Toru Imai

Subregion-specific vulnerability to endoplasmic reticulum stress-induced neurotoxicity in rat hippocampal neurons.

It is well known that in certain disease states, including ischemia and Alzheimers disease, neurodegeneration occurs in the hippocampus and that vulnerability to neuronal death is area dependent. The present study investigated the mechanism of area-dependent vulnerability to neuronal death under endoplasmic reticulum stress conditions induced by tunicamycin (TM), using rat organotypic hippocampal cultures (OHC) and hippocampal slices. Analysis of propidium iodide uptake showed that TM-induced neuronal death in a concentration-dependent manner (20-80 microg/mL) and that the rank order of vulnerability among hippocampal subregions was dentate gyrus (DG)>CA1>CA3. Results of immunohistochemistry using hippocampal slices also showed that procaspase-12-positive cells in area CA3 were significantly fewer than those in area CA1 and the DG. Moreover, procurement of neurons in areas CA1, CA3 and the DG by laser microdissection, followed by Western blot analysis, also revealed that the level of procaspase-12 in area CA3 was significantly lower than those in area CA1 and the DG. Pretreatment with z-ATAD-fmk, a cell-permeable caspase-12-selective inhibitor significantly attenuated the TM-induced increase of PI fluorescence in the CA1 and DG subregion but not in area CA3. These results suggest that TM elicits subregion-specific neuronal toxicity in OHC and that the vulnerability to TM-induced toxicity is at least partly dependent on the expression level of endogenous procaspase-12 in each area of the hippocampus.

Amyloid β-protein potentiates tunicamycin-induced neuronal death in organotypic hippocampal slice cultures

We have assessed amyloid beta protein (Abeta)-induced neurotoxicity, with and without added tunicamycin (TM), an inhibitor of N-glycosylation in the endoplasmic reticulum (ER), in rat organotypic hippocampal slice cultures (OHCs). In the rat OHCs cultured for 3 weeks, there was little neurotoxicity after treatment with Abeta(25-35) (25 microM) alone for 48 h. However, with TM alone, concentration-dependent neuronal death was observed at concentrations between 20 and 80 microg/mL. When amyloid-beta protein was combined with tunicamycin (Abeta+TM), cell death was more acute than with TM alone. Western blot analysis revealed that calpain activity and the active forms of caspase-12 and caspase-3 was increased after exposure to Abeta+TM as compared with exposure to TM alone. In contrast, the levels of glucose regulated protein (GRP)94, GRP78 and C/EBP homologous protein (CHOP) were not changed in the presence of Abeta. Abeta potentiation of TM neurotoxicity was reversibly blocked by S-allyl-L-cysteine (SAC), an organosulfur compound purified from aged garlic extract, and the L-type calcium channel blocker, nifedipine, in a restricted neuronal area of the OHCs. Simultaneously applied SAC also reversed the increases in calpain activity and the active forms of caspase-12 and caspase-3 by Abeta+TM with no change in the increased levels of GRP94, GRP78 and CHOP. These data indicate that Abeta facilitates the calpain-caspase-12-caspase-3 pathway, thus potentiating TM-induced neuronal death in the hippocampus.

Protective effect of S-allyl-L-cysteine against endoplasmic reticulum stress-induced neuronal death is mediated by inhibition of calpain.

Endoplasmic reticulum (ER) stress, implicated in various neurodegenerative processes, increases the level of intracellular Ca2+ and leads to activation of calpain, a Ca2+-dependent cysteine protease. We have shown previously that S-allyl-l-cysteine (SAC) in aged garlic extracts significantly protects cultured rat hippocampal neurons (HPNs) against ER stress-induced neurotoxicity. The neuroprotective effect of SAC was compared with those of the related antioxidant compounds, l-cysteine (CYS) and N-acetylcysteine (NAC), on calpain activity in HPNs and also in vitro. SAC, but not CYS or NAC, reversibly restored the survival of HPNs and increased the degradation of α-spectrin, a substrate for calpain, induced by tunicamycin, a typical ER stress inducer. Activities of μ- and m-calpains in vitro were also concentration dependently suppressed by SAC, but not by CYS or NAC. At submaximal concentration, although ALLN (5xa0pM), which blocks the active site of calpain, and calpastatin (100xa0pM), an endogenous calpain-inhibitor protein, additively inhibited μ-calpain activity in vitro in combination with SAC, the effect of PD150606 (25xa0μM), which prevents interaction of Ca2+ with the Ca2+-binding site of calpain, was unaffected by SAC. In contrast, SAC (1xa0mM) significantly reversed the effect of PD150606 at a concentration that elicited supramaximal inhibition (100xa0μM), but did not affect ALLN (1 nM)- and calpastatin (100 nM)-induced inhibition of μ-calpain activity. These results suggest that the protective effects of SAC against ER stress-induced neuronal cell death are not attributable to antioxidant activity, but to suppression of calpain through interaction with its Ca2+-binding site.

Neuroprotective effect of S-allyl-l-cysteine derivatives against endoplasmic reticulum stress-induced cytotoxicity is independent of calpain inhibition.

S-allyl-l-cysteine (SAC) is known to have neuroprotective properties. We synthesized various SAC derivatives and tested their effects on endoplasmic reticulum stress-induced neurotoxicity in cultured hippocampal neurons (HPNs). Among the compounds tested, S-propyl-l-cysteine (SPC) exhibited the strongest neuroprotective activity in HPNs, followed by S-ethyl-l-cysteine (SEC) and S-methyl-l-cysteine (SMC). Unlike SAC and SMC, SPC and SEC did not have inhibitory activity on μ-calpain, suggesting that the mechanism underlying the protective activity of SPC and SEC differs from that of SAC.

Duration of Systemic Inflammatory Response Syndrome Influences Serum Vancomycin Concentration in Patients With Sepsis

PURPOSEnVancomycin (VCM) is used in the treatment of methicillin-resistant Staphylococcus aureus infection. The dosage of VCM must be adjusted by using therapeutic drug monitoring because of the drugs narrow therapeutic concentration window. Although optimal administration based on population pharmacokinetic (PPK) analysis and/or a Bayesian method has improved prediction accuracy, serum concentrations of VCM in patients with sepsis often deviate significantly from predicted values. We investigated factors influencing prediction errors with PPK analysis in VCM dosing.nnnMETHODSnThis retrospective cohort study included patients treated with VCM. Their clinical data were recorded, and there were 27 nonseptic patients and 68 septic patients. VCM concentrations were predicted by using PPK analysis and data compared with observed concentrations.nnnFINDINGSnPatients with sepsis had a higher mean absolute error than nonseptic patients, indicating a deviation of VCM concentrations from predicted values in the septic patients. To determine factors influencing prediction errors, we classified patients with sepsis into 3 subgroups according to the mean absolute error value (2.17) for the nonseptic patients: lower group (prediction errors, below -2.17), upper group (>2.17), and no change group (-2.17 to 2.17). In a comparison of clinical characteristics of the 3 groups, significant differences were found in the duration of systemic inflammatory response syndrome (SIRS), SIRS score, disseminated intravascular coagulation score, and levels of creatinine clearance (CrCl), hemoglobin, and diastolic blood pressure. Multiple logistic regression analysis identified SIRS duration and CrCl as factors associated with VCM concentrations in the lower and/or upper groups of septic patients. Shorter and longer SIRS duration were associated with VCM concentrations in the lower group and the upper group, respectively, compared with predicted values in patients with sepsis. According to receiver-operating characteristic curve analysis, the optimal cutoff value of SIRS duration for the lower group was 2 days; for the upper group, it was 6 days. VCM clearance in patients with an SIRS duration <2 days was higher than that for patients with an SIRS duration ≥6 days.nnnIMPLICATIONSnSIRS duration was identified as influencing VCM concentration in patients with sepsis. This study has 2 limitations. First, we performed blood sampling only for trough concentrations. Repeated blood sampling for both peak and trough concentrations should be performed for more accurate pharmacokinetic evaluation in critically ill patients. Second, we determined CrCl by using the Cockcroft-Gault formula, which may not be accurate in critically ill patients. Modifying VCM dosing according to SIRS duration will improve prediction accuracy of VCM concentration based on therapeutic drug monitoring.

Pharmaceutical Education Focused on Pharmacotherapy in Emergency Medical Care.

Pharmacists are expected to be active members of the healthcare team in emergency medicine, because many pharmaceuticals are administered to patients with life-threatening conditions. However, adequate education for pharmacists and pharmacy students is not provided. The Emergency Pharmaceutical Sciences course was introduced for the first time in Japan by the Department of Pharmacy, Okayama University, to offer advanced education in emergency medicine and research related to critical care. We offer an emergency pharmaceutical training program with high-performance simulators and have succeeded in improving the clinical skills and confidence of pharmacy students. In this review, we introduce our activities intended to mold pharmacy students into emergency pharmacists who can contribute to emergency medicine.

Evaluation of pharmaceutical lifesaving skills training oriented pharmaceutical intervention.

BackgroundMany pharmacists are participating in team-based medical care in emergency hospitals. Therefore, there is a desperate need to improve the education system. In the present study, we provided a “pharmaceutical lifesaving skills training” to the students in their fifth and sixth year of the pharmaceutical school and evaluated the program’s impact on the students’ learning and confidence in their ability to perform pharmaceutical interventions for emergency patients.MethodsWe conducted a pharmaceutical lifesaving skills training program with 12 participants who were in their fifth and six year of pharmaceutical school. We prepared a fictional scenario in which a patient with cardiac arrest has been rushed into a hospital. We measured the participants’ level of knowledge of pharmaceutical lifesaving procedures and participants’ confidence to perform pharmaceutical interventions before and after the training session. Using the data obtained from type II quantification method, we examined what elements in the content of the pharmaceutical lifesaving skill training attended by pharmacy students will affect the students’ confidence to perform pharmaceutical interventions. In addition, using the correspondence structural analysis, we examined which sections of the content of the pharmaceutical lifesaving skill training should be improved in the future.ResultsWhen we evaluated the level of knowledge acquired in pharmaceutical lifesaving skills training, the post-training overall correct answer rate was significantly higher than the pre-training overall correct answer rate. And also, level of participants confidence to perform pharmaceutical interventions similarly increased after pharmaceutical lifesaving skill training. The influence degree graph indicates that the items likely to have a major impact on the participants’ confidence to perform pharmaceutical interventions was “Selecting medicine”. According to the correspondence structural analysis graph based on the questionnaire survey, one item identified as an improvement required was “Selecting medicine”.ConclusionsOur high-performance patient simulator-based lifesaving skills training program not only increased the participants’ understanding of the training content but also increased their confidence in their ability to perform pharmaceutical interventions. Therefore, the pharmaceutical lifesaving skills training program we developed will contribute to the education of emergency care pharmacists who can perform pharmaceutical interventions for emergency patients.

Assessment of the Relationship between Hypnotics and Delirium Using the Japanese Adverse Drug Event Report (JADER) Database

u3000The Japanese Adverse Drug Event Report (JADER) database was used to examine the risk of delirium and the time of its onset with various hypnotics, including 10 benzodiazepines (BZDs), 3 non-benzodiazepines (non-BZDs), 1 melatonin receptor agonist (MRTA), and 1 orexin receptor inhibitor (OXRI). Data entered in the JADER database between April 1, 2004 and February 1, 2016 were analyzed. The index for safety signal detection, the reporting odds ratio (ROR), was the odds ratio for adverse drug reaction reporting. The ROR for each drug was calculated; a signal was considered present if the lower bound of the 95% confidence interval of the ROR was greater than 1. The time to onset of delirium was calculated for drugs for which the number of days from the start of drug administration to delirium onset was reported. During the period examined in the analysis, a total of 621114 adverse drug reaction reports were seen, and the total number of delirium reports was 1417 after redundant cases were excluded. A signal was detected for 5 of the 10 BZDs and all 3 non-BZDs, with no signal for the MRTA and the OXRI. The time of delirium onset varied widely even for drugs classified as being in the same action duration group, and no correlation was seen for delirium onset time. The results of this study suggested that delirium risk varies depending on the hypnotic. Thus, hypnotics can be selected according to their delirium risk.

Administration of kampo medicine through a tube at an advanced critical care center

n emergency and critical care medical centers, tube administration is employed for patients who have difficulty swallowing oral drugs owing to decreased consciousness or mechanical ventilation. However, tube clogging due to drug injection is a concern. We compared the crushing method with the simple suspension method for the passage of amlodipine, an antihypertensive drug, in combination with rikkunshito, which has been used to treat upper gastrointestinal disorders such as functional dyspepsia and gastroesophageal reflux in emergency and critical care medical centers, to ascertain the effect of Kampo products on the passage of other drugs during tube administration. When the crushing method was employed, poorly water-soluble solid products were formed, while a uniformly dispersed suspension was obtained using the simple suspension method. In addition, the passage rate of amlodipine through the tube was 64% and 93% in the crushing and simple suspension methods, respectively, thereby indicating that the simple suspension method provided more favorable than the crushing method. The results of this study suggested that the passage rate of amlodipine for patients who received Kampo products concurrently was higher when the simple suspension method was used, and an appropriate drug amount might well be able to administered to patients using this method. J. Med. Invest. 65:32-36, February, 2018.

Evaluation of Pharmacotherapy on Emergency and Intensive Care Medicine: The Influence of Intensity and Duration of Invasion

u2003Critically ill patients who receive high-level invasion show physiological changes different from those under more normal conditions, along with variable therapeutic effects and pharmacokinetics. The concept of systemic inflammatory response syndrome (SIRS) has been introduced to describe the clinical state resulting from invasive actions taken under acute circumstances, resulting in an acute-phase systemic response. In particular, dosages of vancomycin (VCM) and phenytoin (PHT) need to be adjusted by therapeutic drug monitoring (TDM) because of their narrow therapeutic concentration windows. However, there are few reports on the pharmacokinetics of VCM and PHT in patients with SIRS. We performed a retrospective cohort study of patients treated with VCM and PHT. These studies suggest that the pharmacokinetics of VCM are affected by SIRS score and duration. Furthermore, the concentration of PHT was also shown to be higher in SIRS patients compared with non-SIRS patients. These findings suggest that the pharmacokinetics of VCM and PHT may be affected by the pathology of SIRS, rather than by other patient characteristics. Modifying dosing according to SIRS will improve the prediction accuracy of drug concentration based on TDM. In this review, I introduce work conducted by pharmacists in the clinical study of critically ill patients, and will be discussing the evaluation of pharmacotherapy in emergency and intensive care medicine.