We investigated the utility of three-dimensional (3D) spheroid cultures of human hepatocytes in discovering drug metabolites. Metabolites of acetaminophen, diclofenac, lamotrigine, midazolam, propranolol and salbutamol were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) to measure enzyme activities in this system cultured for 2 and 7 days. Sequential metabolic reactions by Phase I and then Phase II enzymes were found in diclofenac [CYP2C9 and UDP-glucuronyltransferases (UGTs)], midazolam (CYP3A4 and UGTs) and propranolol (CYP1A2/2D6 and UGTs). Moreover, lamotrigine and salbutamol were metabolized to lamotrigine-N-glucuronide and salbutamol 4-O-sulfate, respectively. These metabolites, which are human specific, could be observed in clinical studies, but not in conventional hepatic culture systems as in previous reports. Acetaminophen was metabolized to glucuronide and sulfate conjugates, and N-acetyl-p-benzo-quinoneimine (NAPQI) and its metabolites were not observed. In addition, mRNA of drug-metabolism enzymes [CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, UGT1A1, UGT2B7, sulfotransferase 1A1 (SULT1A1) and glutathione S-transferase pi 1 (GSTP1)], which were measured by qRT-PCR, were expressed in the human hepatocyte spheroids. In conclusion, these results suggest that human hepatocyte spheroids are useful in discovering drug metabolites.
-The objective of this study was to elucidate the range of abilities of nonclinical safety assessment for predicting adverse drug reactions (ADRs) in humans. The dataset included 1256 ADRs with an incidence rate of 5% or more collected from 142 drugs approved in Japan from 2001 to 2010 (excluding anticancer agents and vaccines). Gastrointestinal, neurological and hepatobiliary ADRs were relatively common, followed by hematological, cutaneous, systemic and cardiovascular ADRs in the dataset. The analysis revealed that 48% of ADRs were predictable based on a comprehensive nonclinical safety assessment considering animal toxicity. Hematological and ocular ADRs, infection, and application site reactions showed a correlation of more than 70%, while musculoskeletal, respiratory and neurological ADRs showed a correlation of less than 30%. In addition to subjective patient perceptions, several laboratory parameters routinely monitored both in animals and humans showed a lower correlation, e.g., abnormalities in hepatobiliary and metabolic parameters, and blood pressure increase. Large molecule drugs showed lower correlation than small molecule drugs; ADRs were observed in various organs and consideration of pharmacological action did not significantly contribute to the prediction. It was also confirmed that the current standard of toxicology testing regarding dosing duration and dose level is adequate to Correspondence: Takako Ohkura
-Drug-induced hepatotoxicity is a common reason for discontinuing the development of candidate clinical drugs. In the present study, we investigated the utility of three-dimensionally cultured human hepatocytes (spheroids) for prediction of hepatotoxicity, using a panel of model drugs: acetaminophen, benzbromarone, chlorpromazine, cyclosporin A, diclofenac, fialuridine, flutamide, imipramine, isoniazid, ticlopidine and troglitazone. Cultured spheroids showed a significant increase of albumin secretion from 2 to 7 days; the secretion started to decrease at 14 days, but continued from 14 days to 21 days. The morphology of the spheroids was well maintained for 21 days. Long-term exposure of spheroids to hepatotoxic drugs resulted in concentration-dependent depression of albumin secretion and elevation of aspartate aminotransferase (AST) leakage. The estimated 50% effective concentration (IC 50 ) values for decrease of albumin secretion changed from 7 days to 14 days, but similar values were obtained at 14 and 21 days, except for diclofenac. Since the IC 50 values and the values of drug concentration inducing 1.2-fold elevation of AST leakage (F1.2) were similar at 14 and 21 days, an incubation period of 14 days was
Sevoflurane is widely used for its rapid onset and offset due to a lower blood/gas coefficient. However, involuntary movements, tachycardia, and hypertension have been observed in some patients despite a continuing constantly delivered concentration of sevoflurane during 1-lung ventilation (OLV), indicating the possibility of insufficient depth of anesthesia. We observed a temporary but obvious decrease in arterial sevoflurane concentration and pulse oximeter readings in a patient during OLV. This may have resulted in the depth of inhaled anesthesia being insufficient during OLV because the arterial sevoflurane concentration was lower than expected in spite of constantly delivered and inspiratory/expiratory sevoflurane concentrations.
SummaryDuring one-lung ventilation, ventilation-perfusion mismatch decreases the arterial concentration of inhaled anaesthetics due to the arterial-to-venous concentration difference. This study tested the hypothesis that in humans, the 'presumed effect-site concentration' (taken as the mid-point between the arterial and superior jugular venous concentrations) of inhaled anaesthetic falls during one-lung (vs two-lung) ventilation. Four patients scheduled for elective prostatectomy (two-lung ventilation) and four patients for elective thoracotomy (one-lung ventilation) were randomly selected and assigned to receive sevoflurane (vaporiser-dial setting, 1.5%). Sevoflurane concentrations were measured periodically from radial artery and superior jugular vein (via a catheter advanced cephalad from the jugular vein). During one-lung ventilation, the end-expiratory sevoflurane concentration was stable at 1.3% but the mean (SD) presumed effect-site concentration declined initially from 58 (6.7) to 43 (4.7) lg.ml )1 (p = 0.011) before slowly recovering. A period of insufficient depth of anaesthesia is thus a risk during one-lung ventilation. Minimum alveolar concentration (MAC) -awake (MAC awake ) is the minimum alveolar concentration of inhaled anaesthetic at which 50% of anaesthetised patients respond appropriately to spoken commands [1]. It is considered an appropriate target for anaesthetic concentration when using a balanced-anaesthesia regimen that incorporates regional or local anaesthesia supplemented with opioids for analgesia and with neuromuscular blocking agents for immobilisation, provided that the concentration of inhaled anaesthetic is constant. However, in a recent report of a single case we noted that during one-lung ventilation (OLV), ventilation-perfusion mismatch caused a decrease in the arterial anaesthetic concentration as a result of the gap it created between the arterial and venous anaesthetic concentrations [2]. This alerted us to the need for a quantitative analysis of changes in the depth of inhalation anaesthesia during OLV.
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