Pharmacokinetics and Intestinal Metabolism of Compound K in Rats and Mice.

We aimed to investigate the plasma concentration, tissue distribution, and elimination of compound K following the intravenous administration of compound K (2 mg/kg) in rats and mice. The plasma concentrations of compound K in mice were much higher (about five-fold) than those in rats. In both rats and mice, compound K was mainly distributed in the liver and underwent biliary excretion. There was 28.4% fecal recovery of compound K in mice and 13.8% in rats, whereas its renal recovery was less than 0.1% in both rats and mice. Relative quantification of compound K and its metabolite protopanaxadiol (PPD) in rat bile and intestinal feces indicated that the metabolism from compound K into PPD occurred in the intestine but not in the plasma. Therefore, PPD detected in the plasma samples could have been absorbed from the intestine after metabolism in control rats, while PPD could not be detected in the plasma samples from bile duct cannulated rats. In conclusion, mice and rats shared common features such as exclusive liver distribution, major excretion pathway via biliary route, and intestinal metabolism to PPD. However, there were significant differences between rats and mice in the plasma concentrations of compound K and the fecal recovery of compound K and PPD.

Simultaneous Determination and Pharmacokinetic Characterization of Glycyrrhizin, Isoliquiritigenin, Liquiritigenin, and Liquiritin in Rat Plasma Following Oral Administration of Glycyrrhizae Radix Extract.

Glycyrrhizae Radix is widely used as herbal medicine and is effective against inflammation, various cancers, and digestive disorders. We aimed to develop a sensitive and simultaneous analytical method for detecting glycyrrhizin, isoliquiritigenin, liquiritigenin, and liquiritin, the four marker components of Glycyrrhizae Radix extract (GRE), in rat plasma using liquid chromatography-tandem mass spectrometry and to apply this analytical method to pharmacokinetic studies. Retention times for glycyrrhizin, isoliquiritigenin, liquiritigenin, and liquiritin were 7.8 min, 4.1 min, 3.1 min, and 2.0 min, respectively, suggesting that the four analytes were well separated without any interfering peaks around the peak elution time. The lower limit of quantitation was 2 ng/mL for glycyrrhizin and 0.2 ng/mL for isoliquiritigenin, liquiritigenin, and liquiritin; the inter- and intra-day accuracy, precision, and stability were less than 15%. Plasma concentrations of glycyrrhizin, isoliquiritigenin, liquiritigenin, and liquiritin were quantified for 24 h after a single oral administration of 1 g/kg GRE to four rats. Among the four components, plasma concentration of glycyrrhizin was the highest and exhibited a long half-life (23.1 ± 15.5 h). Interestingly, plasma concentrations of isoliquiritigenin and liquiritigenin were restored to the initial concentration at 4-10 h after the GRE administration, as evidenced by liquiritin biotransformation into isoliquiritigenin and liquiritigenin, catalyzed by fecal lysate and gut wall enzymes. In conclusion, our analytical method developed for detecting glycyrrhizin, isoliquiritigenin, liquiritigenin, and liquiritin could be successfully applied to investigate their pharmacokinetic properties in rats and would be useful for conducting further studies on the efficacy, toxicity, and biopharmaceutics of GREs and their marker components.

Effect of Red Ginseng Extract on the Pharmacokinetics and Efficacy of Metformin in Streptozotocin-Induced Diabetic Rats.

The purpose of this study was to investigate the effect of red ginseng extract on the pharmacokinetics (PK) and efficacy of metformin in streptozotocin-induced diabetic rats. The diabetes mellitus rat model was established by intraperitoneally administering multiple doses of streptozotocin (30 mg/kg, twice on day 1 and 8), and diabetic rats received metformin 50 mg/kg with or without single or multiple administration of Korean red ginseng extract (RGE, 2 g/kg/day, once or for 1 week). RGE administration did not affect the plasma concentration and renal excretion of metformin. Further, diabetic rats were administered metformin (50 mg/kg) and RGE (2 g/kg) alone or concomitantly for 5 weeks, and both regimens decreased the fasting blood glucose and glycated hemoglobin (Hb-A1c) levels. Furthermore, fasting blood glucose levels were reduced by metformin or RGE administered alone but recovered to the control level following co-administration, suggesting that the effect was additive. However, triglyceride and free fatty acid levels were not different with metformin and RGE treatment alone or in combination. Biochemical parameters such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides, total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol levels were not different among the three treatment groups. In conclusion, RGE and metformin showed an additive effect in glycemic control. However, the co-administration of RGE and metformin did not cause PK interactions or affect biochemical parameters including the free fatty acid, triglyceride, AST, ALT, or cholesterol levels.

Decision-Making Deficits Are Associated With Learning Impairments in Female College Students at High Risk for Anorexia Nervosa: Iowa Gambling Task and Prospect Valence Learning Model.

This study investigated deficits in decision-making ability in female college students at high risk for anorexia nervosa (AN) using the Iowa Gambling Task (IGT) and the prospect valence learning (PVL) model. Based on scores on the Korean version of the Eating Attitude Test-26 (KEAT-26), participants were assigned to either the high risk for AN group (n = 42) or the control group (n = 43). The high risk for AN group exhibited significantly lower total net scores and block net scores on the third, fourth, and fifth blocks of the IGT than the control group did. The high risk for AN group selected cards significantly more often from the disadvantageous A and B decks and less often from the advantageous D deck than the control group did. In addition, the block net scores of the high risk for AN group did not differ across the five blocks, whereas those of the control group increased as the trials progressed. There was a significant negative correlation between IGT total net score and total score on the KEAT-26. The high risk for AN group had significantly lower values than the control group on the learning and response consistency parameters of the PVL model. These results indicate that female college students at high risk for AN have deficits in decision-making ability, and that these deficits are related to difficulties in remembering experience obtained from earlier trials and applying it to later trials. These difficulties further lead them to make decisions randomly.

Decision-making deficits in patients with chronic schizophrenia: Iowa Gambling Task and Prospect Valence Learning model.

PURPOSE: Decision-making is the process of forming preferences for possible options, selecting and executing actions, and evaluating the outcome. This study used the Iowa Gambling Task (IGT) and the Prospect Valence Learning (PVL) model to investigate deficits in risk-reward related decision-making in patients with chronic schizophrenia, and to identify decision-making processes that contribute to poor IGT performance in these patients. MATERIALS AND METHODS: Thirty-nine patients with schizophrenia and 31 healthy controls participated. Decision-making was measured by total net score, block net scores, and the total number of cards selected from each deck of the IGT. PVL parameters were estimated with the Markov chain Monte Carlo sampling scheme in OpenBugs and BRugs, its interface to R, and the estimated parameters were analyzed with the Mann-Whitney U-test. RESULTS: The schizophrenia group received significantly lower total net scores compared to the control group. In terms of block net scores, an interaction effect of group × block was observed. The block net scores of the schizophrenia group did not differ across the five blocks, whereas those of the control group increased as the blocks progressed. The schizophrenia group obtained significantly lower block net scores in the fourth and fifth blocks of the IGT and selected cards from deck D (advantageous) less frequently than the control group. Additionally, the schizophrenia group had significantly lower values on the utility-shape, loss-aversion, recency, and consistency parameters of the PVL model. CONCLUSION: These results indicate that patients with schizophrenia experience deficits in decision-making, possibly due to failure in learning the expected value of each deck, and incorporating outcome experiences of previous trials into expectancies about options in the present trial.

Highly efficient gene transfection by a hyperosmotic polymannitol based gene tranporter through regulation of caveolae and COX-2 induced endocytosis.

The regulation of cellular uptake to cross the cell membrane is one of the key strategies of importance for efficient gene transfection of non-viral vectors. Hyperosmotic activity of polyplexes may facilitate crossing of the membrane barrier by elevating the osmolarity of the extracellular matrix. In this study, we demonstrated that a polymannitol based gene transporter (PMGT) utilizes the hyperosmoticity contributed by the polymannitol backbone leading to accelerated cellular uptake and enhanced gene transfection. Mannitol dimethacrylate (MDM) monomer was synthesized by esterification of mannitol and methacryloyl chloride. The prepared MDM was then cross-linked with low molecular weight (LMW) branched polyethyleneimine (bPEI) by Michael addition reaction to produce PMGT. PMGT provided polyplex stability in serum, low cytotoxicity, and degradability due to the ester linkages present in the polymannitol backbone. Elevated transfection activity and efficiency, both in vitro and in vivo, were achieved by modulating the mode of cellular uptake due to the effect of the hyperosmotic properties of PMGT. Cyclooxygenase-2 (COX-2) inhibition by SC58236 revealed the up-regulation of this osmoprotectant molecule against the hyperosmotic activity of polymannitol, inducing rapid endocytosis of PMGT in order to re-balance the hyperosmotic environment. Various inhibition studies of endocytosis showed caveolae-mediated endocytosis to be the main route of cellular internalization to account for the enhanced transgene expression.

Methylmercury induces caspase-dependent apoptosis and autophagy in human neural stem cells.

Methylmercury (MeHg) is a well-known human neurotoxic agent whose exposure sources are mainly environmental and aquatic-derived food. MeHg is reported to induce central nervous system disability. However, the exact mechanism of MeHg-induced neurotoxicity is still unknown. In this study, to investigate which cell death signaling pathway is related with MeHg-induced cytotoxicity, the effects of MeHg on apoptosis and autophagy were evaluated in HB1.F3 human neural stem cells (NSCs). Human NSCs were treated with 1 µM of MeHg for 48 hr and the effect of MeHg on cell signaling pathway was elucidated. MeHg inhibited Akt1/mTOR signaling that led to induction of caspase-dependent apoptosis and autophagy in the NSCs. Furthermore, retinoic acid (RA)-induced neuronal differentiation was inhibited by MeHg. Taken together, these results suggest that MeHg inhibits the differentiation of human NSCs by induction of caspase-dependent apoptosis and autophagy.

Overexpression of beclin1 induced autophagy and apoptosis in lungs of K-rasLA1 mice.

Beclin1, as a key molecule in controlling autophagy pathway, can activate both cell survival and cell death pathway. As a role of autophagy in cancer progression remains controversial, introduction of beclin1 to the lungs of K-ras(LA1) mice was performed via inhalation. Prolonged autophagy activation was induced by repeated exposure of lentivirus-beclin1, total of 8 times (2 times/week, 4 weeks). By the time of sacrifice, lungs were collected and analyzed for the therapeutic efficacy. Total numbers of tumors on the surface and histopathological tumor progression were reduced in the lungs of K-ras(LA1) mice. Successful delivery of beclin1 induced autophagy and apoptosis in the target organ, which were confirmed by following features; increased autophagic vacuoles in the cytosol, increased number of mitochondria with decreased mitochondrial 12S RNA, and increased protein levels of mitochondria-related apoptosis. Markers for cell proliferation and angiogenesis, PCNA and VEGF, which used for prediction of cancer prognosis, were significantly reduced after introduction of beclin1. Taken together, the results indicate that autophagy regulating gene, beclin1, can be a potential target for lung cancer gene therapy.

Aerosol delivery of lentivirus-mediated O-glycosylation mutant osteopontin suppresses lung tumorigenesis in K-ras (LA1) mice.

BACKGROUND: Osteopontin (OPN) is a secreted glycophosphoprotein that has been implicated in the regulation of cancer development. The function of OPN is primarily regulated through post-translational modification such as glycosylation. As yet, however, the relationship between OPN glycosylation and lung cancer development has not been investigated. In this study, we addressed this issue by studying the effect of a triple mutant (TM) of OPN, which is mutated at three O-glycosylation sites, on lung cancer development in K-ras (LA1) mice, a murine model for human non-small cell lung cancer. METHODS: Aerosolized lentivirus-based OPN TM was delivered into the lungs of K-ras (LA1) mice using a nose-only-inhalation chamber 3 times/wk for 4 wks. Subsequently, the effects of repeated delivery of OPN TM on lung tumorigenesis and its concomitant OPN-mediated signaling pathways were investigated. RESULTS: Aerosol-delivered OPN TM inhibited lung tumorigenesis. In addition, the OPN-mediated Akt signaling pathway was inhibited. OPN TM also decreased NF-κB activity and the phosphorylation of 4E-BP1, while facilitating apoptosis in the lungs of K-ras (LA1) mice. CONCLUSIONS: Our results show that aerosol delivery of OPN TM successfully suppresses lung cancer development in the K-ras (LA1) mouse model and, therefore, warrant its further investigation as a possible therapeutic strategy for non-small cell lung cancer.

Selective stimulation of caveolae-mediated endocytosis by an osmotic polymannitol-based gene transporter.

Controlling the cellular uptake mechanism and consequent intracellular route of polyplexes is important to improve the transfection efficiency of the non-viral gene delivery. Here, we report a new non-viral vector, polymannitol-based gene transporter (PMT), generated by crosslinking low molecular weight polyethylenimine with mannitol diacrylate, which has low cytotoxicity and good transfection efficiency. Interestingly, the uptake pathway of PMT/DNA complexes was shifted into caveolae-mediated endocytosis, avoiding lysosomal degradation. The mechanism of increased caveolae-mediated endocytosis of PMT/DNA complexes was found to be correlated with mechanosensing signal transduction by the hyperosmotic polymannitol part. Our results suggested that PMT, polymannitol-based gene transporter, is a safe and efficient gene delivery system with a well-modulated uptake pathway and intracellular route for gene therapy.

GOLGA2/GM130, cis-Golgi matrix protein, is a novel target of anticancer gene therapy.

Achievement of long-term survival of patients with lung cancer treated with conventional chemotherapy is still difficult for treatment of metastatic and advanced tumors. Despite recent progress in investigational therapies, survival rates are still disappointingly low and novel adjuvant and systemic therapies are urgently needed. A recently elucidated secretory pathway is attracting considerable interest as a promising anticancer target. The cis-Golgi matrix protein, GOLGA2/GM130, plays an important role in glycosylation and transport of protein in the secretory pathway. In this study, the effects of short hairpin RNA (shRNA) constructs targeting GOLGA2/GM130 (shGOLGA2) on autophagy and lung cancer growth were evaluated in vitro and in vivo. Downregulation of GOLGA2/GM130 led to induction of autophagy and inhibition of glycosylation in A549 cells and in the lungs of K-ras(LA1) mice. Furthermore, downregulation of GOLGA2/GM130 decreased angiogenesis and cancer cell invasion in vitro and suppressed tumorigenesis in lung cancer mice model. The tumor specificity of sequence targeting GOLGA2/GM130 was also demonstrated. Taken together, these results suggest that induction of autophagy by shGOLGA2 may induce cell death rather than cell survival. Therefore, downregulation of GOLGA2/GM130 may be a potential therapeutic option for lung cancer.

Suppression of lung cancer progression by biocompatible glycerol triacrylate-spermine-mediated delivery of shAkt1.

BACKGROUND: Polyethylenimine (PEI)-based nonviral gene-delivery systems are commonly employed because of their high transfection efficiency. However, the toxic nature of PEI is a significant obstacle in clinical gene therapy. In this study, we developed biocompatible glycerol triacrylate-spermine (GT-SPE) polyspermine as a nanosized gene carrier for potential lung cancer gene therapy. METHODS: The GT-SPE was synthesized using the Michael addition reaction between GT and SPE. The molecular weight was characterized using gel permeability chromatography multiangle laser light scattering and the composition of the polymer was analyzed using proton nuclear magnetic resonance. RESULTS: The GT-SPE successfully protected the DNA from nucleases. The average particle size of the GT-SPE was 121 nm with a zeta potential of +23.45 mV. The GT-SPE was found to be less toxic than PEI for various cell lines, as well as for a murine model. Finally, our results showed that the GT-SPE/small hairpin Akt1 (shAkt1) complex suppressed lung tumorigenesis in a K-ras(LA1) lung cancer mice model by inducing apoptosis through the Akt signaling pathway and cell cycle arrest. Aerosol delivered GT-SPE/shAkt1, which reduced matrix metalloproteinase-9 activity and suppressed the expression levels of proliferating cell nuclear antigen, as well as vascular endothelial growth factors and CD31, which are known proliferation and angiogenesis markers, respectively. CONCLUSION: Our data suggest that GT-SPE may be a candidate for short hairpin-shaped RNA-based aerosol lung cancer gene therapy.

Aerosol delivery of kinase-deficient Akt1 attenuates Clara cell injury induced by naphthalene in the lungs of dual luciferase mice.

Conventional lung cancer therapies are associated with poor survival rates; therefore, new approaches such as gene therapy are required for treating cancer. Gene therapies for treating lung cancer patients can involve several approaches. Among these, aerosol gene delivery is a potentially more effective approach. In this study, Akt1 kinase-deficient (KD) and wild-type (WT) Akt1 were delivered to the lungs of CMV-LucR-cMyc-IRES-LucF dual reporter mice through a nose only inhalation system using glucosylated polyethylenimine and naphthalene was administrated to the mice via intraperitoneal injection. Aerosol delivery of Akt1 WT and naphthalene treatment increased protein levels of downstream substrates of Akt signaling pathway while aerosol delivery of Akt1 KD did not. Our results showed that naphthalene affected extracellular signal-regulated kinase (ERK) protein levels, ERK-related signaling, and induced Clara cell injury. However, Clara cell injury induced by naphthalene was considerably attenuated in mice exposed to Akt1 KD. Furthermore, a dual luciferase activity assay showed that aerosol delivery of Akt1 WT and naphthalene treatment enhanced cap-dependent protein translation, while reduced cap-dependent protein translation was observed after delivering Akt1 KD. These studies demonstrated that our aerosol delivery is compatible for in vivo gene delivery.

Aerosol delivery of small hairpin osteopontin blocks pulmonary metastasis of breast cancer in mice.

BACKGROUND: Metastasis to the lung may be the final step in the breast cancer-related morbidity. Conventional therapies such as chemotherapy and surgery are somewhat successful, however, metastasis-related breast cancer morbidity remains high. Thus, a novel approach to prevent breast tumor metastasis is needed. METHODOLOGY/PRINCIPAL FINDING: Aerosol of lentivirus-based small hairpin osteopontin was delivered into mice with breast cancer twice a week for 1 or 2 months using a nose-only inhalation system. The effects of small hairpin osteopontin on breast cancer metastasis to the lung were evaluated using near infrared imaging as well as diverse molecular techniques. Aerosol-delivered small hairpin osteopontin significantly decreased the expression level of osteopontin and altered the expression of several important metastasis-related proteins in our murine breast cancer model. CONCLUSION/SIGNIFICANCE: Aerosol-delivered small hairpin osteopontin blocked breast cancer metastasis. Our results showed that noninvasive targeting of pulmonary osteopontin or other specific genes responsible for cancer metastasis could be used as an effective therapeutic regimen for the treatment of metastatic epithelial tumors.

Transcriptional regulation of CD38 expression by tumor necrosis factor-alpha in human airway smooth muscle cells: role of NF-kappaB and sensitivity to glucocorticoids.

The transmembrane glycoprotein CD38 catalyzes the synthesis of the calcium mobilizing molecule cyclic ADP-ribose from NAD. In human airway smooth muscle (HASM) cells, the expression and function of CD38 are augmented by the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), leading to increased intracellular calcium response to agonists. A glucocorticoid response element in the CD38 gene has been computationally described, providing evidence for transcriptional regulation of its expression. In the present study, we investigated the effects of dexamethasone, a glucocorticoid, on CD38 expression and ADP-ribosyl cyclase activity in HASM cells stimulated with TNF-alpha. In HASM cells, TNF-alpha augmented CD38 expression and ADP-ribosyl cyclase activity, which were attenuated by dexamethasone. TNF-alpha increased NF-kappaB expression and its activation, and dexamethasone partially reversed these effects. TNF-alpha increased the expression of IkappaBalpha, and dexamethasone increased it further. An inhibitor of NF-kappaB activation or transfection of cells with IkappaB mutants decreased TNF-alpha-induced CD38 expression. The results indicate that TNF-alpha-induced CD38 expression involves NF-kappaB expression and its activation and dexamethasone inhibits CD38 expression through NF-kappaB-dependent and -independent mechanisms.

Regulation of leukocyte function-associated antigen 1-mediated adhesion by somatostatin and substance P in mouse spleen cells.

BACKGROUND: Interaction of the integrin leukocyte function-associated antigen (LFA)-1 (CD11a/CD18) with its ligands, the intercellular adhesion molecules (ICAM)-1, -2, and -3 (CD54, CD102, and CD50), is pivotal to many leukocyte adhesion events. METHOD: To define the mechanism of the movement of leukocytes to the inflammatory site by somatostatin (SOM) and substance P (SP), we examined the expression of the adhesion molecule LFA-1 and inside-out signals for integrins, protein kinase C (PKC), Ras, Rap1, and phosphoinositide (PI) 3-kinase, in anti-CD3-, anti-CD3+SOM-, anti-CD3+SP-stimulated or unstimulated spleen cells. RESULTS: SOM caused down-regulation of LFA-1 mRNA translation as well as of adhesion-stimulating molecules such as Rap1, Ras, and PI 3-kinase. On the other hand, SP slightly induced LFA-1 mRNA translation and activation signals for integrins. The early-phase alteration of LFA-1 mRNA translation after 3 h of culture may be due to the changes of CD8+ T cells rather than changes of CD4+ cells. In adhesion assays, SOM significantly decreased cell adhesion (p < 0.05). CONCLUSION: These data suggest that SOM treatment of spleen cells, especially in CD8+ T cells, leads to downregulation of LFA-1 mRNA translation, inside-out signaling molecules for integrins (Ras, Rap1 and PI 3-kinase, but not PKC), and consequently to a decrease in the LFA-1-mediated adhesion to ICAM-1.

Effects of benzo[a]pyrene, 2-bromopropane, phenol and 2,3,7,8-tetrachlorodibenzo-p-dioxin on proinflammatory cytokines gene expression by mice spleen cells.

The detrimental effects of environmental pollutants on the health of the individual are generally accepted, although the mechanisms of these effects remain to be incompletely understood. In the present study, we examined the effects of B[a]P, 2-BP, phenol and TCDD on proinflammatory cytokine gene expression in mice spleen cells which were stimulated with anti-CD3. 10(-9)M TCDD increased IFNgamma and TNFalpha gene expression, but suppressed IL-1 gene expression. 10(-6)M phenol inhibited IL-1, IL-6 and TNFalpha gene expression, and 10(-6)M of 2-BP downregulated TNFalpha gene expression. However, 10(-6)M of B[a]P did not influence on IL-1, IL-6, IFNgamma and TNFalpha gene expression. These findings suggest that TCDD may impair the immune functions of mice by enhancing proinflammatory cytokines production, whereas phenol and 2-BP may impair the functions by inhibiting the production of these cytokines.