Decoding predicted musical notes from omitted stimulus potentials.

Electrophysiological studies have investigated predictive processing in music by examining event-related potentials (ERPs) elicited by the violation of musical expectations. While several studies have reported that the predictability of stimuli can modulate the amplitude of ERPs, it is unclear how specific the representation of the expected note is. The present study addressed this issue by recording the omitted stimulus potentials (OSPs) to avoid contamination of bottom-up sensory processing with top-down predictive processing. Decoding of the omitted content was attempted using a support vector machine, which is a type of machine learning. ERP responses to the omission of four target notes (E, F, A, and C) at the same position in familiar and unfamiliar melodies were recorded from 25 participants. The results showed that the omission N1 were larger in the familiar melody condition than in the unfamiliar melody condition. The decoding accuracy of the four omitted notes was significantly higher in the familiar melody condition than in the unfamiliar melody condition. These results suggest that the OSPs contain discriminable predictive information, and the higher the predictability, the more the specific representation of the expected note is generated.

Visual omitted stimulus potentials are not retinotopic.

Omitted stimulus potentials (OSPs) are elicited in response to the omission of expected stimuli and are thought to reflect prediction errors. If prediction errors are signaled in the sensory cortex, OSPs are expected to be generated in the sensory cortex. The present study investigated the involvement of the early visual cortex in the generation of OSPs by testing whether omitted visual stimuli elicit brain responses in a spatially specific manner. Checkerboard pattern stimuli were presented alternately in the upper and lower visual fields, and the stimuli were omitted in 10 % of the trials. Event-related potentials were recorded from 33 participants. While a retinotopic C1 component was evoked by real visual stimuli, omitted stimuli did not produce any response reflecting retinotopy but did elicit a visual mismatch negativity, which was larger for omitted stimuli expected in the lower visual field than for those in the upper visual field. These results suggest that omitted visual stimuli are processed in a different pathway than actual stimuli.

Effects of sensory modality and task relevance on omitted stimulus potentials.

Omitted stimulus potentials (OSPs) occur when a sensory stimulus is unexpectedly omitted. They are thought to reflect predictions about upcoming sensory events. The present study examined how OSPs differ across the sensory modalities of predicted stimuli. Twenty-nine university students were asked to press a mouse button at a regular interval of 1-2 s, which was immediately followed by either a visual or auditory stimulus in different blocks. The stimuli were sometimes omitted (p = 0.2), to which event-related potentials (ERPs) were recorded. The results showed that stimulus omissions in both modalities elicited ERP waveforms consisting of three components, oN1, oN2, and oP3. The peak latencies of these components were shorter in the auditory modality than in the visual modality. The amplitudes of OSPs were larger when participants were told that the omission indicated their poor performance (i.e., they pressed a button at an irregular interval) than when it was irrelevant to their performance. These findings suggest that OSPs occur from around 100 ms in a modality-specific manner and increase in amplitude depending on the task relevance of stimulus omissions.

Liposome-Encapsulated Eribulin Shows Enhanced Antitumor Activity over Eribulin for Combination Therapy with Anti-PD-1 Antibody.

Eribulin is a microtubule dynamics inhibitor with tumor microenvironment modulation activity such as vascular remodeling activity. Here, we investigated antitumor and immunomodulatory activities of eribulin and its liposomal formulation (eribulin-LF) as monotherapies or in combination with anti-programmed death 1 (PD-1) Ab. The antitumor activity of eribulin or eribulin-LF as monotherapy or in combination with anti-PD-1 Ab was examined in a P-glycoprotein-knockout 4T1 model. Eribulin and eribulin-LF showed stronger antitumor activity in immunocompetent mice compared with immunodeficient mice, indicating that they have immunomodulatory activity that underlies its antitumor activity. Combination therapy of eribulin and eribulin-LF with anti-PD-1 Ab showed antitumor activity, and the combination activity of eribulin-LF with anti-PD-1 Ab was observed at a lower dose and longer interval of administration compared with that using eribulin. To examine the immunomodulatory activity of eribulin and eribulin-LF and its underlying mechanisms, we performed flow cytometry, IHC, and gene expression profiling. IHC and flow cytometry revealed that eribulin-LF increased microvessel density and intratumoral populations of cytotoxic T cells and natural killer cells rather than eribulin. Gene expression profiling demonstrated that eribulin-LF induces IFNγ signaling. Furthermore, IHC also showed that eribulin-LF increased infiltration of CD8-positive cells together with increased CD31-positive cells. Eribulin-LF also increased ICAM-1 expression, which is essential for lymphocyte adhesion to vascular endothelial cells. In conclusion, eribulin showed combination antitumor activity with anti-PD-1 Ab via immunomodulation due to its vascular remodeling activity, and the liposomal formulation showed improved antitumor activity over the standard formulation.

Imaging Mass Spectrometry (IMS) for drug discovery and development survey: Results on methods, applications and regulatory compliance.

Imaging mass spectrometry (IMS) is increasingly used for drug discovery and development to understand target enagement, tissue distribution, drug toxicity, and disease mechanisms, etc. However, this is still a relatively new technique that requires further development validation before it will be an acceptable technique to support regulated development of new drugs. Thus, best practices will need to be established to build more confidence and gain wider acceptance by the scientific community, pharmaceutical industry, and regulatory authorities. The Imaging Mass Spectrometry Society (IMSS) and the Japan Association for Imaging Mass Spectrometry (JAIMS) have conducted a thorough survey to gather information on the current state of IMS and to identify key issues. The survey was sent to researchers or managers in the position who are currently using IMS techniques in support of their drug discovery and development efforts and/or who plan to use such tools as best practices are established. The survey probes questions related to details regarding technical aspects of IMS, which includes data acquisition, data analysis and quantitation, data integrity, reporting, applications, and regulatory concerns. This international survey was conducted online through the Survey Monkey (https://www.surveymonkey.com) in both English and Japanese from September 14 through September 30, 2020.

Disposition and Metabolism of [14C]Lemborexant in Healthy Human Subjects and Characterization of Its Circulating Metabolites.

Lemborexant is a novel dual orexin receptor antagonist recently approved for the treatment of insomnia in the United States and Japan. Here, disposition and metabolic profiles were investigated in healthy human subjects. After single oral administration of 10 mg [14C]lemborexant (100 µCi), plasma concentrations of lemborexant and radioactivity peaked at 1 hour postdose and decreased biphasically. Cumulative recovery of the administered radioactivity within 480 hours was 86.5% of the dose, with 29.1% in urine and 57.4% in feces. Unchanged lemborexant was not detected in urine but accounted for 13.0% of the dose in feces, suggesting that the main elimination pathway of lemborexant was metabolism. Metabolite analyses revealed that the major metabolic pathways of lemborexant are oxidation of the dimethylpyrimidine moiety and subsequent further oxidation and/or glucuronidation. In plasma, lemborexant was the dominant component, accounting for 26.5% of total drug-related exposure. M4, M9, M10, and M18 were detected as the major radioactive components; M10 was the only metabolite exceeding 10% of total drug-related exposure. Although M4, M9, and M10 showed binding affinity for orexin receptors comparable to that of lemborexant, their contributions to the sleep-promoting effects of lemborexant are likely low because of the limited brain penetration by P-glycoprotein. Exposure comparison between humans and nonclinical toxicology species confirmed that plasma exposure of M10 was higher in at least one animal species compared with that in humans, indicating that there is no disproportionate metabolite in humans, as defined by International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use M3(R2) and U.S. Food and Drug Administration Metabolite in Safety Testing guidance; therefore, no additional toxicology studies are needed. SIGNIFICANCE STATEMENT: This study provides detailed data of the disposition and metabolism of lemborexant, a novel therapeutic drug for insomnia, in humans, as well as a characterization of the circulating metabolites and assessment of their contributions to efficacy and safety. The information presented herein furthers our understanding of the pharmacokinetic profiles of lemborexant and its metabolites and will promote the safe and effective use of lemborexant in the clinic.

Quantitative and qualitative application of a novel capillary microsampling device, Microsampling Wing™ (MSW), using antiepileptic drugs in rats.

A novel microsampling device, namely, the Microsampling Wing™ (MSW), was evaluated using three anti-epileptic drugs (AEDs): carbamazepine, lamotrigine, and phenytoin. A simultaneous assay method of the three AEDs was developed and qualified via liquid chromatography with tandem mass spectrometry. Using 2.8 µL plasma, the three AEDs were quantifiable from 1 or 2 ng/mL. According to the intra-assay reproducibility assessment and additional validation parameters, the established method is reproducible. To apply the device to a pharmacokinetic (PK) study in rats, a cocktail of the three AEDs was orally administered to rats. Whole blood samples were serially collected using the MSW device and a glass capillary from the tail vein, and plasma samples (each 2.8 µL) from each device were assayed to compare PK parameters. The PK parameters of the three AEDs were similar between the two devices. A metabolite identification study was also conducted after oral administration of carbamazepine to rats. At least seven metabolites were detected in plasma, and the major metabolite was carbamazepine 10,11-epoxide, which is in accordance with the reported results. These findings suggest that the MSW device is a useful microsampling device for PK and metabolite identification studies.

Bioactivation of diclofenac in human hepatocytes and the proposed human hepatic proteins modified by reactive metabolites.

To reveal putative bioactivation pathways of diclofenac, in vitro human liver materials such as microsomal fractions and hepatocytes were used to confirm metabolic activation of diclofenac by 35S-cysteine trapping assay and covalent binding assay. Candidate human liver proteins possibly targeted by 14C-diclofenac via bioactivation were investigated using two-dimensional gel electrophoresis followed by detection of remaining radioactivity on the modified proteins with bio-imaging analyzer.In the 35S-cysteine trapping assay, three and two adducts with 35S-cysteine were observed in NADPH-fortified and UDPGA-fortified human liver microsomes, respectively. In the covalent binding assay using 14C-diclofenac in human hepatocytes, the extent of covalent binding of diclofenac to human hepatic proteins increased time-dependently. Addition of glutathione attenuated the extent of covalent binding of 14C-diclofenac to human liver microsomal proteins.Fifty-nine proteins from human hepatocytes were proposed as the candidate proteins targeted by reactive metabolites of diclofenac. Proteins modified by cytochrome P450-mediated reactive metabolites were identified by using a cytochrome P450 inhibitor, 1-aminobenzyltriazole and seven of the nine radioactive protein spots were removed by 1-aminobenzyltriazole treatment.In contrast, the remaining two radioactive protein spots, mainly containing human serum albumin and heat shock proteins, were not affected by the addition of 1-aminobenzyltriazole, which suggested the involvement of the acyl glucuronide of diclofenac, formed via uridine diphosphate-glucuronosyl transferases, in the covalent modifications induced by diclofenac.

The Effects of Meaning Dominance in the Time-Course of Activation of L2 Lexical Ambiguity Processing.

This paper investigates the effects of meaning dominance in the time-course of activation for ambiguous words out of context in a second language (L2) based on two models: the ordered access model, where the most frequent dominant meaning is always accessed first, and the multiple access model, where dominant and subordinate meanings are activated. Non-native speakers of English (divided into high and low proficiency groups) and native English speakers completed a lexical decision task. While both L2 high and low proficiency groups retrieved multiple meanings of the ambiguous words at different stimulus-onset asynchronies supporting the multiple access model, the move from the ordered access model to the multiple access model was confirmed for the native English speaker group. The findings indicated developmental change of sensitivity to meaning dominance. The results also demonstrated that the rate of facilitation differed among the groups due to slow and more transient L2 activation.

Disposition and metabolism of [14C]lemborexant, a novel dual orexin receptor antagonist, in rats and monkeys.

The disposition and metabolism of lemborexant, a novel dual orexin receptor antagonist currently under development as a therapeutic agent for insomnia disorder, were evaluated after a single oral administration of [14C]lemborexant in Sprague-Dawley rats (10 mg/kg) and cynomolgus monkeys (3 mg/kg). In both species, [14C]lemborexant was rapidly absorbed: radioactivity concentration in blood peaked at 0.83-1.8 h, and decreased with elimination half-life of 110 h. The radioactivity administered was excreted primarily into faeces, with relatively little excreted into urine. Lemborexant was not detected in bile, urine or faeces, indicating that lemborexant administered orally was completely absorbed from the gastrointestinal tract and that the main elimination pathway was metabolism in both species. In rats, lemborexant was found to be minor in plasma (≤5.2% of total radioactivity), and M9 (hydroxylated form) was the major circulating metabolite. In monkeys, the major circulating components were lemborexant, M4 (N-oxide metabolite), M13 (di-oxidised form), M14 (di-oxidised form) and M16 (glucuronide of mono-oxidised form). In both species, lemborexant was metabolised to various metabolites by multiple pathways, the primary of which was oxidation of the dimethylpyrimidine or fluorophenyl moiety.

Semantic Ambiguity Effects in L2 Word Recognition.

The present study examined the ambiguity effects in second language (L2) word recognition. Previous studies on first language (L1) lexical processing have observed that ambiguous words are recognized faster and more accurately than unambiguous words on lexical decision tasks. In this research, L1 and L2 speakers of English were asked whether a letter string on a computer screen was an English word or not. An ambiguity advantage was found for both groups and greater ambiguity effects were found for the non-native speaker group when compared to the native speaker group. The findings imply that the larger ambiguity advantage for L2 processing is due to their slower response time in producing adequate feedback activation from the semantic level to the orthographic level.

Rapid and quantitative screening method for 43 benzodiazepines and their metabolites, zolpidem and zopiclone in human plasma by liquid chromatography/mass spectrometry with a small particle column.

Benzodiazepines and their pharmacologically related drugs, zolpidem and zopiclone are widely prescribed as safe drugs, but these drugs are also abused in cases of crime, suicide and drug-facilitated sexual assault. We developed a rapid and quantitative screening method for 43 benzodiazepines, their metabolites, zolpidem and zopiclone in human plasma by liquid chromatography/mass spectrometry with a small particle column. All drugs were successfully separated within 12 min using combined scan and selected ion recording (SIR) mode. The calibration curves of most drugs were linear in the concentration range 0.5-250 ng/mL with correlation coefficients exceeding 0.99. Within-day precisions (RSD, %) of this method were 1.8-15.6% (10 ng/mL) and 0.6-10.1% (100 ng/mL) and between-day precisions (RSD, %) were 1.6-16.9% (10 ng/mL) and 0.6-16.7% (100 ng/mL). The average recoveries were 70.1% (10 ng/mL) and 87.1% (100 ng/mL). The limit of detection ranged from 0.2 to 8.0 ng/mL in 37 drugs and was below 0.2 ng/mL in 6 drugs. The established method is sensitive and rapid, thus it should be useful in forensic and clinical toxicological analyses.

[Review of the drug analysis system accompanied by forensic autopsy in Finland].

In Japan, drug analyses for forensic autopsies have been traditionally carried out at each laboratory of the Department of Forensic Medicine. However, it is difficult to maintain a high quality of drug analysis in each department due to an insufficient number of staff and lack of equipment. Therefore, the establishment of more advanced toxicology centers which can handle all drugs associated with forensic autopsies is essential. In addition, a systematic system for requesting drug analyses from each department and dealing with the results from the center is needed. The number of forensic autopsies carried out in Finland is as high as that in Japan although the population is 1/24th that of Japan, and toxicological analyses for the entire country are centralized in one place, the Laboratory of Toxicology, Department of Forensic Medicine, University of Helsinki. Since the autopsies and drug analyses are carried out at a University as in Japan, the drug analysis system in Finland can be a good model when considering the future system in Japan. Therefore, a review of the drug analysis system accompanied by forensic autopsy in Finland was carried out with the collaboration of the Departments of Forensic Medicine, University of Helsinki and University of Turku. Based on the above studies and the present situation in Japan, we discuss the future drug analysis system needed in Japan.

Rapid diagnosis of drug intoxication using novel NAGINATA gas chromatography/mass spectrometry software.

In Japan, not only the classical stimulant, methamphetamine, but also a wide variety of illicit drugs and designer drugs are abused by juveniles. It is, however, difficult to screen these drugs in human urine due to the poor availability of high-quality standards. Therefore, it is important to develop a screening method that does not require the use of standard compounds. Furthermore, if we can obtain approximate drug concentrations in biological fluids by the first screening procedure, the subsequent treatment of the patient and forensic diagnosis can be carried out more rapidly and exact quantitative analysis performed more efficiently. We have devised a rapid screening method for the simultaneous semi-quantitative analysis of 30 abused drugs using gas chromatography/mass spectrometry (GC/MS) with a retention time locking technique. Based on this method, an 'abused drugs database' was constructed including retention time (RT), qualifier ion/target ion (QT) percentage and calibration curve (values of slope and intercept) using the novel GC/MS software, NAGINATA. We compared the analytical results obtained by this method using the constructed database with those from conventional methods in six forensic cases. The number of confirmed drugs and concentrations obtained by the established method was comparable with that obtained by conventional methods. We found a significant improvement in the time for data analysis, and qualitative and quantitative information about each drug was obtained without using standards. Therefore, this new screening procedure using NAGINATA has potential for the rapid identification of poisoning and should be useful in clinical and forensic toxicological analyses.

Simultaneous determination of 13 amphetamine related drugs in human whole blood using an enhanced polymer column and gas chromatography-mass spectrometry.

Metamphetamine (MA) is one of the most frequently encountered abused drugs in Japan and the Triage immunoassay kit is often used to screen for this drug. However, immunoassay screening also gives positive results with other structurally related compounds, such as 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), p-methoxyamphetamine (PMA), an ephedrine metabolite and beta-phenethylamine (PEA). Therefore, it is important to develop a simple and reliable method which can determine these drugs simultaneously. This paper describes a simple method for simultaneous identification and quantification of 13 amphetamine related drugs in human whole blood. The method consists of a solid phase extraction using a new polar-enhanced Focus column followed by acetylation and gas chromatography-mass spectrometry in the scan mode. Tetradeuterated MA and trideuterated methylephedrine (ME) were used as internal standards. As the Focus column required only simple extraction steps and provided a clean extract, identification of each drug was feasible even at low concentrations. The calibration curves were linear over the concentration range from 50 to 5000 ng/ml for all drugs with correlation coefficients that exceeded 0.99. The lower limits of detection of the drugs were 5-50 ng/ml. The absolute recoveries for the drugs were 65-95% and 64-89% at concentrations of 100 and 1000 ng/ml, respectively. Accuracy and precision data were satisfactory when using 2 internal standards. The applicability of the assay was proven by the analysis of blood samples in forensic cases. This method should be most useful for confirmation of positive immunoassay results for amphetamines and related drugs.

A fatal case of amoxapine poisoning under the influence of chronic use of psychotropic drugs.

A 43-year-old woman was found dead in a car in the supine position. She had been suffering from depression for 2 years and hesitation wounds on the left forearm and wrist were observed. On microscopic examination, pulmonary congestion and edema were observed with heart failure cells in many alveoli, thereby suggesting not only acute but also chronic heart failure. Drug screening in the blood by gas chromatography-mass spectrometry (GC-MS) revealed the presence of amoxapine and levomepromazine, and their concentrations in tissues were determined by GC-MS with three-step solvent extraction followed by acetylation. The concentration of amoxapine in the blood and liver was 0.86-1.77 and 18.76microg/ml, respectively; the levels were much higher than the therapeutic level but did not reach the lethal level. The concentrations of levomepromazine in tissues were within the therapeutic level. Based on the pathological and toxicological findings, the cause of death was determined to be amoxapine poisoning on the basis of chronic heart failure due to the chronic use of psychotropic drugs.

Rapid screening for and simultaneous semiquantitative analysis of thirty abused drugs in human urine samples using gas chromatography-mass spectrometry.

In Japan, a wide variety of designer drugs became popular among juveniles because of their availability via the Internet and mobile phones. Hence, it is necessary to develop simple and rapid screening method for these drugs. We devised a rapid screening method for and simultaneous semiquantitative analysis of 30 abused drugs, including amphetamines, amphetamine-, piperazine-, tryptamine-, and phenethylamine-derived designer drugs and opiates in human urine. The urine sample was digested with urease, and the drugs were analyzed by gas chromatography-mass spectrometry in the scan mode after solid-phase extraction with a Focus column and acetylation. The retention time obtained with the use of a retention time locking technique and three qualifier ions were used to obtain positive results. As the Focus column requires only simple extraction steps and can retain various drugs of a wide range of polarity, screening of 30 abused drugs was feasible within 3 h. The calibration curves were linear in the concentration range of 100-5,000 ng/mL in most drugs with correlation coefficients exceeding 0.99. The absolute recoveries for all drugs in urine samples were 6.9-125.4% at the concentration 1,000 ng/mL. This method will be most useful to confirm the presence of many abused drugs in urine in clinical and forensic cases.

Methamphetamine-related sudden death with a concentration which was of a 'toxic level'.

We reviewed 32 cases where a forensic autopsy detected methamphetamine in the blood, and all of these autopsies were performed at two institutes between 1991 and 2003. In accordance with several criteria, the blood concentration in 11 cases was classified as above the toxic level, and 10 of these cases were diagnosed as methamphetamine poisoning. In 20 cases (62.5% of total cases), the blood concentration was of a 'toxic level', and 10, 2 and 1 of these cases were diagnosed as methamphetamine poisoning, cardiomyopathy and intracerebral hemorrhage, respectively. Since it is unclear how the effects of methamphetamine may contribute to the death of an individual, a diagnosis of the exact cause of death is often difficult to make in cases where the blood concentration of methamphetamine was of a 'toxic level'. Therefore, a diagnosis has to be carefully made in consideration of the pathological findings, the pharmacological effects of methamphetamine and the process until death in such cases. Additionally, the mechanism of methamphetamine-related death needs to be more fully studied to enable an appropriate diagnosis to be made easily.

Vulnerability of experimentally induced fatty liver to heat stress in rats.

BACKGROUND: The aim of this study was to confirm the vulnerability of fatty liver to heat stress using fatty liver rats from the viewpoint of the induction of apoptosis. METHODS: We exposed rats with and without a fatty liver to heat stress and then looked for apoptotic cells within the liver tissue using two apoptosis detection kits. We also determined the mRNA expression of heat shock protein (HSP) 70, caspase-3, bcl-2, and bax using a quantitative reverse transcription-polymerase chain reaction method. RESULTS: Following heat stress, apoptosis was strongly visible in the fatty liver comparing with that noted in the normal liver. The expression of HSP70 was increased following heat stress in both livers, but the volume of its expression was significantly less in the fatty liver than in the normal liver. The ratio of bcl-2/bax expression tended to increase in the normal liver but decrease in the fatty liver following heat stress. Caspase-3 demonstrated no significant change following heat stress in both livers. CONCLUSIONS: The detection of apoptosis, together with changes in the mRNA expression of HSP70 and the expression ratio bcl-2/bax mRNA may indicate vulnerability of a fatty liver to heat stress and may support the hypothesis that morphologic change is induced in a fatty liver by exposure to heat stress. These results suggest that fatty liver may be more vulnerable to heat stress than normal liver.

Simple and sensitive determination of free and total morphine in human liver and kidney using gas chromatography-mass spectrometry.

We developed a reliable, simple and sensitive method to determine free and total morphine in human liver and kidney, using gas chromatography-mass spectrometry (GC-MS). Free morphine or total morphine obtained by acid hydrolysis from 0.2g tissue sample was extracted using an Extrelut NT column with an internal standard, dihydrocodeine, followed by trimethylsilylation. The derivatized extract was submitted to GC-MS analysis of EI-SIM mode. The calibration curves of morphine in both liver and kidney samples were linear in the concentration range from 0.005 to 5 microg/g. The lower limits of detection of morphine were 0.005 microg/g. This method proved successful when we determined free and total morphine in liver and kidney obtained from an autopsied man who was mis-ingested morphine compound in the hospital, which resulted in the cause of death being morphine intoxication.