Discovery of novel pyridazine derivatives as glucose transporter type 4 (GLUT4) translocation activators.

We report herein the synthesis and structure-activity relationships (SAR) of a series of pyridazine derivatives with the activation of glucose transporter type 4 (GLUT4) translocation. Through a cell-based phenotype screening in L6-GLUT4-myc myoblasts and functional glucose uptake assays, lead compound 1a was identified as a functional small molecule. After further derivatization, the thienopyridazine scaffold as the central ring (B-part) was revealed to have potent GLUT4 translocation activities. Consequently, we obtained promising compound 26b, which showed a significant blood glucose lowering effect in the severe diabetic mice model (10-week aged db/db mice) after oral dosing even at 10 mg/kg, implying that our pyridazine derivatives have potential to become novel therapeutic agents for diabetes mellitus.

Human carboxymethylenebutenolidase as a bioactivating hydrolase of olmesartan medoxomil in liver and intestine.

Olmesartan medoxomil (OM) is a prodrug type angiotensin II type 1 receptor antagonist widely prescribed as an antihypertensive agent. Herein, we describe the identification and characterization of the OM bioactivating enzyme that hydrolyzes the prodrug and converts to its pharmacologically active metabolite olmesartan in human liver and intestine. The protein was purified from human liver cytosol by successive column chromatography and was identified by mass spectrometry to be a carboxymethylenebutenolidase (CMBL) homolog. Human CMBL, whose endogenous function has still not been reported, is a human homolog of Pseudomonas dienelactone hydrolase involved in the bacterial halocatechol degradation pathway. The ubiquitous expression of human CMBL gene transcript in various tissues was observed. The recombinant human CMBL expressed in mammalian cells was clearly shown to activate OM. By comparing the enzyme kinetics and chemical inhibition properties between the recombinant protein and human tissue preparations, CMBL was demonstrated to be the primary OM bioactivating enzyme in the liver and intestine. The recombinant CMBL also converted other prodrugs having the same ester structure as OM, faropenem medoxomil and lenampicillin, to their active metabolites. CMBL exhibited a unique sensitivity to chemical inhibitors, thus, being distinguishable from other known esterases. Site-directed mutagenesis on the putative active residue Cys(132) of the recombinant CMBL caused a drastic reduction of the OM-hydrolyzing activity. We report for the first time that CMBL serves as a key enzyme in the bioactivation of OM, hydrolyzing the ester bond of the prodrug type xenobiotics.

Distribution of KAI-9803, a novel δ-protein kinase C inhibitor, after intravenous administration to rats.

KAI-9803 is composed of a selective δ-protein kinase C (δPKC) inhibitor peptide derived from the δV1-1 portion of δPKC (termed "cargo peptide"), conjugated reversibly to the cell-penetrating peptide 11-amino acid, arginine-rich sequence of the HIV type 1 transactivator protein (TAT47₋57; termed "carrier peptide") via a disulfide bond. KAI-9803 administration at the end of ischemia has been found to reduce cardiac damage caused by ischemia-reperfusion in a rat model of acute myocardial infarction. In the study presented here, we examined the TAT47₋57-mediated distribution of KAI-9803 in rats after a single intravenous bolus administration (1 mg/kg). ¹4C-KAI-9803 was rapidly delivered to many tissues, including the heart (1.21 µg eq/g tissue), while being quickly cleared from the systemic circulation. The microautoradiography analysis showed that ¹4C-KAI-9803 was effectively delivered into various cells, including cardiac myocytes and cardiac endothelial cells within 1 min after dosing. The tissue distribution of ¹²5I-labeled KAI-9803 was compared to that of ¹²5I-labeled cargo peptide; this comparison demonstrated that the distribution of KAI-9803 to tissues such as the liver, kidney, and heart was facilitated by the reversible conjugation to TAT47₋57. In an in vitro cardiomyocyte study, the extent of ¹²5I-KAI-9803 internalization was greater at 37°C than that at 4°C, whereas the internalization of the ¹²5I-cargo peptide at 37°C was not observed, indicating that the uptake of ¹²5I-KAI-9803 into the cardiomyocytes was mediated by the TAT47₋57 carrier. Our studies demonstrated that after a single intravenous administration, KAI-9803 can be delivered into the target cells in the liver, kidney, and heart by a TAT47₋57-mediated mechanism.

Tissue distribution of humanized anti-human Fas monoclonal antibody (R-125224) based on fas antigen-antibody reaction in collagen-induced arthritis monkeys.

R-125224 is a novel humanized anti-human Fas monoclonal antibody prepared from HFE7A, which is a monoclonal mouse IgG anti-Fas antibody, by grafting the mouse complementarity-determining regions to human IgG, presently being developed as a drug for treatment of rheumatoid arthritis. In the present study, we investigated the tissue distribution of radioactivity in cynomolgus monkeys with collagen-induced arthritis at the arm joint (CIA monkeys) after intravenous administration of (125)I-labeled R-125224 ((125)I-R-125224). At 168 h after administration, we observed a high radioactivity in the bone marrow, thymus, lungs, liver, adrenals, spleen, ovaries, axillary lymph node and mesenteric lymph node compared to the radioactivity in the plasma. These tissues and organs in human are reported to express Fas antigen, strongly suggesting a specific binding of (125)I-R-125224 to Fas antigen in cynomolgus monkeys. Semi-micro autoradioluminograms of arm joint showed that radioactivity is detected in pharmacological site, such as the bone marrow and articular cavity at 168 h. The kinetics in binding of R-125224 to activated monkey lymphocytes and hepatocytes was also investigated. K(d) values of activated lymphocytes and hepatocytes were 1.51+/-0.08 and 0.60+/-0.11 nM, respectively, which were similar to those values in human lymphocytes and hepatocytes, demonstrating that R-125224 cross-reacts with the monkey Fas antigen.

Suicide proneness in American and Japanese college students: associations with suicide acceptability and emotional expressivity.

This study considered whether suicide acceptability and emotional expressivity were associated with suicide proneness in American and Japanese women and men. Participants included 417 (283 women, 134 men) American and 396 (243 women, 150 men) Japanese college students. Regression models indicated that suicide acceptability predicted unique variance in suicide proneness for both American and Japanese women and men. However, emotional expressivity contributed to understanding the suicide proneness of American college students only. Culturally appropriate prevention and intervention implications associated with reducing suicide acceptance and cultivating well-being and resiliency are offered.

In SCID mice with transplanted joint tissues from rheumatism patients, a model mice of human rheumatoid arthritis, anti-human fas antibody (R-125224) distributes specifically to human synovium.

PURPOSE: We investigated the tissue distribution of a humanized anti-human Fas monoclonal antibody, R-125224, in SCID mice transplanted with synovial tissues from patients with rheumatoid arthritis (SCID-HuRAg mice). The binding kinetics of R-125224 was also determined, using isolated human synovial cells. MATERIALS AND METHODS: Tissue distribution was assessed at 1, 24 and 168 h after intravenous administration of (125)I-R-125224 to SCID-HuRAg mice (0.4 mg/kg). The in vitro binding of (125)I-R-125224 to isolated human synovial cells was investigated. RESULTS: After intravenous administration of (125)I-R-125224 to SCID-HuRAg mice, the radioactivity distributed to various tissues at 1 h. Thereafter, the radioactivity in the tissues gradually decreased except for the transplanted synovial tissues, in which the radioactivity increased in a time-dependent manner, and at 168 h, the tissue/plasma concentration ratio was about 1. The in vitro binding affinity of (125)I-R-125224 to human synovial cells was high with a dissociation constant of 1.32 +/- 0.62 nM and the binding was inhibited by non-labeled R-125224 in a concentration-dependent manner. CONCLUSION: R-125224, a candidate compound for treating rheumatoid arthritis, specifically distributed to the pharmacological target site, human synovium transplanted in SCID mice, with high affinity.

A humanized anti-human Fas antibody, R-125224, induces apoptosis in type I activated lymphocytes but not in type II cells.

Fas-mediated apoptosis plays an important role in the immune system, including the elimination of autoreactive lymphoid cells. The Fas-mediated signaling pathway is classified into two types, type I and type II, in human lymphoid cell lines. We investigated whether a humanized anti-human Fas mAb, R-125224, has cell selectivity in induction of apoptosis. R-125224 induced apoptosis in H9 cells, SKW6.4 cells and activated human lymphocytes when cross-linked with anti-human IgG. On the other hand, R-125224 did not induce apoptosis in HPB-ALL cells, Jurkat cells or human hepatocytes. By analysis of death-inducing signaling complex formation, it was demonstrated that R-125224 induced apoptosis selectively in type I cells but not in type II cells. Type I cells also expressed more Fas and had more Fas-clustering activity than type II cells. Moreover, co-localization of these clusters and GM1, which is an sphingoglycolipid associated with lipid rafts, was detected. It was also shown that R-125224 treatment could reduce the number of activated human CD3+Fas+ cells in a SCID mouse model in vivo. Thus, we demonstrated that R-125224 induces apoptosis specifically in type I cells in vitro and in vivo.

Influence of coronary artery disease on operative mortality and long-term survival after abdominal aortic aneurysm repair.

PURPOSE: This retrospective study was conducted to evaluate the effects of coronary artery disease (CAD) on short- and long-term survival after abdominal aortic aneurysm (AAA) repair. METHODS: One hundred consecutive patients underwent elective AAA repair between 1991 and 2002. Coronary angiography was performed in all patients, revealing significant coronary artery lesions in 47 (47%). Percutaneous transluminal coronary angioplasty (PTCA) was performed in 11 patients, 20 (median) days before the abdominal surgery. Abdominal aortic aneurysm repair was performed 60 (median) days after coronary artery bypass grafting (CABG) in five patients, and both procedures were performed simultaneously in two patients. RESULTS: The in-hospital mortality rate for AAA repair was 1.0%, but there was no cardiac-related operative morbidity or mortality. The 96 patients discharged were followed up for a mean period of 2.9 years (range 3-143 months). The cumulative survival rates after 1, 2, 3, and 5 years were 98%, 95%, 88%, and 77%, respectively. Only one patient (1%) died of myocardial infarction. There was no significant difference in the long-term survival of the CAD and non-CAD patients. CONCLUSIONS: These results emphasize the importance of routine coronary angiography and subsequent coronary revascularization to improve early and late survival rates after AAA repair.

Metabolic stability and uptake by human hepatocytes of pitavastatin, a new inhibitor of HMG-CoA reductase.

To gain a better understanding of the metabolic stability and transport of pitavastatin (CAS 147526-32-7), a new and potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, experiments were conducted using human hepatocytes and oocytes of Xenopus laevis expressing human organic anion transporting polypeptide-2 (OATP2), respectively. Almost the entire radioactivity was from the unchanged substance or lactone form in human hepatocytes, and the cytochrome P450 (CYP)-mediated metabolism of pitavastatin was negligible. The results suggested that CYPs are not critically involved in determining the metabolic fate of pitavastatin. The hepatic uptake of pitavastatin reached saturation with a Km of 2.99 +/- 0.79 micromol/L. Also, the uptake of pitavastatin was mediated by OATP2 expressed in oocytes with a Km of 5.53 +/- 1.70 micromol/L. These results indicated that OATP2 plays a major role in the distribution of pitavastatin in liver. Furthermore, to elucidate the increase in the plasma concentration of pitavastatin in a clinical setting, the inhibitory effect of ciclosporin (cyclosporin A, CAS 59865-13-3) on the uptake of pitavastatin was examined. The uptake of pitavastatin was inhibited in the presence of cyclosporin A and the apparent IC50 value was 2.91 +/- 0.78 micromol/L. This result may at least partly explain the drug-drug interaction between pitavastatin and cyclosporin A. In conclusion, the characterization of transporters needs to be taken into account to avoid transporter-mediated drug-drug interaction.