Informed consent was obtained from each patient and the study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in the a priori Internal Review Board’s approval. APAP history use was recorded and no subjects had taken APAP within a month of enrollment. Subjects were excluded if they had abnormal liver tests on screening or a history of chronic liver disease. Nine subjects AZD5363 cost were enrolled for 7 days each as inpatients in the General Clinical Research Center at the UNC Hospital. Human overdose subject descriptions have been reported.5 Subjects were placed on a defined liquid diet to assure uniform nutritional intake. The protein source was soy, the fat source was

safflower oil of known composition, and the carbohydrate source was cane or beet sugar. Other ingredients included Metamucil to provide fiber and vanilla. The overall macronutrient composition was 15% of total calories from protein, 30% from fat, and 55% from carbohydrate. Subject’s daily calorie intake, divided into five consistently timed meals per day, was based on the formula 35 kcal/kg actual body weight. On day 4 the subjects were fasting until 2 hours after receiving APAP. Weight was monitored daily and calories adjusted to maintain body weight. On the morning of the fourth day, six subjects received a single dose of 4 g of APAP administered as eight, 500-mg capsules, whereas three

received placebo pills. Blood was collected at 6 AM on each of the clinical days for ALT measurement. PB, 7.5 mL, was drawn into PAXgene (PreAnalytiX/Qiagen, Hilden, Germany) blood RNA Venetoclax ic50 collection tubes (3 tubes at 2.5 mL) immediately before the first dose and at 6, 18, 24, 48, 72, and 96 hours postdosing. Samples were mixed and allowed to remain at room temperature for 2 hours, then frozen at −20°C until RNA isolation. Blood was also collected at 6 AM on each of the clinical days for measurement of clinical chemistries SPTLC1 and complete blood counts (CBCs), performed by the UNC Hospital clinical laboratories. Serum was collected and frozen at −80°C predose

and at the following times postdose: 30 minutes, 60 minutes, 90 minutes, 2, 3, 4, 5, 6, 8, and 12 hours. Upon study completion, APAP and metabolites were assayed in the serum by high-performance liquid chromatography (HPLC).6 In order to measure APAP metabolite excretion, urine was also collected for 24 hours postdosing and stored at −20°C with ascorbic acid (1 g/L). RNA was isolated utilizing the PAXgene blood RNA isolation kit (PreAnalytiX/Qiagen) according to the manufacturer’s protocol, including the optional on-column DNase digestion. RNA quality was assessed with an Agilent Bioanalyzer (Palo Alto, CA) and only samples with intact 18S and 28S ribosomal RNA peaks were used for microarray analysis. Gene expression profiling was conducted using Agilent Human 1A(V2) oligo arrays with ≈20,000 genes represented.