng/mL versus Apo2L/TRAIL IC = 55.4 ng/mL) with negligible activity loss. Furthermore, in vivo pharmacokinetic profiles and tumor distribution demonstrated the superiority of Apo2L/TRAIL-HSA-NPs over Apo2L/TRAIL. The circulating half-life period was significantly prolonged from 9.8 to 90.7 min (9.2-fold enhancement), and drug bioavailability was clearly enhanced on the basis of area under the curve analysis (2.7-fold). And tumor distribution of Apo2L/TRAIL-HSA-NPs was also increased at 1 h after injection, which was about 14-fold (1-h point) over that of Apo2L/TRAIL. These results show that Apo2L/TRAIL-loaded HSA-NPs should be considered as potential long-acting cancer agents. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:482-491, 2011 [ABSTRACT FROM AUTHOR]

Abstract: A purified extract isolated from the dried flower buds of Magnolia fargesii (NDC-052) is currently being evaluated for phase III clinical trials as a new anti-asthma drug. A rapid, sensitive and selective liquid chromatography–tandem mass spectrometric (LC/MS/MS) method for the simultaneous determination of magnolin and epimagnolin A, the major bioactive components of NDC-052, in rat plasma was developed. After liquid–liquid extraction with tolterodine as an internal standard, magnolin and epimagnolin A were separated on a Luna phenyl-hexyl column with the mobile phase of 70% methanol in 10mM ammonium formate. The analytes were detected by an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curves were linear over the concentration range of 50–2500ng/mL for magnolin and epimagnolin A in rat plasma. The intra- and inter-day coefficients of variation and relative errors for magnolin and epimagnolin A at four QC concentrations were 1.5–11.4% and 5.9–12.5%, respectively. The lower limits of quantification for magnolin and epimagnolin A were 50.0ng/mL using 50μL of plasma. This method was successfully applied to the pharmacokinetic study of magnolin and epimagnolin A after an oral administration of NDC-052 in male Sprague–Dawley rats. [Copyright &y& Elsevier]

Abstract: Environmental and physiological stresses such as heat shock, oxidative stress, heavy metals, and pathogenic conditions induce cellular stress response. This response is often mediated by heat shock proteins that function as molecular chaperones. A stress-inducible cochaperone, Sti1/Hop (Hsp organizer protein), functions as an adaptor protein that simultaneously binds with Hsp70 and Hsp90 to transfer client proteins from Hsp70 to Hsp90. However, the biological role of STI-1 in vivo is poorly understood in metazoans. Here, we report the characterization of the Caenorhabditis elegans homolog of Sti1/Hop, which is approximately 56% identical with human STI-1. C. elegans STI-1 (CeSTI-1) is expressed in the pharynx, intestine, nervous system, and muscle from larvae to adults. Analysis of proteins immunoprecipitated with anti-STI-1 antibody by mass spectrometry revealed that CeSTI-1 can bind with both Hsp70 and Hsp90 homologs like its mammalian counterpart. sti-1 expression is elevated by heat stress, and an sti-1(jh125) null mutant shows decreased fertility under heat stress conditions. These mutants also show abnormally high lethality in extreme heat and may be functioning with DAF-16 in thermotolerance. In addition, sti-1(jh125) mutants have a shortened life span. Our results confirm that CeSTI-1 is a cochaperone protein that may maintain homeostatic functions during episodes of stress and can regulate longevity in nematodes. [Copyright &y& Elsevier]

Abstract: For the rapid, selective and sensitive analysis of metoclopramide in human plasma, hydrophilic interaction chromatography with electrospray ionization tandem mass spectrometric (HILIC/MS/MS) method was developed. This method involved liquid–liquid extraction with dichloromethane followed by separation on an Atlantis HILIC silica column using the mobile phase of acetonitrile–ammonium formate (100mM, pH 6.5) (85:15, v/v). Analytes were quantified using electrospray ionization mass spectrometry in the selected reaction monitoring mode. The standard curve was linear (r 2 =0.998) over the concentration range of 2.00–150ng/mL using 50μL of plasma sample. The coefficient of variation and relative error for intra- and inter-assay at four QC levels were 1.8–7.7% and −7.5 to 3.6%, respectively. The matrix effect for metoclopramide and levosulpiride (internal standard) was practically absent. The present method was successfully applied to the pharmacokinetic study of metoclopramide after oral dose of metoclopramide hydrochloride (10mg) to male healthy volunteers. [Copyright &y& Elsevier]

Abstract: Jaceosidin (4′,5,7-trihydroxy–3′,6-dimethoxyflavone), isolated from Artemisia species as well as Eupatorium species, has antiallergic, anticancer, anti-inflammatory and antioxidant activity. A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for the quantification of jaceosidin in rat plasma was developed to characterize the pharmacokinetics of jaceosidin. Jaceosidin and the internal standard, linezolid, were extracted from rat plasma with ethyl acetate at acidic pH and analyzed on a Luna phenyl-hexyl column using the mixture of acetonitrile and 0.1% formic acid (45:55, v/v) as a mobile phase. The analytes were determined using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The calibration curve was linear (r 2 =0.9973) over the concentration range of 2.00–500ng/ml. The lower limit of quantification for jaceosidin was 2.0ng/ml using 50μl of plasma sample. The coefficients of variation of intra- and inter-assay at four QC levels were 2.4–9.6% and the relative errors were −9.1 to 10.0%. The matrix effects for jaceosidin and linezolid were practically absent. The recoveries of jaceosidin and linezolid were 87.0 and 87.7%, respectively. This method was successfully applied to the pharmacokinetic study of jaceosidin in rats. [Copyright &y& Elsevier]

Multiresidual pesticide analysis in hair can provide useful perspectives on the relationship between pesticides and human health. To establish a rapid and simultaneous analytical method using LC-MS/MS and GC-MS/MS, optimization of hair pulverization, extraction solvent and purification with dispersive SPE was performed for 300 pesticides. Hair pulverization was standardized with a ball mill, at 30 Hz for 20 min (10 min twice), using 3-mm diameter beads. For extraction, 0.1% formic acid in acetonitrile was selected, and PSA d-SPE was chosen for clean-up among three different types of solid phase extraction. The limits of quantitation (LOQs) in this method were between 2.5 and 7.5 pg mg-1. In recovery test, fifty milligrams of hair powder were extracted with 0.1% formic acid in acetonitrile, and incubated for three h at 40 ℃. The crude extract was treated using PSA-dSPE, dried under nitrogen gas, and reconstructed with acetonitrile. An aliquot was analyzed with LC- and GC-MS/MS. Recovery ranges were 22.7–131.1%, in LC-MS/MS analysis, and 81.1–151.8% in GC-MS/MS analysis. The validated analysis systems were applied to biomonitoring of ten agricultural workers, and residues of 28 target pesticides were detected in their hair. [Display omitted] • A simultaneous and rapid analysis of 300 pesticide residues in hair using LC-MS/MS and GC-MS/MS. • Optimum pulverization parameters were established for hair. • Acidified extraction solvent and purification with primary secondary amine dSPE were used for efficient hair preparation. • The method successfully measured 28 pesticides in hair of 10 agricultural workers. [ABSTRACT FROM AUTHOR]