Identification of integrative hepatotoxicity induced by lysosomal phospholipase A2 inhibition of cationic amphiphilic drugs via metabolomics.

Drug-induced liver injury (DILI) is a condition caused by drugs that leads to abnormal hepatic function. Hepatotoxicity caused by DILI has been shown to be due to cellular stress, mitochondrial dysfunction, cell necrosis and apoptosis and many types of hepatotoxicity, such as phospholipidosis, steatosis and hepatitis, commonly share intracellular molecular mechanisms. Metabolomics can be useful for mechanism-based toxicity evaluations and has been recently utilized as a scientific technique that can effectively predict the risk factors for chemical substances. To evaluate the key events in hepatotoxicity associated with lysosomal phospholipase A2 (LPLA2) inhibition by cationic amphiphilic drugs (CADs), LPLA2 inhibition assays and phospholipid accumulation assays were performed in HepG2 cells. Additionally, to suggest the integrative molecular mechanisms of hepatotoxicity by CADs, we profiled intracellular metabolites. Cell-based metabolomics was performed using an UPLC-Orbitrap-MS instrument equipped with heated electrospray ionization in positive and negative ion modes. As a result, CADs such as amiodarone, fluoxetine, chlorpromazine and tamoxifen significantly inhibited LPLA2 and accumulated phospholipids. In metabolomics, a total of 17 significant metabolites were identified, and the changed metabolite types were as follows: nucleotide sugars, conjugated bile acids, branched-chain amino acids, polyamine biosynthesis, and long-chain fatty acid and glycerophospholipid metabolism. From these data, it was suggested that the integrative mechanism of DILI could be verified and that a toxicological approach is possible using metabolomics.

Identification of interactions between multiple components in Socheongryong-tang using a plant profiling approach.

Traditional herbal medicine consists of multiple components. There are interactions among the components, which affect both potency and toxicity. The preparation of herbal medicines can be a cause of interactions between multicomponents in herbs. To demonstrate the differences in multiherb interactions based on the preparation methods, the changes in the active components in the different preparations of Socheongryong-tang (SCRT) were evaluated using metabolomics profiling. We performed multicomponent profiling of the decoction of SCRT (SCRTD) and individual herb mixture (SCRTM) using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Active compounds from SCRTD and SCRTM were identified using multivariate analysis, and the activities between the two groups were compared. We also evaluated the anti-inflammatory effect of SCRT through investigating the protein expression of iNOS and COX-2 in lipopolysaccharide-induced macrophage RAW 264.7 cells in both groups. From the multivariate analysis, 53 active compounds that have different intensities between SCRTD and SCRTM were identified. The intensities of those components, such as ephedrines, glycyrrhizic acid, 6-gingerol and (2E,4E,8Z,10E)-N-isobutyl-2,4,8,10-dodecatetraenamide, which is newly identified in Asiasarum heterotropoides, were mostly higher in SCRTD than in SCRTM, which was related to the anti-inflammatory effect. From the iNOS inhibition test, it was found that SCRTD had a stronger anti-inflammatory effect than SCRTM. It was demonstrated that multicomponent interactions can be changed by the preparation method, and finally the anti-inflammatory effect in SCRT can be affected.

Development of GC-MS based cytochrome P450 assay for the investigation of multi-herb interaction.

As drug interactions with cytochrome P450 enzymes become increasingly important in the field of drug discovery, a high-throughput screening method for analysing the effects of a drug is needed. We have developed a simple and rapid simultaneous analytical method using a cocktail approach for measuring the activities of seven cytochrome P450 enzymes (CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4). Human liver microsomes were used as a source for the seven cytochrome P450 enzymes, and a gas chromatography-mass spectrometry (GC-MS) was used for analysing their activities. Kinetic studies and inhibition assays of CYP enzymes were performed using known substrates and inhibitors for validating and comparing the reaction rates and time-dependent activities between methods using each substrate versus a method using a cocktail solution. The optimized cocktail method was successfully applied to evaluate the effects of the decoction of Socheongryong-tang (SCRT) on cytochrome P450 enzymes. Our cocktail method provides a simultaneous high-throughput activity assay using GC-MS for the first time. This method is applicable for analysing the drug interactions of various plant-derived mixtures.

An Asian traditional herbal complex containing Houttuynia cordata Thunb, Perilla frutescens Var. acuta and green tea stimulates hair growth in mice.

BACKGROUND: Houttuynia cordata Thunb (HC) is a traditional herbal medicine widely used in Asia for the treatment of patients with alopecia, usually in combination with other two herbal medicines (Perilla frutescens var. acuta (PFVA) and green tea (GT)). However, the effect of this herbal complex has not been clearly demonstrated. We sought to determine the hair growth-promoting effect of this herbal complex (HC, PFVA, and GT) in the animal model. METHODS: Six-week-old male C57BL/6 mice were randomly divided into four groups (negative control, finasteride (1 mg/kg) as a positive control, and two (200 and 400 mg/kg) concentrations of the herbal complex as experimental groups) and were fed its corresponding medications orally for 25 days. Hair growth was evaluated visually and microscopically. Western blot analysis for insulin-like growth factor (IGF)-1 and transforming growth factor (TGF)-ß1 was performed. RESULTS: The herbal complex exhibited hair growth-promoting activity in C57BL/6 mice. Grossly, the area of hair regrowth was 55.1 (±3.8) %, 70.2 (±6.3) % and 83.5 (±5.7) % in negative control, herbal complex 200 mg/kg and 400 mg/kg group, respectively. In histologic examination, the hair follicle count in deep subcutis was 2.6 (±0.7), 5.8 (±0.7) and 8.6 (±1.2) and the diameter of hair follicles was 11.9 (±5.0) µm, 17.4 (±3.9) µm and 22.8 (±5.2) µm in negative control, herbal complex 200 and 400 mg/kg group, respectively. The expression of IGF-1 was 0.14 (±0.01), 0.23 (±0.02) and 0.24 (±0.01) and the expression of TGF-ß1 was 0.26 (±0.01), 0.19 (±0.02) and 0.15 (±0.01) in negative control, the 200 and 400 mg/kg group, respectively. CONCLUSIONS: This data provides adequate preliminary experimental evidence to support the hair regeneration effect of this herbal complex.

Anti-fatigue Effects of 20(S)-Protopanaxadiol and 20(S)-Protopanaxatriol in Mice.

Ginseng (Panax ginseng C.A. MEYER, Araliaceae), which contains protopanaxadiol-type and protopanaxatriol-type ginsenosides, has been used for inflammation, fatigue, stress, and tumor in Asian countries. Orally administered ginsenosides are metabolized to their aglycones 20(S)-protopanaxadiol (PPD) and 20(S)-protopanaxatriol (PPT) by gut microbiota. However, their anti-fatigue effects have not been studied thoroughly. Therefore, we investigated the anti-fatigue activities of PPD and PPT in mice, using the weight-loaded swimming (WLS) and the rota-rod tests. Ginseng water extract (GW), ginseng saponin fraction (GWS) and ginseng polysaccharide fraction (GWP) at concentrations of 50 and 100 mg/kg and PPD and PPT at 5 and 10 mg/kg were orally administered to mice once daily for 5 d. GW, GWS, and PPT significantly increased the WLS time, however, GWP and PPD did not cause any significant change. PPT induced the most significant increase in WLS time. PPD (10 mg/kg) and PPT (5 and 10 mg/kg) inhibited the WLS-induced increase in corticosterone, lactate, lactate dehydrogenase (LDH), and creatinine levels as well as the reduction in glucose level. PPT increased the riding time in the rota-rod test, and also inhibited corticosterone, lactate, and creatinine levels. These findings suggest that the anti-fatigue effect of ginseng may be attributable to its saponins, particularly PPT, rather than to its polysaccharides.

Anti-stress effects of 20(S)-protopanaxadiol and 20(S)-protopanaxatriol in immobilized mice.

Panax ginseng C.A. MEYER (Araliaceae), which contains ginsenosides as its main components, has been shown to have various biological effects, including anti-inflammatory, anxiolytic, anti-stress, and anti-tumor effects. Orally administered ginsenoside Rb1 and Re are metabolized to 20(S)-protopanaxadiol (PPD) and compound K via ginsenoside Rd and 20(S)-protopanaxatriol (PPT) and ginsenoside Rh1 via ginsenoside Rg1 by gut microbiota, respectively. Therefore, we investigated the anti-stress effects of these metabolites, PPD and PPT, by measuring their anxiolytic and anti-inflammatory effects in immobilized mice. Treatment with PPD and PPT prior to immobilization stress increased the time spent in open arms and open arm entries in the elevated plus-maze (EPM) test. The anxiolytic effects of PPD (10 mg/kg) and PPT (10 mg/kg) were comparable to that of buspirone (1 mg/kg). This observed anxiolytic effect of PPD was significantly blocked by flumazenil or bicuculline, and the effect of PPT was blocked by WAY-100635. Treatment with PPD also potently suppressed immobilization stress-induced serum levels of corticosterone and interleukin (IL)-6 by the enzyme-linked immunosorbent assay. However, PPT treatment did not suppress them. Based on these findings, PPD and PPT may exhibit the anxiolytic effect via γ-aminobutyrateA (GABAA) receptor(s) and serotonergic receptor(s), respectively, and PPD may have an anti-inflammatory effect that is more potent than that of PPT.

The critical role of mast cell-derived hypoxia-inducible factor-1α in human and mice melanoma growth.

Mast cells play an important role in tumorigenesis. Histamine released from mast cells stimulates new vessel formation by acting through the histamine1 (H1) receptor. Despite the evidence of the role of mast cells in tumor growth and angiogenesis, the potential mechanism remains to be elucidated. Therefore, we investigated the role of mast cell-derived HIF-1α in melanoma growth. Here, we identify that the most positive cells for HIF-1α staining are seen in mast cells of human and animal melanoma tissue. The number of the stromal cell types (fibroblasts, macrophages and endothelial cells) was also increased in melanoma tissues. In activated bone marrow-derived mast cells (BMMCs), expressions of HIF-1α and VEGF were increased. Histamine also induced the expressions of HIF-1α and VEGF in BMMCs. H1 receptor antagonists significantly improved overall survival rates and substantially suppressed tumor growth as well as the infiltration of mast cells and levels of VEGF through the inhibition of HIF-1α expression in B16F10 melanoma-bearing mice. Furthermore, the injection of HIF-1α depleted BMMCs markedly inhibited the growth of tumors and migration of mast cells and increased the survival rate of the mice. These findings emphasize that the growth of melanoma can actually be exacerbated by mast cell-derived HIF-1α. In aggregate, our results reveal a novel role for mast cell-derived HIF-1α in the melanoma microenvironment and have important implications for the design of therapeutic strategies.

Ginsenoside Rg1 inhibits the TSLP production in allergic rhinitis mice.

Recent study reports that Korean red ginseng reduces the nasal allergic inflammatory reaction in an allergic murine model. However, the contribution of ginsenoside Rg1 (RG1) and its mechanisms on allergic rhinitis (AR) have not been elucidated. In this study, we evaluated the important activities of RG1 in the ovalbumin (OVA)-induced AR mice. RG1 significantly reduced the levels of thymic stromal lymphopoietin (TSLP) and interleukin (IL)-1ß compared with the AR control mice. Allergic symptom such as rub scores and biomarkers such as spleen weight, histamine, IgE and IgG(1) in the RG1 group were decreased compared with the AR mice. The levels of interferon-γ were enhanced while the levels of IL-4 were reduced in the RG1 group. In the RG1 group, the eosinophils and mast cells infiltration increased by OVA were also decreased. RG1 reduced the levels of inflammation-related protein. RG1 inhibited the caspase-1 activity in nasal mucosa tissue. In addition, RG1 inhibited the production of TSLP and IL-1ß and the activations of caspase-1, receptor interacting protein 2, IκB kinase-ß and nuclear factor-κB/Rel A in activated HMC-1 cells. Our results indicate that RG1 has the inhibitory effect of TSLP production and caspase-1 activity in AR experimental model.

4-EA-NBOMe, an amphetamine derivative, alters glutamatergic synaptic transmission through 5-HT1A receptors on cortical neurons from SpragueDawley rat and pyramidal neurons from C57BL/6 mouse.

New psychoactive substances (NPSs) are compounds designed to mimic illegal recreational drugs. In particular, there are difficulties in legal restrictions because there is no fast NPS detection method to suppress the initial spread of NPS with criminal records; thus, they expose the public to serious health threats, including toxicity and dependence. However, the effects of NPSs on the brain and the related cellular mechanisms are well unknown. One of the recently emerging drugs is 4-ethylamphetamine-NBOMe (4-EA-NBOMe), a member of the 2 C phenylalanine family with a similar structure to methamphetamine (methA). In this study, we tested the effect of methA analogs on the glutamatergic synaptic transmission on primary cultured cortical neurons of SpragueDawley (SD) rats and C57BL/6 mice, and also layer 2/3 pyramidal neurons of the medial prefrontal cortex (mPFC) of C57BL/6 mice. We found that acute treatment with 4-EA-NBOMe inhibits spontaneous excitatory postsynaptic currents (EPSCs) and that withdrawal after chronic inhibition by 4-EA-NBOMe augments glutamatergic synaptic transmission. These modifications of synaptic responses are mediated by 5-HT1A receptors. These findings suggest that 4-EA-NBOMe directly affects the central nervous system by changing the efficacy of glutamatergic synaptic transmission.

IL-32 up-regulation is associated with inflammatory cytokine production in allergic rhinitis.

IL-32 is a described pro-inflammatory cytokine produced by T lymphocytes, natural killer cells, monocytes, and epithelial cells. However, the specific mechanism of IL-32 on allergic rhinitis (AR) has not been elucidated. Here, we report a significant increase of IL-32 protein and mRNA in the nasal mucosa of AR patients. In addition, in nasal mucosa tissue from AR patients, the level of IL-32 production correlated with inflammation, IL-1ß, IL-18, and granulocyte-macrophage colony-stimulating factor (GM-CSF). In an AR animal model, IL-32 significantly increased IgE and inflammatory cytokine levels. IL-32 expression was induced by recombinant human GM-CSF via activation of caspase-1 in eosinophils. In addition, depletion of IL-32 prevents the production of inflammatory cytokines in eosinophils. In conclusion, IL-32 is an important cytokine involved in the inflammation of AR. The regulation of IL-32 expression may form the basis of a new strategy for the treatment of AR.

Alleviation of allergic rhinitis symptoms with Pyeongwee-San extract (KMP6).

CONTEXT: Allergy is characterized by the overreaction of the immune system. Pyeongwee-San is a traditional Korean medicine which has been used for the treatment of the allergic disorder but the mechanism of action is not clear. OBJECTIVE: To investigate the effect of Pyeongwee-San extract (KMP6) and its component, hesperidin (HES) in the allergic rhinitis (AR) animal model. METHOD: We sensitized mice on 1, 5, and 14 days by intraperitoneal injections of 100 µg ovalbumin (OVA) emulsified in 20 mg of aluminum hydroxide and we challenged mice with 1.5 mg OVA. Mice received KMP6 and HES before the intranasal OVA challenge for 10 days. RESULTS: The number of nose rubs after the OVA challenge in the OVA-sensitized mice was significantly higher than that in the OVA-unsensitized mice. The increased number of nose rub was inhibited by the oral administration of KMP6 or HES. The increased levels of IgE and histamine level in serum of the OVA-sensitized mice were reduced by KMP6 or HES administration. The level of interferon-γ was enhanced while the level of IL-4 was reduced on the spleen tissue of the KMP6 or HES-administered AR mice. Inflammatory proteins level was reduced by KMP6 or HES administration in the nasal mucosa tissue of the OVA-sensitized mice. In the KMP6 or HES-administered mice, mast cells and eosinophils infiltration increased by OVA-sensitization was decreased. CONCLUSION: These results indicate that KMP6 and HES ameliorate the allergic inflammatory reactions such as AR.

Distinct effects of imperatorin on allergic rhinitis: imperatorin inhibits caspase-1 activity in vivo and in vitro.

Because imperatorin (IPT), the furanocoumarins exhibits anti-inflammatory activity, we reasoned that IPT might modulate the allergic rhinitis (AR). The aim of this study was to analyze the regulation of AR by IPT. Here, we show the effect and mechanism of IPT in an ovalbumin (OVA)-induced AR model. The number of rubs after the OVA challenge in the OVA-sensitized mice was significantly higher than that in the OVA-unsensitized mice. The increased number of rubs was inhibited by the oral administration of IPT. The increased levels of IgE and histamine in the OVA-sensitized mice were reduced by IPT administration. The levels of interferon-γ were enhanced, whereas the levels of interleukin (IL)-4 were reduced on the spleen tissue of the IPT-administered AR mice. Protein levels of IL-1ß, macrophage inflammatory protein-2, intercellular adhesion molecule-1, and cyclooxygenase-2 were reduced by IPT administration in the nasal mucosa of the OVA-sensitized mice. In the IPT-administered mice, the number of eosinophils and mast cells infiltration increased by OVA-sensitization were also decreased. In addition, IPT inhibited caspase-1 activity in the same nasal mucosa tissue. In activated human mast cells, the receptor-interacting protein 2 (RIP2), IκB kinase (IKK)-ß, nuclear factor-κB (NF-κB)/RelA, and caspase-1 activation were increased, but increased RIP2, IKK-ß, NF-κB/RelA, and caspase-1 activation were inhibited by the treatment of IPT. In addition, IPT inhibited caspase-1 activity and IL-1ß production in IgE-stimulated bone marrow-derived mast cells. We can conclude that IPT exerts significant effects by regulating of caspase-1 activation in AR animal and in vitro models.

Quinacrine directly dissociates amyloid plaques in the brain of 5XFAD transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is the most common type of dementia characterized by the abnormal accumulation of amyloid-ß (Aß) in the brain. Aß misfolding is associated with neuroinflammation and synaptic dysfunction, leading to learning and memory deficits. Therefore, Aß production and aggregation have been one of the most popular drug targets for AD. Failures of drug candidates regulating the aforementioned Aß cascade stimulated development of immunotherapy agents for clearance of accumulated Aß in the brain. Here, we report that quinacrine, a blood-brain barrier penetrating antimalarial chemical drug, dissociates Aß plaques in the brain of AD transgenic mice. When co-incubated with pre-formed Aß fibrils, quinacrine decreased thioflavin T-positive ß-sheets in vitro, on top of its inhibitory function on the fibril formation. We confirmed that quinacrine induced dissociation of high-molecular-weight Aß aggregates into low-molecular-weight species by dot blots in association with size cut-off filtrations. Quinacrine was then administered to adult 5XFAD transgenic mice via weekly intravenous injections for 6 weeks, and we found a significant reduction of Aß plaques and astrocytosis in their cortex and hippocampus. In western blots of quinacrine-administered mouse brains, amelioration of AD-related biomarkers, glial fibrillary acidic protein, postsynaptic protein 95, phosphorylated cAMP response element-binding protein, phosphorylated c-Jun N-terminal kinase were observed. Lastly, quinacrine-stimulated dissociation of misfolded aggregates induced recovery of synaptic function associated with Aß in excitatory post-synaptic current recordings of primary rat cortical neurons treated with Aß aggregates and quinacrine. Collectively, quinacrine can directly dissociate Aß fibrils and alleviate decreased synaptic functions.

Analysis of plasma metabolic profiling and evaluation of the effect of the intake of Angelica keiskei using metabolomics and lipidomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Angelica keiskei contains many bioactive components with anti-oxidative and anti-inflammatory effects. It is also effective for the treatment of diabetes mellitus, hypertension, and arteriosclerosis, but the relationships between these effects and the active components in the herb have not been studied. AIM OF THE STUDY: We aimed to confirm the effects of Angelica keiskei on humans. MATERIALS AND METHODS: A metabolomics and lipidomics study was performed using human plasma samples from 20 subjects after the intake of Angelica keiskei, and the components of Angelica keiskei in the plasma were profiled. UPLC-Orbitrap-MS was used to analyze the plasma and plant extracts, and multivariate analysis and correlation studies between the exogenous components from plant and endogenous metabolite in plasma were performed. RESULTS: The levels of the 14 metabolites including kynurenic acid, prostaglandin E1, chenodeoxycholic acid, lysoPC (18:1), lysoPC (18:2), lysoPC (20:3), lysoPC (20:4), lysoPC (22:6), PC (34:1), PC (34:2), PC (38:3), PC (38:4), PC (38:6) and PC (40:7) in the plasma were changed. By monitoring the components originating from Angelica keiskei in plasma, five components including 5-methoxypsoralen, 8-methoxypsoralen, 4-hydroxyderricin, xanthoangelol B and xanthoangelol F were detected and they reduced the levels of bile acids and fatty acids. CONCLUSIONS: The levels of the metabolites, including bile acids, amino acids, glycerophospholipids and fatty acids, in the plasma were changed, and 14 significantly changed metabolites were closely related to the preventive effect against liver diseases, type 2 diabetes, anemia, obesity, atherosclerosis, depression and anti-inflammatory effects. The five components of Angelica keiskei were related the modulatory activity of reducing the levels of bile acids and fatty acids.

Gene networking in colistin-induced nephrotoxicity reveals an adverse outcome pathway triggered by proteotoxic stress.

Colistin has been widely used for the treatment of infections of multidrug­resistant Gram­negative bacteria, despite the fact that it induces serious kidney injury as a side effect. To investigate the mechanism underlying its nephrotoxicity, colistin methanesulfonate sodium (CMS; 25 or 50 mg/kg) was administered via intraperitoneal injection to Sprague­Dawley rats daily over 7 days. Serum biochemistry and histopathology indicated that nephrotoxicity occurred in the rats administered with CMS. Whole­genome microarrays indicated 894 differentially expressed genes in the group treated with CMS (analysis of variance, false discovery rate <0.05, fold­change ≥1.3). Gene pathway and networking analyses revealed that genes associated with proteotoxic stress, including ribosome synthesis, protein translation, and protein folding, were significantly associated with the nephrotoxicity induced by CMS. It was found that colistin inhibited the expression of the target genes heat shock factor 1 and nuclear factor erythroid­2­related factor­2, which are associated with proteostasis, and that nephrotoxicity of CMS may be initiated by proteotoxic stress due to heat shock response inhibition, leading to oxidative stress, endoplasmic reticulum stress, cell cycle arrest and apoptosis, eventually leading to cell death. A putative adverse outcome pathway was constructed based on the integrated gene networking data, which may clarify the mode of action of colistin­induced nephrotoxicity.

Social support rescues acute stress-induced cognitive impairments by modulating ERK1/2 phosphorylation in adolescent mice.

Social support can relieve stress-induced behavioural outcomes, although its underlying molecular mechanisms are not fully understood. Here, we evaluated whether social interactions can prevent the restraint stress (RS)-induced cognitive impairments in male adolescent mice by utilizing molecular, cellular, and behavioural approaches. Acute RS in adolescent ICR mice impaired the working memory in the Y-maze test and memory consolidation and retrieval in the novel-object-recognition test (NORT). In addition, RS increased the extracellular signal-regulated kinases 1/2 phosphorylation (p-ERK1/2) in the prefrontal cortex (PFC) and corticosterone levels in the plasma. Interestingly, these outcomes were normalized by the presence of a conspecific animal (social support) during RS. RS also significantly upregulated the expression levels of known stress-relevant genes such as Egr1, Crh, and Crhr1, which were normalized by social support. Systemic injection of SL327 (an inhibitor of MEK1/2 that also blocks its downstream signal ERK1/2) prior to RS rescued the working memory impairments and the increased p-ERK1/2 while normalizing the expression of Egr1. Our results suggest that social support can alleviate the RS-induced cognitive impairments partly by modulating ERK1/2 phosphorylation and gene transcription in the PFC, and provide novel insights into the molecular mechanisms of the stress-buffering effects of social support.

Metabolite identification of AZD8055 in Sprague-Dawley rats after a single oral administration using ultra-performance liquid chromatography and mass spectrometry.

AZD8055 is an ATP-competitive specific dual mTOR inhibitor and exhibited potent antitumor activity on several types of solid tumors. However, the metabolism of AZD8055 in the body still remains unknown. In this study, metabolite identification of AZD8055 was performed using ultra high-performance liquid chromatography-ion trap mass spectrometry (UHPLC-IT-MS) through both in vitro and in vivo approaches using rat liver microsomes (RLMs) and rat plasma, urine and feces, respectively. A total of eight putative metabolites (five phase I and three phase II) were identified, and a tentative metabolic pathway was suggested for the first time. Considering the accurate mass and mass fragmentations of the detected metabolites, their plausible structures were suggested. Demethylation, hydroxylation, oxidation and morpholine ring opening were the major biotransformation processes for the phase-I metabolism, while phase-II metabolites were merely generated by the glucuronide conjugation reaction. The cumulative excretion of AZD8055 in urine and feces was 0.13% and 1.11% of the dose, respectively. When the semi-quantitative analysis of the metabolites was performed using UHPLC-MS/MS (ultra-performance liquid chromatography tandem mass spectrometry) to evaluate the overall trend of metabolites formation and excretion, AZD8055 was excreted more in the form of the metabolites than itself and their formation was very fast. Therefore it was presumed that biotransformation was playing a crucial role in its elimination. Ultimately, this study provides novel insights regarding the in vitro and in vivo biotransformations of AZD8055. Further investigations of metabolites of this potent anti-cancer compound could be beneficial for the antitumor drug design and development process.

Standardization of extract mixture of Chaenomeles sinensis and Phyllostachys bambusoides for anti-obesity by HPLC-UV.

JM-101 is a developed functional food formula using water extract of Chaenomeles sinensis and Phyllostachys bambusoides for anti-obesity. Standardization and quality control of herb mixture is more difficult than those of single herb. Additionally, the estimation of mixing ratio is an essential requirement for standardization. This study aimed to develop an efficient analytical method for the standardization of JM-101 based on C. sinensis and P. bambusoides. Protocatechuic acid and p-coumaric acid were selected as marker compounds of JM-101. A mixture of the two medicinal materials (1:1 w/w) was extracted by water and then liquid-liquid extracted (LLE) by ethyl acetate. The supernatant was evaporated to dryness and dissolved in methanol for analysis. The extraction time, material-to-water ratio and ethyl acetate-to-water ratio were optimized by multi-response optimization based on response surface methodology (RSM). The established methods were validated in terms of linearity, precision, accuracy, repeatability, stability and recovery. The novel method based on LLE and RSM provides a sensitive, accurate analysis and excellent extraction efficiency of marker compounds in JM-101, without interruption of other compounds in JM-101. In conclusion, the developed simultaneous analytical method contributes to the standardization of two materials (C. sinensis and P. bambusoides) and JM-101.

Antioxidant and Antifibrotic Effect of a Herbal Formulation In Vitro and in the Experimental Andropause via Nrf2/HO-1 Signaling Pathway.

The Korean herbal formulation Ojayeonjonghwan is used for improving late-onset hypogonadism (LOH) symptoms such as erectile dysfunction (ED). A previous research suggested that a modified Ojayeonjonghwan (KH-204) could be used as an alternative to the treatment for ED. The pharmacological effects were examined in different conditions, including in vitro and in vivo. We measured the survival rate of TM3 Leydig cells under the oxidative stress condition. The s.c. injection of leuprorelin was used to induce androgen deprivation. We measured serum testosterone levels, oxidative stress, and apoptosis. The results of the treatment by KH-204 (1) preserved TM3 cells from oxidative stress by improving the expression of nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1); (2) lowered the expression of transforming growth factor-beta (TGF-ß) 1/SMAD; (3) increased the average of serum testosterone in androgen-deprived male rats; (4) kept the activation of spermatogenesis; (5) upgraded the contents of 8-hydroxy-20-deoxyguanosine (8-OHdG) and degraded the contents of superoxide dismutase (SOD); and (6) reduced apoptosis. We studied that KH-204 improved testicular dysfunction in LOH. It is likely, at least in part, to degrade oxidative stress through the Nrf2/HO-1 pathway. These findings may offer credible evidences for the use of new alternative therapies to treat LOH.

[Two cases of metronidazole-induced encephalopathy].

Metronidazole is a 5-nitroimidazole compound known as an antimicrobial agent widely used for the treatment of protozoal infection, anaerobic infection, Helicobacter pylori infection and hepatic encephalopathy. It may produce a number of neurologic side effects including peripheral neuropathy, seizure, encephalopathy, ataxic gait and dysarthritic speech. There have been ten or more reports of metronidazole-induced encephalopathy in the literatures including a few reports of brain imaging changes by magnetic resonance images (MRI). However, none of the case of metronidazole-induced encephalopathy in patients with hepatic encephalopathy has been reported yet. Recently, we experienced two cases of metronidazole-induced encephalopathy in patients with liver cirrhosis caused by chronic hepatitis B, which were diagnosed by brain MRI and MR spectroscopy. In this report, we present 2 cases of metronidazole-induced encephalopathy with MR imaging and MR spectroscopic changes including follow-up imaging performed after the discontinuation of the metronidazole with a review of the literatures.