Six‐week‐old male Fisher 344 rats (Japan SLC) were treated twice a week for eight weeks with intraperitoneal injections of 0.5 mL/kg chronic carbon tetrachloride (CCl4) (diluted 1:10, Nacalai Tesque) or corn oil only as described previously. 28 After 2 weeks of initial injections with CCl4 or corn oil, the administration of vehicle or lenvatinib was started. First, to optimize in vivo dose of lenvatinib, we administered the different doses of lenvatinib (0.4, 0.8, 1.2, 1.6, 3.2, 6.4 and 9.6 mg/kg) to CCl4‐mediated rats (n = 5) based on the previous reports to evaluate its anticancer effect in HCC xenograft model. 29 , 30 Next, the rats were divided into four groups (n = 10) according to treatment as follows: corn oil injections and vehicle administration (C/O group); CCl4 injections and vehicle administration (CCl4 group); CCl4 injections and low‐dose (0.4 mg/kg) lenvatinib (Ld group); and CCl4 injections and high‐dose (0.8 mg/kg) lenvatinib (Hd group). Because of incomplete water solubility of lenvatinib agent, we used water containing 10% carboxymethyl cellulose to suspend lenvatinib for oral administration to rats. Rats in the Ld and Hd groups received oral administration of lenvatinib by gavage daily throughout the experimental period. Water containing 10% carboxymethyl cellulose was given as vehicle. Rats were killed at the end of the eight‐week experimental period, then body and liver weights were measured and blood was collected from the aorta to measure serum levels of aspartate transaminase (AST), alanine transaminase (ALT), albumin (Alb), alkaline phosphatase (ALP) and bilirubin. Liver specimens were collected and immediately fixed in neutral‐buffered formalin. All animal procedures were performed in compliance with the recommendations of the Guide for Care and Use of Laboratory Animals of the National Research Council, and the study was approved by the ethics committee of Nara Medical University, Kashihara, Japan (No. 12585).

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