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2'O-galloylhyperin, an active flavonol glycoside compound with remarkable anti-immune activity, was isolated from Pyrola [P. incarnata Fisch.]. However, the evidence of anti-inflammatory activity in pulmonary diseases was still not convincing. The aim of the present study was (1) to investigate the effect of 2'O-galloylhyperin on LPS-induced acute lung injury in mice, and (2) to identify the mechanisms of attenuation of inflammatory responses. The results demonstrated that 2'O-galloylhyperin significantly reduced LPS-induced inflammation damage in a dose-dependent manner. After LPS challenge, treatment with 2'O-galloylhyperin reduced the production of pro-inflammatory cytokines and chemokines, and also improved LPS-induced lung histopathology changes. 2'O-galloylhyperin also increased the activities of antioxidant enzymes, including SOD and GSH-Px to maintain cellular redox homeostasis. Furthermore, 2'O-galloylhyperin inhibited translocation of nuclear factor (NF-κB) activation and suppressed phosphorylation of MAPK signaling pathway consisting of p38, ERK, JNK. In addition, 2'O-galloylhyperin enhanced heme oxygenase-1 (HO-1) expression to block LPS-induced inflammation via activating nuclear factor-crythroid 2-related factor (Nrf2). Moreover, 2'O-galloylhyperin induced adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) phosphorylation. 2'O-galloylhyperin attenuated LPS-induced acute lung injury by inhibiting the MAPK and NF-κB signaling pathways, presumably related to up-regulation of the AMPK and Nrf2 signaling pathways. Furthermore, 2'O-galloylhyperin is a potential protective antioxidant to protect lung tissues from the acute injury.

Tacrolimus is one of the most commonly used immunosuppressive agents in animal models of transplantation. However, in these models, oral administration is often problematic due to the lowered compliance associated with highly invasive surgery and due to malabsorption in the intestinal tract. Therefore, we carried out a study to determine the pharmacokinetics of tacrolimus after intramuscular (IM) injection and to determine the optimal IM dosing regimens in primate models. Six male cynomolgus monkeys (Macaca fascicularis) were used in the study. Doses of 0.1 mg/kg and 5 mg were administered via IM injection and oral administration, respectively, once to determine single-dose pharmacokinetics and once daily for 5 days to determine multiple-dose pharmacokinetics. According to pharmacokinetic model estimates, the inter- and intra-individual variabilities in bioavailability following IM injection were remarkably reduced compared with those following oral administration. Monte Carlo simulations revealed that Cpeak, Ctrough and AUC would also have less variability following IM injection compared with oral administration. In this study, we found that the pharmacokinetic characteristics of tacrolimus were more constant following IM injection compared with oral administration. These results suggest that IM injection can be an alternative route of administration fin non-human primate model studies. Keywords: Tacrolimus, Cynomolgus monkey, Intramuscular injection, Intra-individual variability

Due to the growing global population, reduction in arable land and effects of climate change, incongruity between food supply and demand has become increasingly severe. Nowadays, with awareness of the elementary nutrients required for human growth, increasing attention is being paid to the health and medical functions of food. Along with increased food production achieved by modern agricultural techniques, underutilised functional foods are an important strategy for solving food security problems and maintaining the nutritional quality of the human diet. Rosa roxburghii Tratt (RRT) is a natural fruit that contains unique functional and nutritional constituents, which are characterised by a high anti-oxidant potential. This review summarises the biological characteristics, chemical composition, health-promoting properties and development status of RRT products to inspire investigations on the use of RRT fruit as a functional food, dietary supplement and pharmaceutical additive. The nutrients and functional ingredients of RRT fruit are described in detail to provide more reference information for nutritionists and pharmacists.

Objective To investigate the effect of electro-acupuncture (EA) on vasomotor symptoms in rats with acute cerebral infarction, by observing the changes in the expression of factors related to the phosphatidylinositol (PI) system. Methods Forty-two Wistar rats were randomly divided into 3 groups by a random number table: the control group (n=6), the model group (n=18) and the EA group (n=18). The EA group was given EA treatment at Shuigou (GV 26) instantly after modeling with middle cerebral artery occlusion (MCAO) method, while the model and control groups were not given any treatment. The degrees of neurological deficiency were evaluated using neurological severity scores (NSS) and the brain blood flow was evaluated by a laser scanning confocal microscope. Western blot analysis was conducted to detect the expression levels of G-protein subtype (Gq) and calmodulin (CaM). Competition for protein binding was conducted to detect the expression level of inositol triphosphate (IP3). Thin layer quantitative analysis was conducted to detect the expression level of diacylglycerol (DAG). The expression level of intracellular concentration of free calcium ion ([Ca2+]i) was detected by flow cytometry. Results The NSS of the model group was significantly higher than the control group at 3 and 6 h after MCAO (P

Objective SC-E3 is a polyherbal formula that contains five medicinal herbs used frequently in traditional herbal medicine. In our previous study, we demonstrated the antioxidant and anti-inflammatory effects of SC-E3. The present study examined the effects of SC-E3 in a mouse model of type-II collagen-induced arthritis (CIA). Methods In vivo, male DBA/1J mice were immunized by intradermal injection of bovine type-II collagen and complete or incomplete Freund’s adjuvant, to induce arthritis. SC-E3 was orally administered daily for 23 days. In vitro, bone marrow-derived macrophages (BMMs) were treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) in the absence or presence of SC-E3. Results Administrations of SC-E3 were found to have anti-arthritic effects in the joints of CIA mice, as evidenced by reduced paw swelling, bone erosion and deformation, inflammatory cell infiltration, and inflammation in synovial membrane. SC-E3 also reduced serum levels of tumor necrosis factor-α, interleukin-1β, aspartate aminotransferase and alanine aminotransferase. Furthermore, tartrate-resistant acid phosphatase-positive osteoclast numbers in the joints were significantly lower in SC-E3-treated CIA mice than in CIA mice. In addition, the differentiations of BMMs to multinucleated osteoclasts induced by M-CSF and RANKL stimulation were dose-dependently reduced by SC-E3. Conclusion These results suggest that SC-E3 possesses substantial anti-arthritic activity because it inhibits pro-inflammatory cytokines and osteoclastogenesis, and that SC-E3 has potential therapeutic use for the treatment of rheumatoid arthritis.

Allelopathy is regarded as an economic and eco-friendly approach for the control of harmful algal blooms (HABs) because allelochemicals degrade easily and cause less pollution than traditional algicides. We first surveyed the inhibitory effect of the traditional medicinal plant Cerbera manghas L. on the notorious dinoflagellates Alexandrium tamarense, Scrippsiella trochoidea, and Karenia mikimotoi. Then, we identified and quantified the potential algicidal compounds by UPLC-MS and determined their activity. The aqueous extract inhibited algae with EC50–120 h at 0.986, 1.567 and 1.827 g L−1 for A. tamarense, S. trochoidea, and K. mikimotoi, respectively. Three potential allelochemicals were quantified in the stock solution: quinic acid (QA) (28.81 mg L−1), protocatechuic acid (PA) (53.91 mg L−1), and phloridzin (PD) (26.17 mg L−1). Our results illustrated that 1) QA did not have an inhibitory effect, 2) PA had medium toxicity to algae (EC50–120h: 0.22, 0.28, and 0.35 mM for A. tamarense, S. trochoidea, and K. mikimotoi), and 3) PD had low toxicity (EC50–120h > 0.66 mM). These findings suggested that PA might be the main allelopathic compound in the aqueous extract of the studied algae. In addition, PA could have a negative effect on the photosynthesis of S. trochoidea by impeding the reduction of quinone electrons and destroying electron transfer in PSII. In summary, this was the first study to quantify allelochemicals in C. manghas fruit. Moreover, C. manghas and protocatechuic have the potential to be algicides to control and mitigate the HABs caused by dinoflagellates.

Estrogen receptor β (ERβ) plays critical roles in thyroid cancer progression. However, its role in thyroid cancer stem cell maintenance remains elusive. Here, we report that ERβ is overexpressed in papillary thyroid cancer stem cells (PTCSCs), whereas ablation of ERβ decreases stemness-related factors expression, diminishes ALDH+ cell populations, and suppresses sphere formation ability and tumor growth. Screening estrogen-responsive lncRNAs in PTC spheroid cells, we find that lncRNA-H19 is highly expressed in PTCSCs and PTC tissue specimens, which is correlated with poor overall survival. Mechanistically, estradiol (E2) significantly promotes H19 transcription via ERβ and elevates H19 expression. Silencing of H19 inhibits E2-induced sphere formation ability. Furthermore, H19 acting as a competitive endogenous RNA sequesters miRNA-3126-5p to reciprocally release ERβ expression. ERβ depletion reverses H19-induced stem-like properties upon E2 treatment. Appropriately, ERβ is upregulated in PTC tissue specimens. Notably, aspirin attenuates E2-induced cancer stem-like traits through decreasing both H19 and ERβ expression. Collectively, our findings reveal that ERβ-H19 positive feedback loop has a compelling role in PTCSC maintenance under E2 treatment and provides a potential therapeutic targeting strategy for PTC.

Phenolic acids are found in natural plants, such as caffeic acid, rosmarinic acid, and chlorogenic acid. They have long been used as pharmacological actives, owing to their anti-inflammatory and antioxidant activities. Cryptochlorogenic acid (CCGA) is a special isomer of chlorogenic acid; the pharmacological effects and related molecular mechanisms of CCGA have been poorly reported. In the present study, the antioxidant and anti-inflammatory effects of CCGA in RAW 264.7 macrophages and the underlying mechanisms were investigated. The results revealed that CCGA dose-dependently inhibited LPS-induced production of NO, TNF-α, and IL-6 and blocked iNOS, COX-2, TNF-α, and IL-6 expressions. CCGA also significantly increased the GSH/GSSG ratio and SOD activity and reduced the MDA level. Moreover, CCGA suppressed the nuclear translocation of NF-κB by hindering the phosphorylation of IκB kinase (IKK) and degrading IκB. It also downregulated the phosphorylation of MAPKs. Our results indicated that CCGA significantly inhibited NF-κB activation by controlling the expression of pro-inflammatory factors and promoting the nuclear transfer of Nrf2. In conclusion, CCGA could attenuate LPS-induced inflammatory symptoms by modulating NF-κB/MAPK signaling cascades and inhibit LPS-induced oxidative stress via Nrf2 nuclear translocation.

Ethnopharmacological relevance Akebia quinata Decaisne extract (AQE; Lardizabalaceae) is used in traditional herbal medicine for stress- and fatigue-related depression, improvement of fatigue, and mental relaxation. Aim of the study To clarify the effects of AQE on stress-induced fatigue, we investigated the neuroprotective pharmacological effects of A. quinata Decaisne in mice exposed to chronic restraint stress. Materials and methods Seven-week old C57BL/6 mice chronically stressed by immobilization for 3 h daily for 15 d and non-stressed control mice underwent daily oral administration of AQE or distilled water. The open field, sucrose preference, and forced swimming behavioral tests were carried out once weekly, and immunohistochemical analyses of NeuN, brain-derived neurotrophic factor (BDNF), phosphorylated cAMP response element-binding (CREB) protein, and BDNF receptor tropomyosin receptor kinase B (TrkB) in striatum and hippocampus were performed at the end of the experimental period. Brain levels of serotonin, adrenaline, and noradrenaline as well as serum levels of corticosterone were measured. Results Behavioral tests showed that treatment with AQE improved all lethargic behaviors examined. AQE significantly attenuated the elevated levels of adrenaline, noradrenaline, and serotonin in the brain and corticosterone, alanine transaminase, and aspartate transaminase levels in the serum. Histopathological analysis showed that AQE reduced liver injury and lateral ventricle size in restraint-stress mice via inhibition of neuronal cell death. Immunohistochemical analysis showed increased phosphorylation of CREB and expression of BDNF and its receptor TrkB in striatum and hippocampus. Chlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C were identified as the primary components of AQE. All three agents increased expression of BDNF in SH-SY5Y cells and PC12 cells with H2O2-induced neuronal cell damage. Conclusions AQE may have a neuroprotective effect and ameliorate the effects of stress and fatigue-associated brain damage through mechanisms involving regulation of BDNF-TrkB signaling.

Background Chronic fatigue patients experience various neuropsychological symptoms, including fatigue behaviors, chronic pain, and depression. They also display immune system dysregulation. Polygonum aviculare L. extract (PAE) is a traditional herbal medicine used to treat inflammatory diseases by reportedly decreasing pro-inflammatory cytokine production. Hypothesis/Purpose We hypothesized that the anti-inflammatory properties of PAE would attenuate fatigue symptoms in a mouse model of restraint stress. Study design We evaluated the effects of PAE on fatigue using three experimental groups: unstressed, vehicle-treated stressed, and PAE-treated stressed mice. This restraint stress paradigm, comprised of restraint for 3 h daily for 15 days, was used to model chronic fatigue. Methods We compared lethargy-like behavior between our experimental groups using forced-swim, sucrose preference, and open-field tests once per week on days 7 and 14 of restraint stress. We also used histology and western blotting to evaluate pro-inflammatory cytokine expression in the brain and serum, and microglial activation in the brain. Finally, we used liquid chromatography/mass spectroscopy (LC/MS) to identify individual components of PAE, and applied cell culture techniques to test the effects of these components on neuronal cells in vitro. Results In restraint-stressed mice, PAE treatment decreased lethargy-like behavior relative to vehicle-treated animals. PAE treatment also reduced expression of fatigue-related factors such as corticosterone, serotonin, and catecholamines (adrenaline and noradrenaline) in the brain and serum, and decreased expression of CD68, Ibal-1, and the inflammatory cytokines TNF-α, IL-6, and IL-1β in the brain. Together, these data indicate that PAE reduced fatigue and is anti-inflammatory. Furthermore, histopathological analyses indicated that PAE treatment recovered atrophic volumes and hepatic injuries. Finally, LC/MS analysis of PAE identified four individual chemicals: myricitrin, isoquercitrin, avicularin, and quercitrin. In neuronal cell cultures, treatment with these PAE components inhibited TNF-α production, confirming that PAE treatment reduces neuroinflammation. Conclusions PAE treatment may reduce fatigue by suppressing neuroinflammation and the expression of fatigue-related hormones.

To evaluate the potential pharmacokinetic interactions of the anticancer agent gefitinib (Iressa®) and the oriental medications Guipi Decoction (归脾汤, GPD, Guibi-tang in Korean) and Bawu Decoction (八物汤, BWD, Palmul-tang in Korean). Methylcellulose (MC, control), GPD (1,200 mg/kg), or BWD (6,000 mg/kg) was orally administered to rats either as a single dose or multiple doses prior to gefitinib administration. To examine the effects of a single dose of the herbal medicines, gefitinib (10 mg/kg) was orally administered after 5 min or 1 h of MC or the herbal medicine pretreatments. To examine the effects of the multiple doses of the herbal medicines, gefitinib (10 mg/kg) was orally administered following 7 consecutive days of the administration of MC or each herbal medicine. The plasma concentrations of gefitinib were determined with liquid chromatography-tandem mass spectrometry assay. The plasma concentration-time profiles of gefitinib were analyzed with a noncompartmental analysis. Gefitinib was rapidly absorbed and showed a monoexponential decline with an elimination half-life of 3.7–4.1 h. The pharmacokinetics of gefitinib was not affected by GPD pretreatment. However, a significantly lower maximum plasma concentration (Cmax, P

To elucidate how ethanol extract of L. serratum (ELS) could exert anti-migratory effects on glioma with the suppression of nuclear factor kappa B (NF-κB) downstream pathway. Cell viability of ELS on C6 glioma was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Nitric oxide (NO) assay and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) assay were applied to measure NO production and reactive oxygen species (ROS) generation on lipopolysaccharide (LPS)-induced C6 glioma cells. NF-κB, mitogen-activated protein kinase (MAPK), inducible nictric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein were determined by Western blot. Wound healing assay was used to investigate the inhibitory effect of ELS on fetal bovine serum (FBS)-induced migration and matrix metalloproteinase (MMP)-9 and -2 activity was examined by zymography. ELS suppressed LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 through inhibiting the expression of chemokine CCL2 (or monocyte chemoattractant protein-1, MCP-1). In addition, ELS inhibited the expression of iNOS, COX-2, and the production of NO by LPS in C6 glioma cells. ELS also significantly decreased serum-induced migration of C6 glioma cells in scratch wound healing in a dose-dependent manner (P

Anticancer drug targeting to liver asialoglycoprotein receptors (ASGPR) is viewed as a good approach for hepatocellular carcinoma (HCC) treatment. Lactose residue is a promising ASGPR ligand due to its high receptor affinity. Herein, we introduce doxorubicin and paclitaxel co-bound lactosylated albumin (Lac-BSA) nanoparticles (Dox/Pac Lac-BSA NPs) with good liver targetability. Lac-BSA was synthesized by conjugating lactobionic acid to naive BSA then characterized by mass spectrometry. Dox/Pac Lac-BSA NPs were fabricated utilizing high-pressure homogenization and evaporation with Nab® (nanoparticle albumin bound) technology. Dox/Pac Lac-BSA NPs were spherical and well-dispersed, with a 148.7±13.8nm particle size and -54.1±0.7mV zeta potential at a 100% Lac-BSA feed ratio. Combined Dox and Pac synergistic cytotoxicity was confirmed in Hep G2 cells. Specifically, the inhibitory concentration (IC50; 0.21±0.02μg/ml) for Dox/Pac Lac-BSA NPs was 3.2 time lower than plain Dox/Pac BSA NPs (IC50; 0.68±0.04μg/ml). Also, Dox/Pac Lac-BSA NPs exhibited better internalizing in Hep G2 cells (61.8% vs. 14.4% for Dox) and spheroids compared to Dox/Pac BSA NPs. Finally, Dox/Pac Lac-BSA NPs displayed much greater localization into ICR mice livers compared to Dox/Pac BSA NPs. This was indicated by the presence of NP lactose residues revealed by a galactose inhibition study. Based on these results, we suggest that lactose-modified albumin-based nanoparticles fabricated with the Nab® technique can be a potential therapeutic vector for treating HCC via hepatocyte targeting.

For performance assessment of the lipid-based drug delivery systems (LBDDSs), in vitro lipolysis is commonly applied because traditional dissolution tests do not reflect the complicated in vivo micellar formation and solubilization processes. Much of previous research on in vitro lipolysis has mostly focused on rank-ordering formulations for their predicted performances. In this study, we have incorporated in vitro lipolysis with microsomal stability to quantitatively predict the oral bioavailability of a lipophilic antineoplastic drug bexarotene (BEX) administered in LBDDS. Two types of LBDDS were applied: lipid solution and lipid suspension. The predicted oral bioavailability values of BEX from linking in vitro lipolysis with microsomal stability for lipid solution and lipid suspension were 34.2 ± 1.6% and 36.2 ± 2.6%, respectively, whereas the in vivo oral bioavailability of BEX was tested as 31.5 ± 13.4% and 31.4 ± 5.2%, respectively. The predicted oral bioavailability corresponded well with the oral bioavailability for both formulations, demonstrating that the combination of in vitro lipolysis and microsomal stability can quantitatively predict oral bioavailability of BEX. In vivo intestinal lymphatic uptake was also assessed for the formulations and resulted in1% of the dose, which confirmed that liver microsomal stability was necessary for correct prediction of the bioavailability.

Ethnopharmacological relevance Dipsaci Radix, which is the dried root of Dipsacus asperoides C. Y. Cheng and T. M. Ai (Dipsacaceae), is used to treat back pain and blood stasis syndrome in Korean traditional medicine. Aim of the study To understand the mechanisms responsible for the pharmacological activities of D. asperoides, we investigated the inhibitory effect of D. asperoides on lipopolysaccharide (LPS)-induced inflammation in mouse macrophages RAW 264.7 cells. Materials and methods Aqueous extract of D. asperoides (AEDA) was prepared by boiling D. asperoides in water and then administered to LPS treated RAW 264.7 cells. Cell viabilities were measured using an MTT assay, and protein levels were determined by western blotting. The ROS scavenging activity of AEDA was measured using a DCFH-DA assay and levels of nitric oxide (NO) were determined using a NO assay. The nuclear translocations of NF-κB and Nrf2 were investigated immunocytochemically, and pro-inflammatory cytokines in supernatant were evaluated by ELISA. Results Treatment with AEDA suppressed the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW 264.7 macrophages. AEDA also reduced ROS, pro-inflammatory cytokine (IL-6 and IL-1β) levels, and iNOS-derived NO and COX-2-derived prostaglandin E2 release to medium, and suppressed the phosphorylation and degradation of IκB and the activation of NF-κB in macrophages. Furthermore, treatment with AEDA inhibited the ERK1/2 pathway but not the JNK or p38 MAPK pathways. In addition, AEDA significantly promoted Nrf2 translocation from cytoplasm to nucleus and up-regulated the expression of HO-1. Conclusion These results suggest that AEDA has anti-inflammatory and anti-oxidative effects through the inhibition of NF-κB and ERK1/2 and the activation of Nrf2/HO-1 in macrophages.

The intestinal lymphatic system plays an important role in the pathophysiology of multiple diseases including lymphomas, cancer metastasis, autoimmune diseases, and human immunodeficiency virus (HIV) infection. It is thus an important compartment for delivery of drugs in order to treat diseases associated with the lymphatic system. Lipophilic prodrug approaches have been used in the past to take advantage of the intestinal lymphatic transport processes to deliver drugs to the intestinal lymphatics. Most of the approaches previously adopted were based on very bulky prodrug moieties such as those mimicking triglycerides (TG). We now report a study in which a lipophilic prodrug approach was used to efficiently deliver bexarotene (BEX) and retinoic acid (RA) to the intestinal lymphatic system using activated ester prodrugs. A range of carboxylic ester prodrugs of BEX were designed and synthesised and all of the esters showed improved association with chylomicrons, which indicated an improved potential for delivery to the intestinal lymphatic system. The conversion rate of the prodrugs to BEX was the main determinant in delivery of BEX to the intestinal lymphatics, and activated ester prodrugs were prepared to enhance the conversion rate. As a result, an 4-(hydroxymethyl)-1,3-dioxol-2-one ester prodrug of BEX was able to increase the exposure of the mesenteric lymph nodes (MLNs) to BEX 17-fold compared to when BEX itself was administered. The activated ester prodrug approach was also applied to another drug, RA, where the exposure of the MLNs was increased 2.4-fold through the application of a similar cyclic activated prodrug. Synergism between BEX and RA was also demonstrated in vitro by cell growth inhibition assays using lymphoma cell lines. In conclusion, the activated ester prodrug approach results in efficient delivery of drugs to the intestinal lymphatic system, which could benefit patients affected by a large number of pathological conditions.

Pungent spice constituents such as piperine, capsaicin and [6]-gingerol consumed via daily diet or traditional Chinese medicine, have been reported to possess various pharmacological activities. These dietary phytochemicals have also been reported to inhibit P-glycoprotein (P-gp) in vitro and act as an alternative to synthetic P-gp modulators. However, the in vivo effects on P-gp inhibition are currently unknown. This study aimed to test the hypothesis that phytochemical P-gp inhibitors, i.e., piperine, capsaicin and [6]-gingerol, modulate the in vivo tissue distribution of doxorubicin, a representative P-gp substrate. Mice were divided into four groups and each group was pretreated with intraperitoneal injections of control vehicle, piperine, capsaicin, or [6]-gingerol and doxorubicin (1 mg/kg) was administered via the penile vein. The concentrations of the phytochemicals and doxorubicin in the plasma and tissues were determined by LC-MS/MS. The overall plasma concentration-time profiles of doxorubicin were not significantly affected by piperine, capsaicin, or [6]-gingerol. In contrast, doxorubicin accumulation was observed in tissues pretreated with piperine or capsaicin. The tissue to plasma partition coefficients, Kp, for the liver and kidney were higher in the piperine-pretreated group, while the Kp for kidney, brain and liver were higher in the capsaicin-pretreated group. [6]-Gingerol did not affect doxorubicin tissue distribution. The data demonstrated that the phytochemicals modulated doxorubicin tissue distribution, which suggested their potential to induce food-drug interactions and act as a strategy for the delivery of P-gp substrate drugs to target tissues and tumors.

Lrp4 (low-density lipoprotein receptor-related protein 4) is predominantly expressed in astrocytes, where it regulates glutamatergic neurotransmission by suppressing ATP release. Here, we investigated Lrp4's function in ischemia/stroke-induced brain injury response, which includes glutamate-induced neuronal death and reactive astrogliosis.The brain-specific Lrp4 conditional knockout mice (Lrp4Lrp4, largely expressed in astrocytes, was increased in response to ischemia/stroke. Both Lrp4The astrocytic Lrp4 plays an important role in ischemic brain injury response. Lrp4 deficiency in astrocytes seems to be protective in response to ischemic brain injury, likely because of the increased ATP release and adenosine-A

This study describes the development of a sensitive high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the quantification of avobenzone in rat plasma and skin layers. Separations were performed on a Zorbax SB C8 column using a binary gradient mobile phase composed of acetonitrile and 0.1% formic acid in water. The assay achieved LLOQ of 0.5ng/ml for plasma, 5ng/ml for stratum corneum, and 10ng/ml for epidermis and dermis. This method was applied to a percutaneous absorption study of avobenzone in rats. At 12h following topical application of emulsion and lotion (applied amount of avobenzone 11.7mg/kg), avobenzone was found primarily in the stratum corneum (16.3-17.8%) followed by epidermis (2.0-3.4%) and dermis (0.11-0.15%). Avobenzone was not quantifiable in the plasma samples collected over a 12h sampling period. Given the excellent plasma assay sensitivity, this study provides evidence that the systemic absorption of avobenzone is insignificant, if any, after topical application.

This study aimed to evaluate the potential of α-cedrene as a new anti-obesity drug by characterizing absorption, metabolism and pharmacokinetics in rats. α-Cedrene was administered intravenously (10 and 20 mg/kg) and orally (50 and 100 mg/kg) to female and male Sprague-Dawley rats. Blood, tissues, urine, and feces were collected at predetermined times. α-Cedrene concentrations were determined by a validated gas chromatography-tandem mass spectrometry (GC-MS/MS). A gas chromatography-mass selective detection (GC-MSD) method was used to identify the major metabolite. After i.v. injection, α-cedrene exhibited a rapid clearance (98.4-120.3 ml/min/kg), a large distribution volume (35.9-56.5 l/kg), and a relatively long half-life (4.0-6.4 h). Upon oral administration, it was slowly absorbed (Tmax = 4.4 h) with bioavailability of 48.7-84.8%. No gender differences were found in its pharmacokinetics. Upon oral administration, α-cedrene was highly distributed to tissues, with the tissue-to-plasma partition coefficients (Kp) far greater than unity for all tissues. In particular, its distribution to lipid was notably high (Kp = 132.0) compared to other tissues. A mono-hydroxylated metabolite was identified as a preliminary metabolite in rat plasma. These results suggest that α-cedrene has the favorable pharmacokinetic characteristics to be further tested as an anti-obesity drug in clinical studies.

Background SC-E1 is a novel herbal formula consisting of five oriental medicinal herbs used frequently in traditional herbal medicine for the treatment of inflammatory diseases in Korea. This study examined the effects of SC-E1 on lipopolysaccharide (LPS)-stimulated macrophages and the molecular mechanism involved. Methods The cytotoxic effect of the SC-E1 extract was evaluated in RAW 264.7 cells by MTT assay. The effects of SC-E1 on the free radical scavenging and generation of intracellular reactive oxygen species were measured using DPPH and DCFH-DA, respectively. The effects of SC-E1 on the production of pro-inflammatory cytokines, inflammatory mediators, and related products were determined by ELISA and western blotting. The molecular mechanism and the nuclear translocation of nuclear factor-kappa B (NF-κB) and NF-E2-related factor 2 (Nrf2) were examined by western blot analysis and immunocytochemistry. Results SC-E1 exhibited strong anti-oxidant activity and inhibited LPS-induced NO secretion as well as iNOS expression and the production of pro-inflammatory cytokines, without affecting the cell viability. SC-E1 also suppressed the LPS-induced NF-κB activation and the mitogen-activated protein kinase (MAPK) pathway. Moreover, SC-E1 induced heme oxygenase-1 (HO-1) expression via the nuclear translocation of Nrf2. The inhibitory effects of SC-E1 on the production of pro-inflammatory cytokines were abrogated by treatment with SnPP, an HO-1 inhibitor. Conclusion These results suggest that SC-E1 exerts its anti-oxidant and anti-inflammatory effects through the inhibition of NF-κB and MAPK as well as Nrf2-mediated HO-1 induction in macrophages. These findings provide evidences for SC-E1 to be considered as a new prescription for treating inflammatory diseases.

Bexarotene is currently marketed for treatment of cutaneous T-cell lymphoma and there has been growing interest in its therapeutic effectiveness for other cancers. Neuroprotective effects of bexarotene have also been reported. In this study, a simple, sensitive and cost-efficient bioanalytical method for determination of bexarotene in rat plasma was developed and fully validated. The method utilises protein precipitation with acetonitrile and liquid-liquid extraction with n-hexane-ethyl acetate (10:1, v/v). An HPLC-UV system with a Waters Atlantis C18 column and a mobile phase of acetonitrile-ammonium acetate buffer (10 mM, pH 4.1) at a ratio of 75:25 (v/v), flow rate 0.2 mL/min was used. Chromatograms were observed by a UV detector with wavelength set to 259 nm. Intra- and inter-day validations were performed and sample stability tests were conducted at various conditions. The applicability of the method was demonstrated by a pharmacokinetic study in rats. Intravenous bolus dose of 2.5 mg/kg was administered to rats and samples were obtained at predetermined time points. As a result, pharmacokinetic parameters of AUCinf (4668 ± 452 h ng/mL), C0 (6219 ± 1068 ng/mL) and t1/2 (1.15 ± 0.02 h) were obtained. In addition, the developed method was further applied to human and mouse plasma to assess the suitability of the method for samples from other species.

1. The objectives of this study were to evaluate the pharmacokinetics and metabolism of fimasartan in rats. 2. Unlabeled fimasartan or radiolabeled [(14)C]fimasartan was dosed by intravenous injection or oral administration to rats. Concentrations of unlabeled fimasartan in the biological samples were determined by a validated LC/MS/MS assay. Total radioactivity was quantified by liquid scintillation counting and the radioactivity associated with the metabolites was analyzed by using the radiochemical detector. Metabolite identification was conducted by product ion scanning using LC/MS/MS. 3. After oral administration of [(14)C]fimasartan, total radioactivity was found primarily in feces. In bile duct cannulated rats, 58.8 ± 14.4% of the radioactive dose was excreted via bile after oral dosing. Major metabolites of fimasartan including the active metabolite, desulfo-fimasartan, were identified, yet none represented more than 7.2% of the exposure of the parent drug. Fimasartan was rapidly and extensively absorbed and had an oral bioavailability of 32.7-49.6% in rats. Fimasartan plasma concentrations showed a multi-exponential decline after oral administration. Double peaks and extended terminal half-life were observed, which was likely caused by enterohepatic recirculation. 4. These results provide better understanding on the pharmacokinetics of fimasartan and may aid further development of fimasartan analogs.

Prunetin is an O-methylated isoflavone, which is a type of flavonoid. There are a limited number of reports detailing the biological activities of prunetin. Although an anti-inflammatory effect of prunetin has been reported in vitro, to our knowledge, there have been no reports on anti-adipogenic effects of prunetin in obese animals. The aims of this study were to determine whether prunetin suppresses high-fat diet (HFD)-induced adipogenesis in the liver and visceral adipose tissues of mice, and to explore the underlying mechanisms mediating the actions of prunetin. To this end, mice were fed a HFD for 10 weeks to induce obesity, and prunetin (10 μg/kg or 20 μg/kg) was administered in the last 3 weeks. Compared to saline-treated mice, mice treated with prunetin showed significantly reduced body weight gain, visceral fat pad weights, and plasma glucose levels. We found that prunetin significantly inhibited the HFD-induced upregulation of the expression of important adipogenic genes (PPARγ, C/EBPα, SREBP, aP2, LPL adiponectin, and leptin), and suppressed HFD-mediated increase in expression of lipid metabolism-related genes (SREBP, PPARγ, LXR, and HMG-CoA) in the liver tissues. Furthermore, prunetin induced expression of adiponectin receptors 1 and 2 (adipoR1, adipoR2), as well as that of AMP-activated protein kinase (AMPK) in the liver and adipose tissue. These results suggest that prunetin mediates anti-obesity/adipogenesis effects by suppressing obesity-related transcription through a feedback mechanism that regulates the expression of adiponectin, adipoR1, adipoR2, and AMPK.

Cinnamaldehyde, an active compound of cinnamon, has been reported to exert various biological functions such as anti-inflammatory and anti-tumor activities. Previously, we showed that 2'-hydroxycinnamaldehyde (HCA) has an inhibitory effect on nitric oxide (NO) production through the inhibition of NF-κB signaling. In an effort to find a more effective anti-atherosclerotic agent, here we evaluated the anti-inflammatory effect of 2'-benzoyloxycinnamaldehyde (BCA) in RAW 264.7 murine macrophage cells. We showed that BCA more effectively inhibited NO production than HCA with less cytotoxicity. We also demonstrated that BCA inhibited the lipopolysaccharide (LPS)-induced expression of iNOS in a concentration-dependent manner. Signal transduction studies showed that BCA significantly inhibited the phosphorylation of SAPK/JNK and AP-1-dependent reporter gene activity. LPS-induced expression levels of JunB, c-Jun and c-Fos were also decreased by BCA treatment. Moreover, the LPS-induced DNA binding activity of AP-1 was markedly inhibited by BCA. The direct injection of BCA into mice inhibited the LPS-induced increase in plasma nitrite levels, confirming the anti-inflammatory effect of BCA in vivo. Overall, these observations suggest that BCA has the potential for use as an anti-atherosclerotic agent.

Establishing a level A in vitro-in vivo correlation (IVIVC) for a drug with complex absorption kinetics is challenging. The objective of the present study was to develop an IVIVC approach based on population pharmacokinetic (POP-PK) modeling that incorporated physiologically relevant absorption kinetics. To prepare three extended release (ER) tablets of loxoprofen, three types of hydroxypropyl methylcellulose (HPMC 100, 4000, and 15000 cps) were used as drug release modifiers, while lactose and magnesium stearate were used as the diluent and lubricant, respectively. An in vitro dissolution test in various pH conditions showed that loxoprofen dissolution was faster at higher pH. The in vivo pharmacokinetics of loxoprofen was assessed following oral administration of the different loxoprofen formulations to Beagle dogs (n = 22 in total). Secondary peaks or shoulders were observed in many of the individual plasma concentration vs time profiles after ER tablet administration, which may result from secondary absorption in the intestine due to a dissolution rate increase under intestinal pH compared to that observed at stomach pH. In addition, in vivo oral bioavailability was found to decrease with prolonged drug dissolution, indicating site-specific absorption. Based on the in vitro dissolution and in vivo absorption data, a POP-PK IVIVC model was developed using S-ADAPT software. pH-dependent biphasic dissolution kinetics, described using modified Michaelis-Menten kinetics with varying V

Background Medicinal herb-derived drug development has become important in the relief of liver pathology. Amomun cardamomum is traditionally used therapeutically in Korea to treat various human ailments including dyspepsia, hiccupping, and vomiting. We investigated to assess the protective effect of A. cardamomum on carbon tetrachloride (CCl4)-induced liver damage through antioxidant activity in hepatic tissues of Sprague–Dawley rats. Methods Antioxidant properties of different fractions from A. cardamomum from ethanol extracts were evaluated by an in vitro free radical scavenging systems. The protective effect of the ethyl acetate fraction from A. cardamomum (EAAC) against CCl4-induced cytotoxicity was determined by a cell viability assay using HepG2 hepatocarcinoma cells. In vivo study, the influence of EAAC concentrations of 100 and 200 mg/kg following CCl4-induced hepatic injury was assessed. Serum levels of glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), and alkaline phosphatase (ALP) were determined, as was lipid peroxidation (malondialdehyde, MDA). Effect of EAAC on liver detoxification enzymes including superoxide dismutase (SOD), total glutathione (GSH), and glutathione S-transferase (GST) activity was measured in rat liver homogenates. Liver cytochrome P450 (CYP2E1) expression level was determined by quantification of mRNA. Results Phytochemical analysis of A. cardamomum indicated that EAAC was enriched in total polyphenol and total flavonoid. Most of the tannins were confined to the hexane fraction. Hepatoprotective properties of EAAC were evident, with significantly reduced serum levels of GOT, GPT, and ALP compared with the control group. Improved hepatic antioxidant status was evident by increased SOD, GSH, and GST enzymes in rat liver tissue. Liver lipid peroxidation induced by CCl4 was apparent by increased intracellular MDA level. EAAC suppressed lipid peroxidation as evidenced by the significant decrease in MDA production. Expression of CYP2E1 was also significantly decreased at the higher concentration of EAAC, indicating the hepatoprotective efficacy of EAAC on acute liver damage. Conclusion These results indicated that EAAC has a significant hepatoprotective activity on CCl4-induced acute hepatic injury in rats, which might be derived from its antioxidant properties and CYP2E1 downregulation.

This study reports the pharmacokinetics and tissue distribution of a novel histone deacetylase and DNA methyltransferase inhibitor, psammaplin A (PsA), in mice. PsA concentrations were determined by a validated LC-MS/MS assay method (LLOQ 2 ng/mL). Following intravenous injection at a dose of 10 mg/kg in mice, PsA was rapidly eliminated, with the average half-life (t(1/2, λn)) of 9.9 ± 1.4 min and the systemic clearance (CL(s)) of 925.1 ± 570.1 mL/min. The in vitro stability of PsA was determined in different tissue homogenates. The average degradation t(1/2) of PsA in blood, liver, kidney and lung was found relatively short (≤ 12.8 min). Concerning the in vivo tissue distribution characteristics, PsA was found to be highly distributed to lung tissues, with the lung-to-serum partition coefficients (K(p)) ranging from 49.9 to 60.2. In contrast, PsA concentrations in other tissues were either comparable with or less than serum concentrations. The high and specific lung targeting characteristics indicates that PsA has the potential to be developed as a lung cancer treatment agent.

Bicyclol is a novel anti-hepatitis drug used for the treatment of chronic hepatitis B. Bicyclol is insoluble in water and poorly absorbed after oral administration. To date, formulation development studies to improve the in vitro dissolution profiles of bicyclol and the in vivo oral absorption characteristics have not been performed.To overcome problems associated with the poor solubility and low oral bioavailability of bicyclol, a microemulsion system was prepared and evaluated in vitro and in vivo.The solubility of bicyclol in various cosurfactants was determined. The optimized premicroemulsion concentrate consisted of transcutol, Tween 20, Cremophor RH 40, propylene glycol monocaprylate and bicyclol (ratio, 50:150:100:150:3). The in vitro solubility and dissolution profiles were determined, and the in vivo oral absorption pharmacokinetics were evaluated in rats (dose, equivalent to 25 mg/kg of bicyclol) in comparison with bicyclol suspended in 0.5% calcium-carboxymethylcellulose (Ca-CMC).Of various cosurfactants tested, transcutol provided the most significantly increased solubility of bicyclol (20 mg/ml). Bicyclol was rapidly dissolved from the premicroemulsion concentrate (approximately 80% within 10 min). Consistent with the improved in vitro profiles, the oral absorption of bicyclol was significantly increased for the premicroemulsion concentrate, i.e. AUC and C(max) were increased by 7.7- and 7.2-fold, respectively, over control values. These findings demonstrate that the microemulsion may be a useful drug delivery system to improve the oral bioavailability of bicyclol.

Background: This study assessed the population pharmacokinetics and metabolic conversion of a novel histone deacetylase (HDAC) inhibitor, SD-2007, into its active metabolite, apicidin, in rats. Methods: SD-2007 was given to rats by intravenous injection (4 mg/kg) and oral administration (40 mg/kg). Serum concentrations of SD-2007 and apicidin were determined by LC-MS/MS. All concentrations were analyzed using a population pharmacokinetic model with 9 compartments in S-ADAPT. Results: The area under the curve for apicidin was 96 ± 16 mg·h/ml after 4 mg/kg administered intravenously and 2,455 ± 1,211 mg·h/ml after 40 mg/kg given orally. The population pharmacokinetic model described all profiles well. After oral administration of SD-2007, the median absolute bioavailability of SD-2007 was 6.67% (range 3.83–9.89) and the median apparent bioavailability was 22.3% (range 15.7–35.8) for apicidin, whereas only a median of 8.85% (range 7.57–9.34) of an intravenous SD-2007 dose was converted to apicidin. Conclusions: Oral SD-2007 displayed a substantial presystemic metabolism to active apicidin. The high serum concentrations of apicidin after oral administration of SD-2007 may cause significant HDAC inhibition.

The objectives of this study were to develop physiologically based models for the pharmacokinetics (PK) and organ distribution of apicidin in rats and mice and to predict human PK in blood and organs. The PK of apicidin was characterized in rats and mice after i.v. bolus injection, and distribution to various tissues was determined in rats following i.v. infusions at steady state. The developed models were prospectively validated within rat and within mouse and by scaling from rat to mouse using data after multiple i.v. injections. Human PK was predicted by the physiologically based modeling using intrinsic clearance data for humans from in vitro experiments. The Cls predicted for human (9.8 ml/min/kg) was lower than those found in mice (116.9 ml/min/kg) and rats (61.6 ml/min/kg), and the Vss predicted for human (1.9 l/kg) was less than in mice (2.0 l/kg) and rats (2.5 l/kg). Consequently, the predicted t 1/2 was longer in human (2.3 h) than in mice and rats (0.4 and 0.9 h, respectively). The highest concentrations of apicidin were predicted in liver followed by adipose tissue, kidney, lung, spleen, heart, arterial blood, venous blood, small intestine, stomach, muscle, testis, and brain. The developed models adequately described the PK of apicidin in rats and mice and were applied to predict human PK. These models may be useful in predicting human blood and tissue concentrations of apicidin under different exposure conditions.

The objectives of this study were to (1) develop physiologically based pharmacokinetic (PBPK) models for zearalenone following intravenous (i.v.) and oral (p.o.) dosing in rats and (2) predict concentrations in humans via interspecies scaling. The model for i.v. dosing consisted of vein, artery, lung, liver, spleen, kidneys, heart, testes, brain, muscle, adipose tissue, stomach, and small intestine. To describe the secondary peak phenomenon observed after p.o. administration, the absorption model was constructed to reflect glucuronidation, biliary excretion, enterohepatic recirculation, and fast and slow absorption processes from the lumenal compartment. The developed models adequately described observed concentration-time data in rats after i.v. or p.o. administration. Upon model validation in rats, steady-state zearalenone concentrations in blood and tissues were simulated for rats after once daily p.o. exposures (0.1 mg/kg/d). The average steady-state blood zearalenone concentration predicted in rat was 0.014 ng/ml. Subsequently, a daily human p.o. dose needed to achieve the same steady-state blood concentration found in rats (0.014 ng/ml) was determined to be 0.0312 mg/kg/d or 2.18 mg/70 kg/d. The steady-state zearalenone concentration-time profiles in blood and tissues were also simulated for human after multiple p.o. administrations (dose 0.0312 mg/kg/d). The developed PBPK models adequately described the pharmacokinetics in rats and may be useful in predicting human blood and tissue concentrations for zearalenone under different p,o, exposure conditions.

This study was conducted to examine the oral bioavailability and the possibility of enterohepatic recirculation of otilonium bromide in rats. A sensitive LC/MS/MS assay (LLOQ 0.5 ng/mL) was developed for the determination of otilonium and applied to i.v. and oral administration studies in bile duct cannulated (BDC) and non-BDC rats. After i.v. injection to BDC rats (1 mg/ kg as otilonium), average t(1/2), CL, Vz and AUC were 7.9 +/- 1.9 h, 8.7 +/- 3.1 mL/min/kg, 5.7 +/- 1.4 L/kg and 2,088 +/- 676 ng h/mL, respectively, and these values were comparable to those found in non-BDC rats. The percentages of i.v. dose excreted unchanged in bile and urine in BDC rats were 11.6 +/- 3.0 and 3.1 +/- 0.7%, respectively. Upon oral administration to non-BDC rats (20 mg/kg as otilonium), t(1/2), Cmax, Tmax and AUC were 6.4 +/- 1.3 h, 182.8 +/- 44.6 ng/mL, 1.9 +/- 1.6 h and 579 +/- 113 ng h/mL, respectively. The absolute oral bioavailability was low (1.1%), while the drug was preferentially distributed to gastrointestinal tissues. A secondary peak was observed in the serum concentration-time profiles in non-BDC rats following both i.v. and oral administration, indicating that otilonium bromide was subject to enterohepatic recirculation.

This study examined the absorption and pharmacokinetic disposition of 125I-GST-TatdMt, a recombinant Tat protein possessing potent anti-obesity activity, in mice after vascular and extravascular administration. GST-TatdMt was over-expressed in E. coli, purified, and radioiodinated using the IODO-GEN method. 125I-GST-TatdMt was administered to mice by i.v., i.p. and oral administration at doses of 652.7 nCi (102.3 microg). Upon i.v. injection, the average terminal elimination half-life (t1/2,lambdaz), AUC and AUMC were 6.4 h, 318.2 nCixh/mL and 2518 nCixh2/ mL, respectively. The highest radioactivity was observed in lung followed by liver, spleen, heart and kidney. The t1/2lambdaz values obtained from i.v., i.p., and oral administration were comparable from each other (range 5.8-6.4 h). The absolute bioavailability of 125I-GST-TatdMt was 42.8% and 60.5% after p.o. and i.p. administration, respectively. Given the cell-penetrating nature, 125I-GST-TatdMt may be absorbed into the systemic circulation to a relatively high extent after extravascular administration.

Di(2-ethylhexyl) phthalate (DEHP) is a well-known hepatic and reproductive toxicant whose toxicity may be mediated by peroxisome proliferators-activated receptor (PPAR). This study examined the effects of DEHP on the expression of PPAR-regulated genes involved in testicular cells apoptosis. Sprague-Dawley male rats were treated orally with 250, 500, or 750 mg/kg/d DEHP for 28 d, while control rats were given corn oil. The levels of cell cycle regulators (pRb, cyclins, CDKs, and p21) and apoptosis-related proteins were analyzed by Western blot analysis. The role of PPAR-gamma (PPAR-gamma), class B scavenger receptor type 1 (SR-B1), and ERK1/2 was further studied to examine the signaling pathway for DEHP-induced apoptosis. Results showed that the levels of pRB, cyclin D, CDK2, cyclin E, and CDK4 were significantly lower in rats given 500 and 750 mg/kg/d DEHP, while levels of p21 were significantly higher in rat testes. Dose-dependent increases in PPAR-gamma and RXRalpha proteins were observed in testes after DEHP exposure, while there was a significant decrease in RXRgamma protein levels. In addition to PPAR-gamma, DEHP also significantly increased SR-B1 mRNA and phosphorylated ERK1/2 protein levels. Furthermore, DEHP treatment induced pro-caspase-3 and cleavage of its substrate protein, poly(ADP-ribose) polymerase (PARP), in a dose-dependent manner. Data suggest that DEHP exposure may induce the expression of apoptosis-related genes in testes through induction of PPAR-gamma and activation of the ERK1/2 pathway.

Background: Fimasartan (FMS) is a potent angiotensin receptor blocker for the treatment of mild to moderate hypertension. This study aimed to evaluate the transfer of FMS to fetus and breast milk in rats. Methods: In order to study the transfer to the fetus and nursing pup, pregnant and nursing maternal rats were administered with FMS by a constant intravenous infusion to reach target plasma concentrations of 200 ng/mL and 100 ng/mL. The concentrations of FMS in plasma, placenta, amniotic fluid, fetus, and milk were determined by a validated LC-MS/MS assay. Results: Upon constant intravenous infusion, the plasma FMS concentration reached the target steady state concentrations (C-ss = 200 ng/mL and 100 ng/mL) in 24 h. The tissue-to-plasma partition coefficients (K-p) for placenta, amniotic fluid, and milk were obtained based on the observed FMS concentrations in the tissues and C-ss. The K-p values for all tissues were not different between high (C-ss = 200 ng/mL) and low (C-ss = 100 ng/mL) dose groups. While the mean K-p of the placenta was 44.6-59.0 %, the mean K-p was 1.3-1.7 % for the amniotic fluid and 14.9-17.0 % for fetus. The mean K-p of milk was 10.4-15.2 %. Conclusions: Placental transfer and milk excretion of FMS was relatively lower compared to other angiotensin receptor blockers.

This study describes the development of a sensitive liquid chromatography-electrospray-tandem mass spectrometry method for the simultaneous determination of benzyl butyl phthalate (BBP) and its major metabolites, monobenzyl phthalate (MBzP) and monobutyl phthalate (MBuP), in rat plasma, urine, and 10 different tissues. The method was validated with regard to the specificity, linearity, precision, accuracy, lower limit of quantification (LLOQ), recovery, and stability by using the matrix-matched quality control samples. The assay achieved LLOQ of 1 ng/ml of BBP for plasma and urine, 4 ng/g for kidney and liver, 10 ng/g for fat, and 20 ng/g for all other tissues. For MBzP and MBuP, the assay achieved LLOQ of 5 ng/ml for plasma and urine, 10 ng/g for fat, and 20 ng/g for all other tissues. The disposition of BBP was characterized by a large volume of distribution (71.1–82.9 l/kg) and a high clearance (838.7–871.0 ml/min/kg). It was extensively metabolized to MBzP and MBuP, with their levels consistently exceeding the BBP levels. The distribution of BBP, MBzP, and MBuP to tissues of kidney, liver, stomach, small intestine, large intestine, spleen, brain, testis, thyroid, and fat was determined under steady-state conditions. For BBP, the steady-state tissue-to-plasma partition coefficient (K p) was the highest for fat (25.0) followed by small intestine (2.6), thyroid (2.0), and stomach (1.1). In contrast, for MBzP and MBuP, it was the highest for kidney (2.0 and 4.3, respectively) and liver (4.3 and 2.1, respectively) but was less than unity for all other tissues. The developed assay method and findings of this study may be useful to evaluate the exposure and toxic potential of BBP and its metabolites in risk assessment.

Ethnopharmacological relevance Adipocyte lipid accumulation due to impaired fatty acid oxidation causes adipocyte hypertrophy and adipose tissue increment, leading to obesity. The aim of this study was to determine the antiobesity effects of the herbal composition Gambigyeongsinhwan (4) (GGH(4)) composed of Curcuma longa L. (Zingiberaceae), Alnus japonica (Thunb.) Steud. (Betulaceae), and the fermented traditional Korean medicine Massa Medicata Fermentata. Materials and methods The effects of GGH(4) and the individual components on lipid accumulation in 3T3-L1 adipocytes and body weight gain in Otsuka Long-Evans Tokushima Fatty (OLETF) rats were examined using Oil red O staining, hematoxylin and eosin staining, quantitative real-time PCR, and peroxisome proliferator-activated receptor α (PPARα) transactivation assay. Results GGH(4), individual components, and an active principle of Curcuma longa curcumin inhibited lipid accumulation and mRNA levels of adipocyte-specific genes (PPARγ, aP2, and C/EBPα) in 3T3-L1 adipocytes compared with control cells. Treatment with GGH(4), the individual components or curcmumin increased mRNA levels of mitochondrial (CPT-1, MCAD, and VLCAD) and peroxisomal (ACOX and thiolase) PPARα target genes. GGH(4) and the individual components also increased PPARα reporter gene expression compared with control cells. These effects were most prominent in GGH(4)-treated cells. However, the PPARα antagonist GW6471 reversed the inhibitory effects of GGH(4) on adipogenesis. An in vivo study showed that GGH(4) decreased body weight gain, adipose tissue mass, and visceral adipocyte size with increasing mRNA levels of adipose tissue PPARα target genes in OLETF rats. Conclusions These results demonstrate that GGH(4) has an antiobesity effects through the inhibition of adipocyte lipid accumulation, and this process may be mediated in part through adipose PPARα activation.

This study describes the development of a simple LC-ESI-MS/MS method with polarity switching for the simultaneous analysis of phenoxyethanol (PE) and its major metabolite, phenoxyacetic acid (PAA), in rat plasma, urine, and 7 different tissues. The assay was validated to demonstrate the linearity, precision, accuracy, LLOQ, recovery, and stability by using the matrix matched QC samples. The assay achieved the LLOQ of 10 and 20 ng/mL of PE and PAA, respectively, for plasma samples and the LLOQ of 20 and 50 ng/mL of PE and PAA, respectively, for urine and tissue samples. This method was successfully applied to the percutaneous absorption, distribution, metabolism, and excretion studies in rats. The absolute topical bioavailability of PE was 75.4% and 76.0% for emulsion and lotion, respectively. Conversion of PE to PAA was extensive, with the average AUCPAA-to-AUCPE ratio being 4.4 and 5.3 for emulsion and lotion, respectively. The steady-state tissue-to-plasma PE concentration ratio (Kp) was higher than unity for kidney, spleen, heart, brain, and testis and was lower (≤0.6) for lung and liver, while the metabolite Kp ratio was higher than unity for kidney, liver, lung, and testis and was lower (≤0.3) for other tissues. Findings of this study may be useful to evaluate the relationship between exposure and toxic potential of PE in risk assessment.

Objectives This study was conducted to examine the tissue distribution of human recombinant epidermal growth factor (EGF) after multiple intravenous and subcutaneous injections in mice. Methods Male BALB/c mice were divided into (1) EGF 1 mg/kg intravenous dose, (2) EGF 5 mg/kg intravenous dose, (3) drug-free intravenous control, (4) EGF 1 mg/kg subcutaneous dose, (5) EGF 5 mg/kg subcutaneous dose and (6) drug-free subcutaneous control groups. EGF and drug-free dosing solutions were injected by intravenous and subcutaneous injections once a day for 3 days. EGF concentrations in serum and tissues of kidney, liver, lung, small intestine and tongue were determined by ELISA. Key findings As the intravenous and subcutaneous doses were increased from 1 to 5 mg/kg, serum Cmax and area under the concentration–time curve (AUC) values were increased dose-proportionally. In lung, tongue and small intestine, increases in AUC were dose-proportional after intravenous injections, but greater than dose-proportional after subcutaneous injections. The fold-increases in Cmax and AUC values were lowest in liver and highest in kidney. Conclusion Based on Cmax and AUC data, the systemic exposure achieved by subcutaneous injections was comparable with that achieved by intravenous injections.

Peptides like salmon calcitonin (sCT) are subjected to aggressive proteolytic attack by various intestinal enzymes, and fractions that enter the systemic circulation via the intestinal route are rapidly inactivated by tissue accumulation and glomerular filtration. Here, we describe the beneficial effects of the Lys(18)-amine specific PEGylation of sCT on the intestinal delivery of sCT. Two key properties were enhanced by the PEGylation process: (i) the resistance of sCT to intestinal enzymes and (ii) the systemic clearance of sCT that had entered the circulation. Initially, we evaluated the cAMP-secreting activities of PEG(2K)-sCT isomers substituted at Cys(1)-, Lys(11)- or Lys(18)-amine position in T47D cells, and found that sCT PEGylated at Lys(18)-amine (Lys(18)-PEG(2K)-sCT) had the highest bioactivity. We then investigated the stability of Lys(18)-PEG(2K)-sCT in the presence of intestinal enzymes, its abilities to traverse the intestinal membrane, its pharmacokinetic behavior and in vivo hypocalcemic efficacy. Results show that Lys(18)-PEG(2K)-sCT has significantly increased resistance to pancreatic peptidases and brush-border peptidases. Despite the molecular size increase caused by PEGylation, Lys(18)-PEG(2K)-sCT was found to have an intestinal permeability similar to that of unmodified sCT (p0.59) over an apical concentration range 12.5-100 microM in a Caco-2 cell monolayer transport system. In particular, tissue distribution results showed that (125)I-labeled Lys(18)-PEG(2K)-sCT markedly resists liver accumulation and glomerular filtration; levels were reduced by 75% and 50% vs. sCT. Finally, the hypocalcemic efficacy of intestinally administered Lys(18)-PEG(2K)-sCT, measured as total serum calcium in a rat model, was 5.8 and 3.0 times that of sCT at 100 and 200 IU/kg (p0.025). Our findings suggest that this site-specific conjugation of peptides with PEG of proper size enhances pharmacokinetic properties by increasing their abilities to resist both proteolysis and systemic clearance without significantly reducing their membrane permeabilities or bioactivities. We believe that this concept, namely, dual effects by PEGylation, has great potential value because it presents a practical means of enhancing the efficacies of the peroral/intestinal pharmacologic route.

Chemopreventive and cytotoxic effect of genistein against human breast cancer cell lines was investigated. Genistein inhibited cell proliferation in estrogen receptor-positive (MCF-7) and estrogen receptor-negative (MDA-MB-231) human breast carcinoma cell lines. Cytochrome P450 (CYP) 1A1-mediated ethoxyresorufin O-deethylase (EROD)activity was inhibited by genistein in a concentration-dependent manner. Genistein significantly inhibited 12-Otetradecanoylphorbol-13-acetate (TPA)-induced cyclooxygenase-2 activity and protein expression at the concentrations of 10 (p < 0.05), 25 (p < 0.05) and 50 mM (p < 0.01). In addition, ornithine decarboxylase (ODC) activity was reduced to 53.8 % of the control after 6 h treatment with 50 mM genistein in MCF-7 breast cancer cells. These results suggest that genistein could be of therapeutic value in preventing human breast cancer.

This study examined the effect of combined aqueous extracts (BHR) of Ginko biloba, Mentha arvensis var. piperascens, Citrus unshiu, and Pueraria lobata var. chinensis on oral absorption of alcohol in rats. The rats were pretreated with BHR, placebo solution identical to BHR without the herbal extract, and isotonic saline. Alcohol was administered orally at 1- and 3-g/kg doses and the absorption profiles were compared. After oral administration of 1-g/kg doses, mean area under the curve (AUC) and C(max) values were significantly reduced in BHR-treated rats (16.1 +/- 10.0 and 0.3 +/- 0.1 mg/ml, respectively) as compared with saline-treated (37.9 +/- 14.4 and 0.7 +/- 0.7 mg/ml, respectively) and placebo solution-treated (63.0 +/- 46.4 and 0.7 +/- 0.4 mg/ml, respectively) rats. Similarly, after administration of 3-g/kg doses, mean AUC and C(max) values in BHR-treated rats (188.1 +/- 119.7 mg(.)min/ml and 1.0 +/- 0.4 mg/ml) were significantly reduced over those in saline-treated rats (571.4 +/- 512.4 mg(.)min/ml and 1.8 +/- 0.9 mg/ml, respectively). The relative oral bioavailability of alcohol calculated as the ratio of AUC(BHR)/AUC(Saline) was 42.5% and 32.9% at 1- and 3-g/kg doses, respectively. The reduced serum alcohol levels as well as the reduced AUC and C(max) after pretreatment with BHR appear to be a result of a reduced systemic absorption not due to an increased metabolic clearance.

This study was conducted to examine the effects of aloe and aloesin on the weight gain and blood chemistry as well as the pharmacokinetics of benzo[a]pyrene (BaP) and 3-OH-BaP in rats. The rats treated with multiple doses of aloe and aloesin (100 mg/kg every 12 h for 14-19 d) did not show any significant changes in the weight gain and blood biochemical parameters. In addition, the effects of oral treatment with aloe, aloesin, and propolis on the absorption and pharmacokinetics of benzo[a]pyrene (BaP) and its metabolite, 3-OH-BaP, were studied in rats. The treatment with a single oral dose (200 mg/kg) of aloe, aloesin, and propolis did not alter the concentration-time profiles of BaP and 3-OH-BaP after iv and oral administration of BaP. At higher oral doses (500 mg/kg), the biliary excretion of BaP and the urinary excretion of 3-OH-BaP were significantly increased, but the urinary excretion of BaP and the fecal excretion of 3-OH-BaP remained unaltered. Whether high doses of aloe increase the overall elimination of BaP deserves further investigation.

Ricin A-chain, which exhibits excellent cytotoxicity to tumor cells, has been widely used as an immunotoxin source. However, it has the fatal shortcoming of poor pharmacokinetics due to the tremendous liver uptake via carbohydrate-mediated recognition. Modification of proteins with polyethylene glycol, PEGylation, has the advantages of shielding the specific sites and prolonging the biological half-life. In this study, the carbohydrate-specific PEGylation of ricin A-chain was considered to be a novel approach to overcome this limitation. The carbohydrate group of ricin A-chain was oxidized by sodium m-periodate and further specifically conjugated with hydrazide-derivatized PEG. For a comparative study, the PEGylated ricin A-chain at amino groups was prepared using the hydroxysuccinimide ester-derivatized PEG. The carbohydrate-specifically PEGylated ricin A-chain showed a markedly lower liver uptake and systemic clearance compared with the amine-directly PEGylated ricin A-chain as well as the unmodified ricin A-chain. Furthermore, carbohydrate-specifically PEGylated ricin A-chain showed a significantly higher in vitro ribosome-inactivating activity than the amine-directly PEGylated ricin A-chain. These findings clearly demonstrate that the carbohydrate-specificity as well as PEGylation plays an important role in improving the in vivo pharmacokinetic properties and in vitro bioactivity. Therefore, these results suggest that the therapeutic use of immunotoxins constructed using this carbohydrate-specifically PEGylated ricin A-chain has potential as a cancer therapy.

This study first examined the pharmacokinetic disposition of GST-TatdMt, a recombinant Tat protein possessing potent anti-obesity activity, in rats after i.v. injection. GST-TatdMt was over-expressed in E. coli, purified, and radioiodinated using the IODO-GEN method. The radioiodinated 125I-GST-TatdMt was administered to rats at doses of 9 microg (1640 nCi), 18 mug (3388 nCi), and 35 microg (6420 nCi). Upon administration, the total radioactivity in serum declined bi-exponentially, with the average terminal elimination half-life ranging from 13.7 to 15.7 h. There was a linear relationship between dose and AUC(INF) (r2=1.000) and between dose and Co (r2=0.999). The fraction of administered radioactivity excreted in feces was low (mean range 1.5-2.8%), while the majority of the radioactivity was excreted in urine (mean range 54.9-61.4%). The radioactivity found in the liver, lungs, spleen, and kidneys were higher than in serum, but the tissue-to-serum ratios were relatively low (

Apicidin, a fungal metabolite isolated from Fusarium pallidoroseum, is a cyclic tetrapeptide that exhibits potent anti-protozoal and anti-angiogenic activities. Although extensive studies have been recently conducted to examine the biological and pharmacological action, no information is available on the quantitative analysis of apicidin. To our knowledge, this study is the first to describe a rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay method for the quantification of apicidin in rat serum. The method was validated to demonstrate the specificity, linearity, recovery, lower limit of quantification (LLOQ), accuracy, and precision. The multiple reaction monitoring was based on the transitions m/z 624.7 84.3 and 372.1 176.1 for apicidin and trazodone, respectively. The assay utilized a single liquid-liquid extraction and isocratic elution, and the LLOQ was 0.5 ng/mL using 0.1 mL of rat serum. The assay was linear over a range from 0.5–1000 ng/mL, with correlation coefficients >0.9994. The mean intra- and inter-day assay accuracy ranged from 99.9–101.5% and 94.8–102.1%, respectively, and the mean intra- and inter-day precision was between 2.7–5.9% and 1.6–11.5%, respectively. The developed assay method was applied to a pharmacokinetic study after intravenous injection of apicidin in rats at a dose of 1 mg/kg. Copyright © 2005 John Wiley & Sons, Ltd.