High-Sensitivity and High-Throughput Quantification of Everolimus in Human Whole Blood Using Ultrahigh-Performance Liquid Chromatography Coupled With Tandem Mass Spectrometry.

BACKGROUND: Rigorous dose adjustment by therapeutic drug monitoring (TDM) is recommended when everolimus (EVR) is administered for immunosuppression. In this study, the authors developed a highly sensitive ultrahigh-performance liquid chromatography coupled with the tandem mass spectrometry (UHPLC-MS/MS) method for measuring EVR concentrations in whole blood using a high-throughput solid-phase extraction method for sample pretreatment. Furthermore, the blood EVR concentrations in routine TDM samples from patients who underwent renal transplantation measured using the established UHPLC-MS/MS method were compared with those measured using the latex agglutination turbidimetric immunoassay (LTIA). METHODS: Blood samples were pretreated by solid-phase extraction using a 96-well HLB µElution plate. The clinical application of the newly developed method was evaluated using 87 blood samples from 19 patients who underwent kidney transplant. RESULTS: The calibration curve showed good linearity over a wide range of 0.1-50 ng/mL, with relative error ≤15% obtained from the back calculation of calibrators, and ≤20% for the lower limit of quantification. Within-batch and batch-to-batch accuracies and precisions fulfilled the acceptance criteria of the US Food and Drug Administration guidelines for bioanalytical method validation. The extraction recovery rates were good (≥65.2%), and almost no matrix effects were found in any of the quality control samples. Blood EVR concentrations measured by UHPLC-MS/MS were positively correlated with those measured by LTIA. A Bland-Altman plot indicated that the UHPLC-MS/MS method yielded better measurements than the LTIA method, regardless of the concentration. CONCLUSIONS: Therefore, the authors succeeded in developing a novel high-sensitivity and high-throughput method for measuring blood EVR concentration by UHPLC-MS/MS using a µElution plate for sample pretreatment.

Oligomerization mechanism of tea catechins during tea roasting.

Roasting of green tea causes oligomerization of tea catechins, which decreases the astringency. The aim of this study was to elucidate the oligomerization mechanism. The 13C NMR spectrum of the oligomer fraction showed signals arising from catechin and sugar residues. Heating of epigallocatechin-3-O-gallate with 13C-labeled glucose (150 °C for 2 h) suggested that condensation of sugars with catechin A-rings caused the oligomerization. The dimeric product obtained by heating for a shorter period (30 min) suggested cross-linking occurred between sugars and catechin A-rings. Furthermore, heating of phloroglucinol, a catechin A-ring mimic, with glucose, methylglyoxal, and dihydroxyacetone, confirmed that the basic mechanism included reaction of the catechin A-ring methine carbons with carbonyl carbons of glucose and their pyrolysis products.

Ignavine: a novel allosteric modulator of the µ opioid receptor.

Processed Aconiti tuber (PAT) is used to treat pain associated with various disorders. Although it has been demonstrated that the κ opioid receptor (KOR) signaling pathway is a mediator of the analgesic effect of PAT, active components affecting opioid signaling have not yet been identified. In this study, we explored candidate components of PAT by pharmacokinetic analysis and identified ignavine, which is a different structure from aconitine alkaloids. A receptor binding assay of opioid receptors showed that ignavine specifically binds the µ opioid receptor (MOR), not the KOR. Receptor internalization assay in MOR-expressing cell lines revealed that ignavine augmented the responses produced by D-Ala(2)-N-Me-Phe(4)-Gly-ol(5)-enkephalin (DAMGO), a representative MOR agonist, at a low concentration and inhibited it at a higher concentration. Ignavine also exerted positive modulatory activity for DAMGO, endomorphin-1 and morphine in cAMP assay. Additionally, ignavine alone showed an analgesic effect in vivo. In silico simulation analysis suggested that ignavine would induce a unique structural change distinguished from those induced by a representative MOR agonist and antagonist. These data collectively suggest the possibility that ignavine could be a novel allosteric modulator of the MOR. The present results may open the way for the development of a novel pain management strategy.

Preventive effect of oral goshajinkigan on chronic oxaliplatin-induced hypoesthesia in rats.

Oxaliplatin, a widely used chemotherapeutic agent, induces peripheral neuropathy that manifests itself as two distinct phases: acute cold hyperesthesia and chronic peripheral hypoesthesia/dysesthesia. The latter is a serious dose-limiting side effect that can often lead to withdrawal of treatment. We have developed a rat model expressing both phases and used the model to investigate the action of goshajinkigan (GJG), a traditional Japanese herbal medicine, which was reported to ameliorate oxaliplatin-induced neuropathy in a placebo-controlled double-blind randomized phase II study. In this study, neuropathy was induced by injection of oxaliplatin twice weekly for 8 wks. The maximum level of cold hyperesthesia was observed at 4 wks with heat hypoesthesia developing later. Microscopy studies revealed atrophy of axons of myelinated sciatic nerve fibers in oxaliplatin-treated rats at 8 wks. Co-administration of GJG ameliorated both abnormal sensations as well as histological damage to the sciatic nerve. A pharmacokinetic study revealed numerous neuroprotective components of GJG that are rapidly absorbed into the blood. GJG and some of its components attenuated the generation of oxaliplatin-induced reactive oxygen species, which is a possible mechanism of oxaliplatin-induced neurotoxicity. The present study provides a useful animal model for oxaliplatin-induced neurotoxicity as well as a promising prophylactic agent.

Goshajinkigan, a traditional Japanese medicine, prevents oxaliplatin-induced acute peripheral neuropathy by suppressing functional alteration of TRP channels in rat.

The acute peripheral neuropathy induced by oxaliplatin treatment occurs very frequently and is aggravated by exposure to cold. Goshajinkigan (GJG), a traditional Japanese (kampo) medicine, was recently shown to be effective against oxaliplatin-induced acute neuropathy. However, because the effects of GJG and its mechanism in relation to those of its ingredients and its mechanism are not well understood, we examined the effects of GJG on acute neuropathy. Further, we investigated whether GJG affects the functions and gene expressions of transient receptor potential (TRP) channels using a rat model of oxaliplatin-induced neuropathy. Administration of oxaliplatin increased withdrawal responses from cold stimulation, and GJG or calcium gluconate/magnesium sulfate significantly inhibited the oxaliplatin-induced cold hypersensitivity. Application of menthol, a TRPA1/TRPM8 agonist, or allyl isothiocyanate (AITC), a selective TRPA1 agonist, to the hind paw of oxaliplatin-treated rats enhanced the nocifensive behaviors evoked by each agonist, whereas oxaliplatin had no significant effect on nocifensive behaviors evoked by capsaicin, a TRPV1 agonist. GJG treatment reduced menthol- or AITC-evoked withdrawal responses potentiated by oxaliplatin. Furthermore, GJG suppressed the increase of TRPA1 and TRPM8 mRNA expression induced by oxaliplatin in dorsal root ganglia. These findings suggest that GJG prevented oxaliplatin-induced acute peripheral neuropathy by suppressing functional alteration of TRP channels, especially TRPA1 and TRPM8.

Multitargeted effects of hangeshashinto for treatment of chemotherapy-induced oral mucositis on inducible prostaglandin E2 production in human oral keratinocytes.

OBJECTIVE: Chemotherapy-induced oral mucositis (COM) is characterized by painful inflammation with prolonged damage that involves the pathological pain-evoking prostaglandin E2 (PGE2). We previously found that gargling with hangeshashinto (HST), a traditional Japanese medicine, was effective for the treatment of COM. However, little is known regarding the mechanisms. Our aim was to identify the active ingredients and clarify the characteristic effects of HST on the PGE2 system. METHODS: Prostanoids produced by human oral keratinocytes (HOK) stimulated with IL-1ß were measured by enzyme immunoassay. Active ingredients that regulate PGE2 production were identified and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and a culture system of HOK cells. RESULTS: Inducible PGE2, PGD2, and PGF2α, metabolites of cyclooxygenase (COX) pathways, were reduced by HST (10-300 µg/mL) without inducing cytotoxicity. The active ingredients of HST were quantified by LC-MS/MS, and [6]-shogaol, [6]-gingerol, wogonin, baicalein, baicalin, and berberine were shown to reduce PGE2 production. A mixture of these 6 ingredients at concentrations equal to 300 µg/mL of HST strongly suppressed PGE2 production to the same level as HST. [6]-Shogaol and [6]-gingerol did not decrease COX-2 mRNA expression and mostly inhibited PGE2 metabolic activity in an assay using intact HOK cells, suggesting that they regulate PGE2 synthesis at the posttranscriptional level. Wogonin, baicalin, and berberine inhibited expression of COX-2 mRNA without affecting PGE2 metabolic activity. Moreover, wogonin, but not [6]-shogaol, suppressed phosphorylation of mitogen-activated protein kinases (p38s and JNKs). CONCLUSIONS: These lines show that HST includes several PGE2-regulating ingredients that have different mechanisms and can function as a multicomponent and multitarget agent for treatment of COM, indicating that HST may be beneficial in a new medical strategy for COM treatment.

Free radical scavenging and hepatoprotective actions of the medicinal herb, Crassocephalum crepidioides from the Okinawa Islands.

Free radical scavenging and protective actions against chemically induced hepatotoxicity of Crassocephalum crepidioides were investigated. A water extract of C. crepidioides strongly scavenged superoxide anion, hydroxyl radical and also stable radical 1,1-diphenyl-2-picrylhydrazyl. Galactosamine (GalN, 400 mg/kg) and lipopolysaccharide (LPS, 0.5 microg/kg) induced hepatotoxicity of rats as seen by an elevation of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and of lipid peroxidation in liver homogenates was significantly depressed when the herbal extract was given intraperitoneally 1 and 15 h before GalN and LPS treatment. Similarly, carbon tetrachloride (CCl4) induced liver injury as evidenced by an increase in AST and ALT activities in serum was also inhibited by the extract pretreatment. Isochlorogenic acids, quercetin and kaempferol glycosides were identified as active components of C. crepidioides with strong free radical scavenging action. These results demonstrate that C. crepidioides is a potent antioxidant and protective against GalN plus LPS- or CCl4-induced hepatotoxicity.

Genetic and phenotypic characterization of pyrazinamide-resistant mycobacterium tuberculosis complex isolates in Japan.

The pncA gene mutations associated with pyrazinamide (PZA) resistance in Mycobacterium tuberculosis complex were determined in 26 PZA-resistant isolates in Japan. Of the 26 PZA-resistant isolates included, 21 were negative for pyrazinamidase (PZase). Of these, 20 isolates had various pncA mutations, resulting in alteration of primary amino acid sequence. However, 1 PZase-negative isolate did not have any mutation on pncA gene. The remaining 5 PZA-resistant isolates were positive for PZase and had identical pncA alleles with PZA-susceptible isolates. IS6110 RFLP analysis demonstrated various distinct IS6110 types and 5 pairs of isolates were very close to each other (>90% identical pattern). This study demonstrates that most of the PZA resistance is a result of various mutations on pncA resulting in loss of PZase activity. Further investigation, particularly on PZase-positive but PZA-resistant isolates and a PZase-negative isolate with no mutation on pncA, should be urgently done.

[Hospital-acquired Streptococcus pneumoniae infection].

Three elderly patients were consecutively found to harbor or to become infected with intermediate-level penicillin-resistant Streptococcus pneumoniae in a hospital respiratory ward. All the isolates from the respective patients produced mucoid-type colonies on sheep blood agar plates and were found to have an identical antibiogram, indicating that those were resistant against erythromycin, clarithromycin, clindamycin and minocycline. Pulse-field gel electrophoresis of genomic DNA digested Sma I and Apa I demonstrated homology among the isolates, which may suggest person-to-person spread in a hospital setting. With this, it is an urgent to establish the institution-based infection control precautions against S. pneumoniae.

Dimerumic acid as an antioxidant from the mold, Monascus anka: the inhibition mechanisms against lipid peroxidation and hemeprotein-mediated oxidation.

This study aimed to investigate the antioxidant mechanism of dimerumic acid isolated as the active component with a radical scavenging action from the mold Monascus anka, traditionally used for the fermentation of foods. Dimerumic acid inhibited NADPH- and iron(II)-dependent lipid peroxidation (LPO) of rat liver microsomes at 20 and 200 microM, respectively. When ferrylmyoglobin was incubated with dimerumic acid, the myoglobin was scavenged and an electron spin resonance (ESR) signal with nine peaks was observed. The spin adduct was identified as a nitroxide radical by analysis of hyperfine structure. Similar ESR signal was also detected by incubation of dimerumic acid with peroxyl radicals. Thus, it was clarified that the antioxidant action of dimerumic acid is due to one electron donation of the hydroxamic acid group in the dimerumic acid molecule toward oxidants resulting in formation of nitroxide radical.

[Interpretive compatibility of antimycobacterial susceptibility for Mycobacterium tuberculosis determined by proportion test method on egg-based Ogawa media and broth microdilution test, BrothMIC MTB].

The antimycobacterial susceptibility test method newly proposed by the Japanese Society for Tuberculosis, a proportion method on egg-based Ogawa media, was evaluated in comparison with microdilution test for Mycobacterium tuberculosis complex, BrothMIC MTB-1 (Kyokuto Pharmaceutical Inc., Tokyo). In the evaluation, five antimicrobial agents, streptomycin, ethambutol, kanamycin, isoniazid and rifampicin were included. Through repeated testings of the three reference strains against five antimicrobial agents, both test methods were found to be highly precise. All the minimum inhibitory concentrations (MICs) determined by BrothMIC MTB fell within 3 log2 dilutions, however a total of 11 MICs resulted in indeterminate(I) interpretations. Whereas, all the test results by a proportion method on Ogawa media were comparable to the expected interpretations. However, three of 48 testings resulted in undeterminable interpretations due to insufficient growth on the growth control media. A total of 127 clinical isolates of M. tuberculosis complex were tested by both methods, and 89 to 90% of the test results were comparable with each other in category interpretations. However, 7.1 to 9.4% of MICs determined by BrothMIC MTB resulted in indeterminate(I), and 0.8 to 3.1% of discrepant interpretations were observed. In conclusion, both test methods were highly precise and comparable in determining antimycobacterial susceptibility for M. tuberculosis complex. Several advantages and disadvantages in each test method were discussed.