Kyorin University

BACKGROUND: The course of autosomal dominant polycystic kidney disease (ADPKD) is often associated with pain, hypertension, and kidney failure. Preclinical studies indicated that vasopressin V(2)-receptor antagonists inhibit cyst growth and slow the decline of kidney function. METHODS: In this phase 3, multicenter, double-blind, placebo-controlled, 3-year trial, we randomly assigned 1445 patients, 18 to 50 years of age, who had ADPKD with a total kidney volume of 750 ml or more and an estimated creatinine clearance of 60 ml per minute or more, in a 2:1 ratio to receive tolvaptan, a V(2)-receptor antagonist, at the highest of three twice-daily dose regimens that the patient found tolerable, or placebo. The primary outcome was the annual rate of change in the total kidney volume. Sequential secondary end points included a composite of time to clinical progression (defined as worsening kidney function, kidney pain, hypertension, and albuminuria) and rate of kidney-function decline. RESULTS: Over a 3-year period, the increase in total kidney volume in the tolvaptan group was 2.8% per year (95% confidence interval [CI], 2.5 to 3.1), versus 5.5% per year in the placebo group (95% CI, 5.1 to 6.0; P<0.001). The composite end point favored tolvaptan over placebo (44 vs. 50 events per 100 follow-up-years, P=0.01), with lower rates of worsening kidney function (2 vs. 5 events per 100 person-years of follow-up, P<0.001) and kidney pain (5 vs. 7 events per 100 person-years of follow-up, P=0.007). Tolvaptan was associated with a slower decline in kidney function (reciprocal of the serum creatinine level, -2.61 [mg per milliliter](-1) per year vs. -3.81 [mg per milliliter](-1) per year; P<0.001). There were fewer ADPKD-related adverse events in the tolvaptan group but more events related to aquaresis (excretion of electrolyte-free water) and hepatic adverse events unrelated to ADPKD, contributing to a higher discontinuation rate (23%, vs. 14% in the placebo group). CONCLUSIONS: Tolvaptan, as compared with placebo, slowed the increase in total kidney volume and the decline in kidney function over a 3-year period in patients with ADPKD but was associated with a higher discontinuation rate, owing to adverse events. (Funded by Otsuka Pharmaceuticals and Otsuka Pharmaceutical Development and Commercialization; TEMPO 3:4 ClinicalTrials.gov number, NCT00428948.).

BACKGROUND: A single, high priming dose of tremelimumab (anti-cytotoxic T lymphocyte–associated antigen 4) plus durvalumab (anti–programmed cell death ligand-1), an infusion regimen termed STRIDE (Single Tremelimumab Regular Interval Durvalumab), showed encouraging clinical activity and safety in a phase 2 trial of unresectable hepatocellular carcinoma. METHODS: In this global, open-label, phase 3 trial, the majority of the patients we enrolled with unresectable hepatocellular carcinoma and no previous systemic treatment were randomly assigned to receive one of three regimens: tremelimumab (300 mg, one dose) plus durvalumab (1500 mg every 4 weeks; STRIDE), durvalumab (1500 mg every 4 weeks), or sorafenib (400 mg twice daily). The primary objective was overall survival for STRIDE versus sorafenib. Noninferiority for overall survival for durvalumab versus sorafenib was a secondary objective. RESULTS: In total, 1171 patients were randomly assigned to STRIDE (n=393), durvalumab (n=389), or sorafenib (n=389). The median overall survival was 16.43 months (95% confidence interval [CI], 14.16 to 19.58) with STRIDE, 16.56 months (95% CI, 14.06 to 19.12) with durvalumab, and 13.77 months (95% CI, 12.25 to 16.13) with sorafenib. Overall survival at 36 months was 30.7%, 24.7%, and 20.2%, respectively. The overall survival hazard ratio for STRIDE versus sorafenib was 0.78 (96.02% CI, 0.65 to 0.93; P=0.0035). Overall survival with durvalumab monotherapy was noninferior to sorafenib (hazard ratio, 0.86; 95.67% CI, 0.73 to 1.03; noninferiority margin, 1.08). Median progression-free survival was not significantly different among all three groups. Grade 3/4 treatment-emergent adverse events occurred for 50.5% of patients with STRIDE, 37.1% with durvalumab, and 52.4% with sorafenib. CONCLUSIONS: STRIDE significantly improved overall survival versus sorafenib. Durvalumab monotherapy was noninferior to sorafenib for patients with unresectable hepatocellular carcinoma. (Funded by AstraZeneca; ClinicalTrials.gov number, NCT03298451.)

BACKGROUND The efficacy of ustekinumab, an antagonist of the p40 subunit of interleukin-12 and interleukin-23, as induction and maintenance therapy in patients with ulcerative colitis is unknown. METHODS We evaluated ustekinumab as 8-week induction therapy and 44-week maintenance therapy in patients with moderate-to-severe ulcerative colitis. A total of 961 patients were randomly assigned to receive an intravenous induction dose of ustekinumab (either 130 mg [320 patients] or a weight-range–based dose that approximated 6 mg per kilogram of body weight [322]) or placebo (319). Patients who had a response to induction therapy 8 weeks after administration of intravenous ustekinumab were randomly assigned again to receive subcutaneous maintenance injections of 90 mg of ustekinumab (either every 12 weeks [172 patients] or every 8 weeks [176]) or placebo (175). The primary end point in the induction trial (week 8) and the maintenance trial (week 44) was clinical remission (defined as a total score of ≤2 on the Mayo scale [range, 0 to 12, with higher scores indicating more severe disease] and no subscore &gt;1 [range, 0 to 3] on any of the four Mayo scale components). RESULTS The percentage of patients who had clinical remission at week 8 among patients who received intravenous ustekinumab at a dose of 130 mg (15.6%) or 6 mg per kilogram (15.5%) was significantly higher than that among patients who received placebo (5.3%) (P&lt;0.001 for both comparisons). Among patients who had a response to induction therapy with ustekinumab and underwent a second randomization, the percentage of patients who had clinical remission at week 44 was significantly higher among patients assigned to 90 mg of subcutaneous ustekinumab every 12 weeks (38.4%) or every 8 weeks (43.8%) than among those assigned to placebo (24.0%) (P=0.002 and P&lt;0.001, respectively). The incidence of serious adverse events with ustekinumab was similar to that with placebo. Through 52 weeks of exposure, there were two deaths (one each from acute respiratory distress syndrome and hemorrhage from esophageal varices) and seven cases of cancer (one each of prostate, colon, renal papillary, and rectal cancer and three nonmelanoma skin cancers) among 825 patients who received ustekinumab and no deaths and one case of cancer (testicular cancer) among 319 patients who received placebo. CONCLUSIONS Ustekinumab was more effective than placebo for inducing and maintaining remission in patients with moderate-to-severe ulcerative colitis.

A cDNA was isolated from rat C6 glioma cells by expression cloning which encodes a novel Na+-independent neutral amino acid transporter designated LAT1. For functional expression in Xenopus oocytes, LAT1 required the heavy chain of 4F2 cell surface antigen (CD98), a type II membrane glycoprotein. When co-expressed with 4F2 heavy chain, LAT1 transported neutral amino acids with branched or aromatic side chains and did not accept basic amino acids or acidic amino acids. The transport via LAT1 was Na+-independent and sensitive to a system L-specific inhibitor 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid. These functional properties correspond to those of the classically characterized amino acid transport system L, a major nutrient transporter. In in vitro translation, LAT1 was shown to be a nonglycosylated membrane protein consistent with the property of 4F2 light chain, suggesting LAT1 is at least one of the proteins formerly referred to as 4F2 light chain. LAT1 exhibits relatively low but significant amino acid sequence similarity to mammalian cationic amino acid transporters and amino acid permeases of bacteria and yeasts, indicating LAT1 is a new member of the APC superfamily. Because of highly regulated nature and high level of expression in tumor cell lines, LAT1 is thought to be up-regulated to support the high protein synthesis for cell growth and cell activation. The cloning of LAT1 is expected to facilitate the research on the protein-protein interaction in the transporter field and to provide a clue to the search for still unidentified transporters.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide, and the burden of this devastating cancer is expected to increase further in coming years. The collection and analysis of epidemiologic HCC data will play a critical role in guiding future disease prevention strategies and optimizing patient management. Previous epidemiologic studies have highlighted striking global variations in the incidence of HCC, which is particularly high in much of east Asia and sub-Saharan Africa, and lower, but on the increase, in North America and most of Europe. This variation appears to be related to the complex etiology of HCC, with different risk factors, primarily infection with hepatitis B or hepatitis C virus, responsible for driving HCC incidence rates in different regions. Although previous studies have contributed considerably to the knowledge of HCC epidemiology, there are limitations associated with the currently available data, which arise from studies performed at different times in the past, using varying methodologies, and with diverse patient populations. A new and global approach to the study of HCC epidemiology is required if HCC disease prevention and treatment strategies are to be adequately directed and supported in coming years.

Background: Single-agent nivolumab showed durable responses, manageable safety, and promising survival in patients with advanced hepatocellular carcinoma in the phase 1-2 CheckMate 040 study. We aimed to investigate nivolumab monotherapy compared with sorafenib monotherapy in the first-line setting for patients with advanced hepatocellular carcinoma.&#13;\n&#13;\nMethods: In this randomised, open-label, phase 3 trial done at medical centres across 22 countries and territories in Asia, Australasia, Europe, and North America, patients at least 18 years old with histologically confirmed advanced hepatocellular carcinoma not eligible for, or whose disease had progressed after, surgery or locoregional treatment; with no previous systemic therapy for hepatocellular carcinoma, with Child-Pugh class A and Eastern Cooperative Oncology Group performance status score of 0 or 1, and regardless of viral hepatitis status were randomly assigned (1:1) via an interactive voice response system to receive nivolumab (240 mg intravenously every 2 weeks) or sorafenib (400 mg orally twice daily) until disease progression or unacceptable toxicity. The primary endpoint was overall survival assessed in the intention-to-treat population. Safety was assessed in all patients who received at least one dose of study drug. This completed trial is registered with ClinicalTrials.gov, NCT02576509.&#13;\n&#13;\nFindings: Between Jan 11, 2016, and May 24, 2017, 743 patients were randomly assigned to treatment (nivolumab, n=371; sorafenib, n=372). At the primary analysis, the median follow-up for overall survival was 15·2 months (IQR 5·7-28·0) for the nivolumab group and 13·4 months (5·7-25·9) in the sorafenib group. Median overall survival was 16·4 months (95% CI 13·9-18·4) with nivolumab and 14·7 months (11·9-17·2) with sorafenib (hazard ratio 0·85 [95% CI 0·72-1·02]; p=0·075; minimum follow-up 22·8 months); the protocol-defined significance level of p=0·0419 was not reached. The most common grade 3 or worse treatment-related adverse events were palmar-plantar erythrodysaesthesia (1 [&lt;1%] of 367 patients in the nivolumab group vs 52 [14%] of patients in the sorafenib group), aspartate aminotransferase increase (22 [6%] vs 13 [4%]), and hypertension (0 vs 26 [7%]). Serious treatment-related adverse events were reported in 43 (12%) patients receiving nivolumab and 39 (11%) patients receiving sorafenib. Four deaths in the nivolumab group and one death in the sorafenib group were assessed as treatment related.&#13;\n&#13;\nInterpretation: First-line nivolumab treatment did not significantly improve overall survival compared with sorafenib, but clinical activity and a favourable safety profile were observed in patients with advanced hepatocellular carcinoma. Thus, nivolumab might be considered a therapeutic option for patients in whom tyrosine kinase inhibitors and antiangiogenic drugs are contraindicated or have substantial risks.

When drug reactions resembling allergy occur, they are called drug hypersensitivity reactions (DHRs) before showing the evidence of either drug-specific antibodies or T cells. DHRs may be allergic or nonallergic in nature, with drug allergies being immunologically mediated DHRs. These reactions are typically unpredictable. They can be life-threatening, may require or prolong hospitalization, and may necessitate changes in subsequent therapy. Both underdiagnosis (due to under-reporting) and overdiagnosis (due to an overuse of the term ‘allergy’) are common. A definitive diagnosis of such reactions is required in order to institute adequate treatment options and proper preventive measures. Misclassification based solely on the DHR history without further testing may affect treatment options, result in adverse consequences, and lead to the use of more-expensive or less-effective drugs, in contrast to patients who had undergone a complete drug allergy workup. Several guidelines and/or consensus documents on general or specific drug class-induced DHRs are available to support the medical decision process. The use of standardized systematic approaches for the diagnosis and management of DHRs carries the potential to improve outcomes and should thus be disseminated and implemented. Consequently, the International Collaboration in Asthma, Allergy and Immunology (iCAALL), formed by the European Academy of Allergy and Clinical Immunology (EAACI), the American Academy of Allergy, Asthma and Immunology (AAAAI), the American College of Allergy, Asthma and Immunology (ACAAI), and the World Allergy Organization (WAO), has decided to issue an International CONsensus (ICON) on drug allergy. The purpose of this document is to highlight the key messages that are common to many of the existing guidelines, while critically reviewing and commenting on any differences and deficiencies of evidence, thus providing a comprehensive reference document for the diagnosis and management of DHRs.

The adipocyte-derived hormone adiponectin has been shown to play important roles in the regulation of energy homeostasis and insulin sensitivity. In this study, we analyzed globular domain adiponectin (gAd) transgenic (Tg) mice crossed with leptin-deficient ob/ob or apoE-deficient mice. Interestingly, despite an unexpected similar body weight, gAd Tg ob/ob mice showed amelioration of insulin resistance and beta-cell degranulation as well as diabetes, indicating that globular adiponectin and leptin appeared to have both distinct and overlapping functions. Amelioration of diabetes and insulin resistance was associated with increased expression of molecules involved in fatty acid oxidation such as acyl-CoA oxidase, and molecules involved in energy dissipation such as uncoupling proteins 2 and 3 and increased fatty acid oxidation in skeletal muscle of gAd Tg ob/ob mice. Moreover, despite similar plasma glucose and lipid levels on an apoE-deficient background, gAd Tg apoE-deficient mice showed amelioration of atherosclerosis, which was associated with decreased expression of class A scavenger receptor and tumor necrosis factor alpha. This is the first demonstration that globular adiponectin can protect against atherosclerosis in vivo. In conclusion, replenishment of globular adiponectin may provide a novel treatment modality for both type 2 diabetes and atherosclerosis.

BACKGROUND: Lipoinjection is a promising treatment but has some problems, such as unpredictability and a low rate of graft survival due to partial necrosis. METHODS: To overcome the problems with lipoinjection, the authors developed a novel strategy known as cell-assisted lipotransfer (CAL). In CAL, autologous adipose-derived stem (stromal) cells (ASCs) are used in combination with lipoinjection. A stromal vascular fraction (SVF) containing ASCs is freshly isolated from half of the aspirated fat and recombined with the other half. This process converts relatively ASC-poor aspirated fat to ASC-rich fat. This report presents the findings for 40 patients who underwent CAL for cosmetic breast augmentation. RESULTS: Final breast volume showed augmentation by 100 to 200 ml after a mean fat amount of 270 ml was injected. Postoperative atrophy of injected fat was minimal and did not change substantially after 2 months. Cyst formation or microcalcification was detected in four patients. Almost all the patients were satisfied with the soft and natural-appearing augmentation. CONCLUSIONS: The preliminary results suggest that CAL is effective and safe for soft tissue augmentation and superior to conventional lipoinjection. Additional study is necessary to evaluate the efficacy of this technique further.

BACKGROUND: A British randomised study of gemcitabine plus cisplatin (GC) combination showed promising results in biliary tract cancer (BTC) patients. In our study, we evaluated the efficacy and safety of this combination compared with gemcitabine alone (G) in Japanese BTC patients. METHODS: Overall, 84 advanced BTC patients were randomised to either cisplatin 25 mg m(-2) plus gemcitabine 1000 mg m(-2) on days 1, 8 of a 21-day cycle (GC-arm), or single-agent gemcitabine 1000 mg m(-2) on days 1, 8 and 15 of a 28-day cycle (G-arm). Treatments were repeated for at least 12 weeks until disease progression or unacceptable toxicity occurred, up to a maximum of 48 weeks. RESULTS: A total of 83 patients were included in the analysis. For the GC and G-arms, respectively, the 1-year survival rate was 39.0 vs 31.0%, median survival time 11.2 vs 7.7 months, median progression-free survival time 5.8 vs 3.7 months and overall response rate 19.5 vs 11.9%. The most common grade 3 or 4 toxicities (GC-arm/G-arm) were neutropenia (56.1%/38.1%), thrombocytopenia (39.0%/7.1%), leukopenia (29.3%/19.0%), haemoglobin decrease (36.6%/16.7%) and gamma-GTP increase (29.3%/35.7%). CONCLUSIONS: Gemcitabine plus cisplatin combination therapy was found to be effective and well tolerated, suggesting that it could also be a standard regimen for Japanese patients.

OBJECTIVE: This trial compared the efficacy and safety of transarterial chemoembolisation (TACE) plus sorafenib with TACE alone using a newly established TACE-specific endpoint and pre-treatment of sorafenib before initial TACE. DESIGN: Patients with unresectable hepatocellular carcinoma (HCC) were randomised to TACE plus sorafenib (n=80) or TACE alone (n=76). Patients in the combination group received sorafenib 400 mg once daily for 2-3 weeks before TACE, followed by 800 mg once daily during on-demand conventional TACE sessions until time to untreatable (unTACEable) progression (TTUP), defined as untreatable tumour progression, transient deterioration to Child-Pugh C or appearance of vascular invasion/extrahepatic spread. Co-primary endpoints were progression-free survival (PFS), which is not a conventional one but defined as TTUP, or time to any cause of death plus overall survival (OS). Multiplicity was adjusted by gatekeeping hierarchical testing. RESULTS: Median PFS was significantly longer in the TACE plus sorafenib than in the TACE alone group (25.2 vs 13.5 months; p=0.006). OS was not analysed because only 73.6% of OS events were reached. Median TTUP (26.7 vs 20.6 months; p=0.02) was also significantly longer in the TACE plus sorafenib group. OS at 1 year and 2 years in TACE plus sorafenib group and TACE alone group were 96.2% and 82.7% and 77.2% and 64.6%, respectively. There were no unexpected toxicities. CONCLUSION: TACE plus sorafenib significantly improved PFS over TACE alone in patients with unresectable HCC. Adverse events were consistent with those of previous TACE combination trials. TRIAL REGISTRATION NUMBER: NCT01217034.

IL-33, a member of the IL-1-related cytokines, is considered to be a proallergic cytokine that is especially involved in Th2-type immune responses. Moreover, like IL-1α, IL-33 has been suggested to act as an "alarmin" that amplifies immune responses during tissue injury. In contrast to IL-1, however, the precise roles of IL-33 in those settings are poorly understood. Using IL-1- and IL-33-deficient mice, we found that IL-1, but not IL-33, played a substantial role in induction of T cell-mediated type IV hypersensitivity such as contact and delayed-type hypersensitivity and autoimmune diseases such as experimental autoimmune encephalomyelitis. Most notably, however, IL-33 was important for innate-type mucosal immunity in the lungs and gut. That is, IL-33 was essential for manifestation of T cell-independent protease allergen-induced airway inflammation as well as OVA-induced allergic topical airway inflammation, without affecting acquisition of antigen-specific memory T cells. IL-33 was significantly involved in the development of dextran-induced colitis accompanied by T cell-independent epithelial cell damage, but not in streptozocin-induced diabetes or Con A-induced hepatitis characterized by T cell-mediated apoptotic tissue destruction. In addition, IL-33-deficient mice showed a substantially diminished LPS-induced systemic inflammatory response. These observations indicate that IL-33 is a crucial amplifier of mucosal and systemic innate, rather than acquired, immune responses.

Numerous drugs and endogenous compounds are efficiently excreted from the renal proximal tubule via carrier-mediated pathways. Transepithelial excretion of organic anions occurs via their accumulative transport from the blood into the proximal tubule cells across the basolateral membrane and subsequent secretion into the urine through the apical membrane. Here we report on the isolation of a novel complementary DNA from rat kidney that encodes a 551-amino acid residue protein (OAT1) with 12 putative membrane-spanning domains. When expressed in Xenopus laevisoocytes, OAT1 mediated sodium-independent para-aminohippurate (PAH) uptake (K m = 14.3 ± 2.9 μm). The uptake rate of PAH was increased by the outwardly directed dicarboxylate gradient, consisting with the idea that OAT1 is an organic anion/dicarboxylate exchanger. OAT1 displayed remarkably wide substrate selectivity, covering endogenous substrates such as cyclic nucleotides, a prostaglandin and uric acid, and a variety of drugs with different structures (e.g. antibiotics, a nonsteroidal anti-inflammatory drug, diuretics, an antineoplastic drug, and a uricosuric drug). The Northern blot analysis and in situhybridization revealed that OAT1 is exclusively expressed in the particular segment of the proximal tubule in the kidney. These data suggest that OAT1 is a multispecific organic anion transporter at the basolateral membrane of the proximal tubule. Isolation of OAT1 will facilitate elucidation of the molecular basis of drug kinetics and the development of new drugs lacking unwanted side effects. Numerous drugs and endogenous compounds are efficiently excreted from the renal proximal tubule via carrier-mediated pathways. Transepithelial excretion of organic anions occurs via their accumulative transport from the blood into the proximal tubule cells across the basolateral membrane and subsequent secretion into the urine through the apical membrane. Here we report on the isolation of a novel complementary DNA from rat kidney that encodes a 551-amino acid residue protein (OAT1) with 12 putative membrane-spanning domains. When expressed in Xenopus laevisoocytes, OAT1 mediated sodium-independent para-aminohippurate (PAH) uptake (K m = 14.3 ± 2.9 μm). The uptake rate of PAH was increased by the outwardly directed dicarboxylate gradient, consisting with the idea that OAT1 is an organic anion/dicarboxylate exchanger. OAT1 displayed remarkably wide substrate selectivity, covering endogenous substrates such as cyclic nucleotides, a prostaglandin and uric acid, and a variety of drugs with different structures (e.g. antibiotics, a nonsteroidal anti-inflammatory drug, diuretics, an antineoplastic drug, and a uricosuric drug). The Northern blot analysis and in situhybridization revealed that OAT1 is exclusively expressed in the particular segment of the proximal tubule in the kidney. These data suggest that OAT1 is a multispecific organic anion transporter at the basolateral membrane of the proximal tubule. Isolation of OAT1 will facilitate elucidation of the molecular basis of drug kinetics and the development of new drugs lacking unwanted side effects. The kidney plays an essential role in the elimination of numerous organic anions including endogenous compounds, xenobiotics, and their metabolites (1Pritchard J.B. Miller D.S. Physiol. Rev. 1993; 73: 765-796Crossref PubMed Scopus (464) Google Scholar, 2Ullrich K.J. Rumrich G. Am. J. Physiol. 1988; 254: F453-F462PubMed Google Scholar, 3Moller J.V. Sheikh M.I. Pharmacol. Rev. 1983; 34: 315-358Google Scholar). The proximal tubule cells actively secrete them into the urine. The first step of this secretion is the extraction of organic anion from the peritubular blood plasma by the proximal tubule cells through the basolateral membrane. This basolateral uptake of organic anions has been extensively investigated using para-aminohippuric acid (PAH) 1The abbreviations used are: PAH, para-aminohippuric acid; MES, 4-morpholineethanesulfonic acid; kb, kilobase(s); oatp, organic anion transporting polypeptide. as a test substrate. The most striking feature of this organic anion transport system is its extremely wide substrate selectivity, covering not only endogenous anionic substrates but also a number of clinically important drugs (1Pritchard J.B. Miller D.S. Physiol. Rev. 1993; 73: 765-796Crossref PubMed Scopus (464) Google Scholar, 3Moller J.V. Sheikh M.I. Pharmacol. Rev. 1983; 34: 315-358Google Scholar). Because of its importance in renal physiology and pharmacology, cloning of the organic anion transporter has been attempted by many investigators using different approaches; however, the molecular structure of the responsible transporter has not yet determined. For the last decade it has been proposed that the basolateral uptake of organic anion is mediated by organic anion/dicarboxylate exchanger (1Pritchard J.B. Miller D.S. Physiol. Rev. 1993; 73: 765-796Crossref PubMed Scopus (464) Google Scholar,4Shimada H. Moewes B. Burckhardt G. Am. J. Physiol. 1987; 253: F795-F801PubMed Google Scholar). According to this model, outwardly directed dicarboxylate gradient is essential to express the transport activity of this exchanger. In the present study, we isolated first rat sodium dicarboxylate transporter (rNaDC-1) and then co-expressed it together with rat kidney poly(A)+ RNA in Xenopus oocytes to energize organic anion transport in oocytes. We describe here functional expression cloning of an organic anion/dicarboxylate exchanger (OAT1) and its characteristics as a multispecific organic anion transporter. A nondirectional cDNA library was prepared from rat kidney poly(A)+ RNA using Superscript Choice system (Life Technologies, Inc.) and was ligated to λZipLox EcoRI arms (Life Technologies, Inc.). A polymerase chain reaction product corresponding to nucleotides 1323–1763 of the rabbit sodium dicarboxylate transporter (NaDC-1) (5Pajor A.M. J. Biol. Chem. 1995; 270: 5779-5785Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) was labeled with [32P]dCTP. A rat cDNA library was screened with this probe at low stringency. Hybridization was done overnight in the hybridization solution at 37 °C, and filters were washed finally at 37 °C in 0.1 × SSC, 0.1% SDS. The hybridization solution contains 5 × SSC, 3 × Denhardt's solution, 0.2% SDS, 10% dextran sulfate, 50% formamide, 0.01% Antifoam B, 0.2 mg/ml denatured salmon sperm DNA, 2.5 mm sodium pyrophosphate, and 25 mm MES, pH 6.5. cDNA inserts in positive λZipLox phage were recovered in plasmid pZL1 by in vitro excision and further subcloned into pBluescript II SK− (Stratagene) for sequencing and in vitro transcription. Xenopus laevis oocyte expression studies and uptake measurements were performed as described elsewhere (6Kanai Y. Hediger M.A. Nature. 1992; 360: 467-471Crossref PubMed Scopus (1202) Google Scholar). Defolliculated oocytes were injected with in vitro transcribed cRNA and/or rat kidney poly(A)+ RNA as indicated in each experiment. In vitro transcription was done by using T7 RNA polymerase in the presence of cap analog. After incubation of oocytes at 18 °C for 2–3 days, uptake studies were performed in sodium uptake solution (96 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, 5 mm HEPES, pH 7.4) containing radiolabeled substrates as indicated in each experiment. Four-hundred μg of rat kidney poly(A)+ RNA was size-fractionated as described elsewhere (6Kanai Y. Hediger M.A. Nature. 1992; 360: 467-471Crossref PubMed Scopus (1202) Google Scholar) using preparative gel electrophoresis (Bio-Rad, model 491 Prep cell). Then we co-injected poly(A)+ RNAs of each fraction together with rNaDC-1 cRNA into oocytes. Before the uptake study, the oocytes were routinely preincubated for 2 h in sodium uptake solution containing 1 mm glutarate. [14C]PAH (50 μm) uptake was measured in sodium uptake solution without glutarate for 1 h. A directional cDNA library was constructed from fractions showing the highest rate of [14C]PAH uptake (1.8–2.4-kilobase (kb) poly(A)+ RNA) using Superscript Plasmid system (Life Technologies, Inc.) and was ligated into theSalI and NotI site of pSPORT 1. Recombinants were electroporated into Electro Max DH10B competent cells (Life Technologies, Inc.). Approximately 500 colonies were grown on nitrocellulose membrane. Plasmid DNA was purified from colonies of each plate. Capped cRNA was synthesized in vitro after linearization of each plasmid DNA with NotI. Then we co-injected cRNA synthesized from each filter together with 2 ng of rNaDC-1 cRNA into oocytes. When [14C]PAH uptake was detected on a particular group, it was subdivided into several groups and further screened. Eight-thousand clones were screened until a single clone was isolated. Deleted clones obtained by Kilo-Sequence deletion kit (Takara, Japan), or specially synthesized oligonucleotide primers were used for sequencing of rNaDC1 and OAT1 cDNA. rNaDC1 and OAT1 were sequenced by dideoxytermination method using Sequenase version 2.0 (Amersham Corp.) or dye primer cycle sequencing kit (Applied Biosystems). The functional characterization of OAT1 was analyzed without co-expression of rNaDC-1 except for the experiment of which the results are shown in Fig.1 c. In experiments of Fig. 1, d and e, and Fig. 4, a and b, oocytes expressed with OAT1 were preincubated with 1 mm glutarate for 2 h before uptake studies were performed. For the determination of the sodium dependence of OAT1 transport activity, [14C]PAH uptake was measured either in sodium uptake solution or in choline chloride uptake solution (96 mm NaCl of sodium solution was replaced by 96 mm choline chloride, and pH was adjusted to 7.4 with Tris). For kinetic analysis, [14C]PAH influx was measured for 3 min. For inhibition study, 2 μm[14C]PAH uptake via OAT1 was measured in the absence or presence of 2 mm nonradiolabeled compounds in sodium uptake solution.Figure 4Substrate selectivity of OAT1. a, inhibition of OAT1-mediated [14C]PAH uptake by various drugs and endogenous substrates. [14C]PAH (2 μm) uptake in the presence of nonradiolabeled test substrates (2 mm) is expressed as percent of the control [14C]PAH uptake in the absence of other substrates (mean ± S.E.; n = 5–8 oocytes). b, OAT1-mediated uptake of radiolabeled drugs and endogenous substrates. The amounts of radiolabeled substrates (2 μm[3H]methotrexate, 1 μm[3H]cAMP, 1 μm [3H]cGMP, 60 nm [3H]prostaglandin E2, 100 μm [14C]urate, 5 μm[14C]α-ketoglutarate) taken up by control (open column) or OAT1-expressed (closed column) oocytes were measured for 1 h (mean ± S.E.; n = 5–8).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Three μg of poly(A)+RNA prepared from various rat tissues were electrophoresed on a 1% agarose/formaldehyde gel and transferred to a nitrocellulose filter. The filter was hybridized at 42 °C overnight in the hybridization solution with full-length OAT1 cDNA labeled with [32P]dCTP. The filter was washed finally in 0.1 × SSC, 0.1% SDS at 65 °C. In situ hybridization was performed as described elsewhere (7Kanai Y. Stelzner M.G. Lee W.S. Wells R.G. Brown D. Hediger M.A. Am. J. Physiol. 1992; 263: F1087-F1092PubMed Google Scholar) with some modifications. Briefly, after perfusion fixation with 4% paraformaldehyde, rat kidney was excised and postfixed in 4% paraformaldehyde. Five-μm cryostat sections of rat kidney were used in situ hybridization.35S-Labeled sense and antisense cRNA were synthesized from the full-length OAT1 cDNA (in pBlueScript SK−) using T7 or T3 RNA polymerase after linearization of plasmid DNA withSpeI or XhoI, respectively. RNA probe was degraded by partial hydrolysis for 45 min. The cryosections were hybridized with the probe overnight in the hybridization solution and washed to a final stringency of 0.1 × SSC at 37 °C for 30 min. Using an approach based on homology to rabbit sodium dicarboxylate transporter (NaDC-1) (5Pajor A.M. J. Biol. Chem. 1995; 270: 5779-5785Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), we isolated rNaDC-1 (rat sodium-dependent dicarboxylate transporter-1: accession number for the nucleotide sequence of cDNA: AB001321; DDBJ, EBI, and GenBank™ nucleotide sequence data bases). When expressed inX. laevis oocytes, rNaDC-1 mediates sodium-dependent [14C]glutarate uptake (Fig.1 a). Then we co-injected X. laevisoocytes with rat kidney poly(A)+ RNA and cRNA of rNaDC-1 and preincubated them with 1 mm glutarate to induce generation of an outwardly directed dicarboxylate gradient. These “co-expressed” oocytes exhibited [14C]PAH uptake, whereas oocytes injected with only rat kidney poly(A)+ RNA exhibited no detectable [14C]PAH uptake (Fig.1 b). Using a functional expression method together with this co-expression system, we isolated a 2294-base pair cDNA encoding an organic anion/dicarboxylate exchanger (OAT1: organic anion transporter 1). Fig. 1 c shows the dependence of OAT1-mediated [14C]PAH uptake on the intracellular dicarboxylate (glutarate) concentration. The rate of [14C]PAH uptake by oocytes via OAT1 is increased by preincubation of the oocytes with 1 mm glutarate. When oocytes co-expressing rNaDC-1 and OAT1 are preincubated with glutarate, they showed a further increase in the rate of [14C]PAH uptake. Thistrans-stimulative effect of glutarate indicates that OAT1 is an organic anion/dicarboxylate exchanger. As shown in Fig.1 d, replacement of extracellular sodium with choline had no effect on the rate of [14C]PAH uptake. Because [14C]PAH (1 mm) uptake via OAT1 was linearly increased until 5 min under this condition (data not shown), we performed the kinetic study for 3 min. [14C]PAH uptake via OAT1 follows Michaelis-Menten kinetics (Fig. 1 e), and the estimated K m value (14.3 ± 2.9 μm: mean ± S.E., n = 3) is similar to that previously reported for the basolateral organic anion transport system (80 μm) (2Ullrich K.J. Rumrich G. Am. J. Physiol. 1988; 254: F453-F462PubMed Google Scholar). OAT1 cDNA consists of 2294 nucleotides, and contains an open reading frame encoding a 551-amino acid residue protein (Fig.2 a). Kyte-Doolittle hydropathy analysis (8Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17296) Google Scholar) of OAT1 predicts 12 putative membrane-spanning domains (Fig.2 b). Four N-glycosylation sites are predicted in the first hydrophilic loop. There are four putative protein kinase C-dependent phosphorylation sites in the hydrophilic loop between sixth and seventh transmembrane domains. Under high stringency Northern blot analysis of poly(A)+RNA from rat various tissues, a strong 2.4-kb mRNA band and two bands corresponding to longer transcripts (3.9 and 4.2 kb) were detected predominantly in the kidney (Fig.3 a). Upon longer exposure, a faint 2.4-kb mRNA band was detected in the brain. No hybridization signals were obtained with mRNA isolated from other tissues. In the kidney, expression of OAT1 mRNA is strong in the cortex and outer medulla (cortex > outer medulla) and very weak in the inner medulla. In situ hybridization of rat kidney coronal sections (Fig.3 b) revealed that OAT1 mRNA is expressed in renal cortex and outer medulla, especially in the medullary rays of the cortex. Expression of OAT-1 was not found in the inner medulla. No significant hybridization signal was detected in control experiments using sense OAT1 cRNA as a probe (data not shown). This overall pattern of in situ hybridization suggests that OAT1 is most strongly expressed in the middle portion of the proximal tubule (S2). This intrarenal localization is in good agreement with the previous studies, which demonstrate that the highest PAH transport activity were seen in S2 of rat proximal tubule (1Pritchard J.B. Miller D.S. Physiol. Rev. 1993; 73: 765-796Crossref PubMed Scopus (464) Google Scholar). To evaluate the substrate selectivity of OAT1, we examined the levels of the inhibition of OAT1-mediated [14C]PAH uptake by various compounds (Fig. 4 a).cis-Inhibitory effects were observed for structurally unrelated drugs. Cephaloridine (β-lactam antibiotic), nalidixic acid (“old” quinolone), furosemide and ethacrynic acid (diuretics), indomethacin (nonsteroidal anti-inflammatory drug), probenecid (uricosuric drug), and valproic acid (antiepileptic drug) potently inhibited (>85%) OAT1-mediated [14C]PAH uptake by the oocytes. An antineoplastic drug, methotrexate, moderately inhibited the [14C]PAH uptake. Endogenous compounds, such as prostaglandin E2, cyclic AMP, cyclic GMP, and uric acid also inhibited [14C]PAH uptake. We examined several radiolabeled compounds in terms of whether they are taken up into oocytes via OAT1. As Fig. 4 b shows, [3H]methotrexate, [3H]cAMP, [3H]cGMP, [3H]prostaglandin E2, [14C]urate and [14C]α-ketoglutarate were transported into the oocytes. No uptake of [14C]tetraethylammonium and [3H]taurocholic acid were detected (data not shown). The present study describes the isolation of cDNA encoding a rat renal organic anion transporter, OAT1. The results indicate that OAT1 possesses the same characteristics as the predicted organic anion/dicarboxylate exchanger responsible for multispecific organic anion transport at the basolateral membrane of renal proximal tubules. The anion exchange model of the renal organic anion transporter has been proposed within the last decade (1Pritchard J.B. Miller D.S. Physiol. Rev. 1993; 73: 765-796Crossref PubMed Scopus (464) Google Scholar, 4Shimada H. Moewes B. Burckhardt G. Am. J. Physiol. 1987; 253: F795-F801PubMed Google Scholar). According to this model, organic anions are transported into the cell in exchange for intracellular dicarboxylates, which are subsequently returned into the cell via sodium-dependent dicarboxylate transporter. In the present report, we directly demonstrate the validity of this exchange model. Accumulated glutarate via sodium-dependent dicarboxylate transporter (rNaDC-1) stimulates [14C]PAH uptake via anion exchanger (OAT1) in Xenopus oocytes. These data explain indirect sodium coupling of renal organic anion transporter. Unexpectedly, a search of EBI/GenBank™ data base (January 31, 1997) revealed that the amino acid sequence of OAT1 shows weak (38%) identity to organic “cation” transporter (OCT1) (9Grundemann D. Gorboulev V. Gambaryan S. Veyhl M. Koepsell H. Nature. 1994; 372: 549-552Crossref PubMed Scopus (611) Google Scholar). The sequence of OAT1 shows no significant similarity with the members of the inorganic anion exchanger (AE) family. OCT1 is considered to be a facilitated transporter, and substrates transported by OCT1 are different from those of OAT1. Although OAT1 and OCT1 transport substrates having opposite charges, there is a common feature in which these two transporters interact with molecules possessing a certain size of hydrophobic cores. This common aspect may be relevant to the weak homology between OAT1 and OCT1. Structure function analysis of OAT1 and OCT1 may provide some clues as to the sites of proteins that recognize the charge(s) of the substrates. To date, at least two multispecific transporter families were identified. One is the ABC superfamily, which includes P-glycoprotein (10Gottesman M.M. Pastan I. Annu. Rev. Biochem. 1993; 62: 385-427Crossref PubMed Scopus (3567) Google Scholar), a multidrug resistance associated protein (11Cole S.P. Bhardwaj G. Gerlach J.H. Mackie J.E. Grant C.E. Almquist K.C. Stewart A.J. Kurz E.U. Duncan A.M. Deeley R.G. Science. 1992; 258: 1650-1654Crossref PubMed Scopus (3022) Google Scholar, 12Zaman M. J. S. 1994; PubMed Scopus Google Scholar) and a multispecific organic anion transporter M. Science. PubMed Scopus Google Scholar). P-glycoprotein and multidrug resistance associated protein a of drugs from cells via hydrolysis and multidrug resistance on is considered to excretion of organic however, its functional characteristics not yet been analyzed in is the anion transporting superfamily, which includes a isolated from rat B. B. S. 1994; PubMed Scopus Google Scholar), prostaglandin transporter Y. Science. 1995; PubMed Scopus Google Scholar), and H. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). mediates transport of and and B. B. S. 1994; PubMed Scopus Google Scholar, X. M. B. J. Pharmacol. Google Scholar). We found no significant sequence similarity between OAT1 and other multispecific organic anion including oatp, and prostaglandin transporter. The and transport characteristics of OAT1 are from those of the members of these two multispecific transporter is for of the of the members of these multispecific transporter in drug to and elimination from the to the in the kidney, anionic substrates be transported in other including and brain. In high stringency Northern blot analysis, however, no signals were detected except for the kidney and the brain. The plays a role in the of many endogenous and are transported into and extensively by and As a compounds are which be from the the multispecific of anionic compounds Am. J. Physiol. 1995; Google Scholar, Rev. Physiol. Biochem. Pharmacol. 1994; PubMed Google Scholar). and may certain in organic anion transport in the however, the of drug transport has been 1994; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). organic anion/dicarboxylate exchange in the and been B. Am. J. Physiol. 1995; Google Scholar, J. Pharmacol. Google Scholar), and a search of a data base revealed the of a protein that is structurally to OAT1 but function has not been K.J. Y. V. J. 1994; Google Scholar). of OAT1 as organic anion transporters in several of of OAT1 in other will facilitate the of drug and excretion system of the and provide for drugs that in the The role of OAT1 is to be of transport of endogenous anionic compounds, such as cyclic nucleotides, prostaglandin E2, and uric A variety of anionic xenobiotics, such as drugs and are synthesized and to the which be Because of its wide substrate selectivity, OAT1 the role of a transporter and as an essential multispecific anionic drug transporter. expression of OAT1 in cell is to be for the development of in vitro for drug elimination and such be used for of drugs for which is of the most in drug to the drugs that renal such as antibiotics, diuretics, and nonsteroidal anti-inflammatory drugs G. The and Scholar), are transported by the organic anion transport One such drugs are may be to their via OAT1. Isolation of OAT1, will facilitate elucidation of the molecular basis of and

Poorly differentiated (PD) thyroid carcinomas lie both morphologically and behaviorally between well-differentiated and undifferentiated (anaplastic) carcinomas. Following the original description of this entity, different diagnostic criteria have been employed, resulting in wide discrepancies and confusion among pathologists and clinicians worldwide. To compare lesions occurring in different geographic areas and the diagnostic criteria applied in those countries, we designed a study with a panel of internationally recognized thyroid pathologists to develop consensus diagnostic criteria for PD carcinomas. Eighty-three cases were collected from Europe, Japan, and the United States, and circulated among 12 thyroid pathologists. Diagnoses were made without any knowledge of the clinical parameters, which were subsequently used for survival analysis. A consensus meeting was then held in Turin, Italy, where an agreement was reached concerning the diagnostic criteria for PD carcinoma. These include (1) presence of a solid/trabecular/insular pattern of growth, (2) absence of the conventional nuclear features of papillary carcinoma, and (3) presence of at least one of the following features: convoluted nuclei; mitotic activity >or=3 x 10 HPF; and tumor necrosis. An algorithmic approach was devised for practical use in the diagnosis of this tumor.

BACKGROUND AND PURPOSE: The effect of ebselen, a seleno-organic compound with antioxidant activity through a glutathione peroxidase-like action, on the outcome of acute ischemic stroke was evaluated in a multicenter, placebo-controlled, double-blind clinical trial. METHODS: Patients diagnosed as having acute ischemic stroke who could receive drug treatment within 48 hours of stroke onset were enrolled. Oral administration of ebselen granules suspended in water (150 mg BID) or placebo was started immediately after admission and was continued for 2 weeks. The major end points were the Glasgow Outcome Scale scores at 1 month and 3 months after the start of treatment. The modified Mathew Scale and modified Barthel Index scores at 1 month and 3 months were also studied as secondary outcome measures. RESULTS: Three hundred two patients were enrolled in the trial. Intent-to-treat analysis of 300 patients (151 given ebselen and 149 given placebo) revealed that ebselen treatment achieved a significantly better outcome than placebo at 1 month (P = .023, Wilcoxon rank sum test) but not at 3 months (P = .056, Wilcoxon rank sum test). The improvement was significant in patients who started ebselen within 24 hours of stroke onset but not in those who started treatment after 24 hours. There was a corresponding improvement in the modified Mathew Scale and modified Barthel Index scores. CONCLUSIONS: Early treatment with ebselen improved the outcome of acute ischemic stroke. Ebselen may be a promising neuroprotective agent.

Colorectal cancer is a major cause of death in Japan, where it accounts for the largest number of deaths from malignant neoplasms among women and the third largest number among men. Many new methods of treatment have been developed during recent decades. The Japanese Society for Cancer of the Colon and Rectum Guidelines 2014 for treatment of colorectal cancer (JSCCR Guidelines 2014) have been prepared as standard treatment strategies for colorectal cancer, to eliminate treatment disparities among institutions, to eliminate unnecessary treatment and insufficient treatment, and to deepen mutual understanding among health-care professionals and patients by making these guidelines available to the general public. These guidelines have been prepared as a result of consensuses reached by the JSCCR Guideline Committee on the basis of careful review of evidence retrieved by literature searches and taking into consideration the medical health insurance system and actual clinical practice in Japan. They can, therefore, be used as a guide for treating colorectal cancer in clinical practice. More specifically, they can be used as a guide to obtaining informed consent from patients and choosing the method of treatment for each patient. As a result of the discussions of the Guideline Committee, controversial issues were selected as clinical questions, and recommendations were made. Each recommendation is accompanied by a classification of the evidence and a classification of recommendation categories, on the basis of consensus reached by Guideline Committee members. Here we present the English version of the JSCCR Guidelines 2014.

The complexity of human DNA has been affected by aerobic metabolism, including endurance exercise and oxygen toxicity. Aerobic endurance exercise could play an important role in the evolution of Homo sapiens, and oxygen was not important just for survival, but it was crucial to redox-mediated adaptation. The metabolic challenge during physical exercise results in an elevated generation of reactive oxygen species (ROS) that are important modulators of muscle contraction, antioxidant protection, and oxidative damage repair, which at moderate levels generate physiological responses. Several factors of mitochondrial biogenesis, such as peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), mitogen-activated protein kinase, and SIRT1, are modulated by exercise-associated changes in the redox milieu. PGC-1α activation could result in decreased oxidative challenge, either by upregulation of antioxidant enzymes and/or by an increased number of mitochondria that allows lower levels of respiratory activity for the same degree of ATP generation. Endogenous thiol antioxidants glutathione and thioredoxin are modulated with high oxygen consumption and ROS generation during physical exercise, controlling cellular function through redox-sensitive signaling and protein-protein interactions. Endurance exercise-related angiogenesis, up to a significant degree, is regulated by ROS-mediated activation of hypoxia-inducible factor 1α. Moreover, the exercise-associated ROS production could be important to DNA methylation and post-translation modifications of histone residues, which create heritable adaptive conditions based on epigenetic features of chromosomes. Accumulating data indicate that exercise with moderate intensity has systemic and complex health-promoting effects, which undoubtedly involve regulation of redox homeostasis and signaling.

atic duct and diffuse enlargement of the entire pancreas due to lymphocyte infiltration. In 1995, Japanese investigators 3 firstly proposed a concept of "autoimmune pancreatitis (AIP)", in which the patients showed diffusely enlarged pancreas, narrowing pancreatogram, increased serum IgG, presence of autoantibodies, fibrotic changes with lymphocytic infiltration and steroidal efficacy. Thereafter, many AIP cases have been reported from Japan, and AIP has been accepted as a new clinical entity. The histopathological findings of AIP show massive infiltration of lymphoplasmacytes with fibrosis, which is consistent with lymphoplasmacytic sclerosing pancreatitis (LPSP). 6 Many Japanese investigators have paid great attention to AIP, especially with regard to its unique pancreatic images, 2 IgG4, 7 diseaseassociated autoantibodies, 8 extrapancreatic lesions, 6,9-14 and steroidal efficacy. urrently in Japan, diagnosis of AIP is based on the "diagnostic criteria 2002 of autoimmune pancreatitis" 16 proposed by the Japan Pancreas Society. However, the accumulation of many AIP cases shows that the

For the last 20 years, a great amount of evidence has accumulated through epidemiological studies that most of the dry eye disease encountered in daily life, especially in video display terminal (VDT) workers, involves short tear film breakup time (TFBUT) type dry eye, a category characterized by severe symptoms but minimal clinical signs other than short TFBUT. An unstable tear film also affects the visual function, possibly due to the increase of higher order aberrations. Based on the change in the understanding of the types, symptoms, and signs of dry eye disease, the Asia Dry Eye Society agreed to the following definition of dry eye: "Dry eye is a multifactorial disease characterized by unstable tear film causing a variety of symptoms and/or visual impairment, potentially accompanied by ocular surface damage." The definition stresses instability of the tear film as well as the importance of visual impairment, highlighting an essential role for TFBUT assessment. This paper discusses the concept of Tear Film Oriented Therapy (TFOT), which evolved from the definition of dry eye, emphasizing the importance of a stable tear film.

IMPORTANCE: Aside from the multikinase inhibitor sorafenib, there are no effective systemic therapies for the treatment of advanced hepatocellular carcinoma. OBJECTIVE: To assess the efficacy of everolimus in patients with advanced hepatocellular carcinoma for whom sorafenib treatment failed. DESIGN, SETTING, AND PARTICIPANTS: EVOLVE-1 was a randomized, double-blind, phase 3 study conducted among 546 adults with Barcelona Clinic Liver Cancer stage B or C hepatocellular carcinoma and Child-Pugh A liver function whose disease progressed during or after sorafenib or who were intolerant of sorafenib. Patients were enrolled from 17 countries between May 2010 and March 2012. Randomization was stratified by region (Asia vs rest of world) and macrovascular invasion (present vs absent). INTERVENTIONS: Everolimus, 7.5 mg/d, or matching placebo, both given in combination with best supportive care and continued until disease progression or intolerable toxicity. Per the 2:1 randomization scheme, 362 patients were randomized to the everolimus group and 184 patients to the placebo group. MAIN OUTCOMES AND MEASURES: The primary end point was overall survival. Secondary end points included time to progression and the disease control rate (the percentage of patients with a best overall response of complete or partial response or stable disease). RESULTS: No significant difference in overall survival was seen between treatment groups, with 303 deaths (83.7%) in the everolimus group and 151 deaths (82.1%) in the placebo group (hazard ratio [HR], 1.05; 95% CI, 0.86-1.27; P = .68; median overall survival, 7.6 months with everolimus, 7.3 months with placebo). Median time to progression with everolimus and placebo was 3.0 months and 2.6 months, respectively (HR, 0.93; 95% CI, 0.75-1.15), and disease control rate was 56.1% and 45.1%, respectively (P = .01). The most common grade 3/4 adverse events for everolimus vs placebo were anemia (7.8% vs 3.3%, respectively), asthenia (7.8% vs 5.5%, respectively), and decreased appetite (6.1% vs 0.5%, respectively). No patients experienced hepatitis C viral flare. Based on central laboratory results, hepatitis B viral reactivation was experienced by 39 patients (29 everolimus, 10 placebo); all cases were asymptomatic, but 3 everolimus recipients discontinued therapy. CONCLUSIONS AND RELEVANCE: Everolimus did not improve overall survival in patients with advanced hepatocellular carcinoma whose disease progressed during or after receiving sorafenib or who were intolerant of sorafenib. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01035229.