Kazunori Fujimoto

Angiopoietin-Like Protein3 Regulates Plasma HDL Cholesterol Through Suppression of Endothelial Lipase

Objectives—A low level of high-density lipoprotein (HDL) in plasma has been recognized as an aspect of metabolic syndrome and as a crucial risk factor of cardiovascular events. However, the physiological regulation of plasma HDL levels has not been completely defined. Current studies aim to reveal the contribution of angiopoietin-like protein3 (angptl3), previously known as a plasma suppressor of lipoprotein lipase, to HDL metabolism. Methods and Results—Angptl3-deficient mice showed low plasma HDL cholesterol and HDL phospholipid (PL), and which were increased by ANGPTL3 supplementation via adenovirus. In vitro, ANGPTL3 inhibited the phospholipase activity of endothelial lipase (EL), which hydrolyzes HDL-PL and hence decreases plasma HDL levels, through a putative heparin-binding site in the N-terminal domain of ANGPTL3. Post-heparin plasma in Angptl3-knockout mice had higher phospholipase activity than did that in wild-type mice, suggesting that the activity of endogenous EL is elevated in Angptl3-deficient mice. Furthermore, we established an ELISA system for human ANGPTL3 and found that plasma ANGPTL3 levels significantly correlated with plasma HDL cholesterol and HDL-PL levels in human subjects. Conclusions—Angptl3 acts as an inhibitor of EL and may be involved in the regulation of plasma HDL cholesterol and HDL-PL levels in humans and rodents.

Identification of genes related to heart failure using global gene expression profiling of human failing myocardium.

Although various management methods have been developed for heart failure, it is necessary to investigate the diagnostic or therapeutic targets of heart failure. Accordingly, we have developed different approaches for managing heart failure by using conventional microarray analyses. We analyzed gene expression profiles of myocardial samples from 12 patients with heart failure and constructed datasets of heart failure-associated genes using clinical parameters such as pulmonary artery pressure (PAP) and ejection fraction (EF). From these 12 genes, we selected four genes with high expression levels in the heart, and examined their novelty by performing a literature-based search. In addition, we included four G-protein-coupled receptor (GPCR)-encoding genes, three enzyme-encoding genes, and one ion-channel protein-encoding gene to identify a drug target for heart failure using in silico microarray database. After the in vitro functional screening using adenovirus transfections of 12 genes into rat cardiomyocytes, we generated gene-targeting mice of five candidate genes, namely, MYLK3, GPR37L1, GPR35, MMP23, and NBC1. The results revealed that systolic blood pressure differed significantly between GPR35-KO and GPR35-WT mice as well as between GPR37L1-Tg and GPR37L1-KO mice. Further, the heart weight/body weight ratio between MYLK3-Tg and MYLK3-WT mice and between GPR37L1-Tg and GPR37L1-KO mice differed significantly. Hence, microarray analysis combined with clinical parameters can be an effective method to identify novel therapeutic targets for the prevention or management of heart failure.

Characterization of phenotypes in Gstm1-null mice by cytosolic and in vivo metabolic studies using 1,2-dichloro-4-nitrobenzene.

Glutathione S-transferase Mu 1 (GSTM1) has been regarded as one of the key enzymes involved in phase II reactions in the liver, because of its high expression level. In this study, we generated mice with disrupted glutathione S-transferase Mu 1 gene (Gstm1-null mice) by gene targeting, and characterized the phenotypes by cytosolic and in vivo studies. The resulting Gstm1-null mice appeared to be normal and were fertile. Expression analyses for the Gstm1-null mice revealed a deletion of Gstm1 mRNA and a small decrease in glutathione S-transferase alpha 3 mRNA. In the enzymatic study, GST activities toward 1,2-dichloro-4-nitrobenzene (DCNB) and 1-chloro-2,4-dinitrobenzene (CDNB) in the liver and kidney cytosols were markedly lower in Gstm1-null mice than in the wild-type control. Gstm1-null mice had GST activities of only 6.1 to 21.0% of the wild-type control to DCNB and 26.0 to 78.6% of the wild-type control to CDNB. After a single oral administration of DCNB to Gstm1-null mice, the plasma concentration of DCNB showed larger AUC0–24 (5.1–5.3 times, versus the wild-type control) and higher Cmax (2.1–2.2 times, versus the wild-type control), with a correspondingly lower level of glutathione-related metabolite (AUC0–24, 9.4–17.9%; and Cmax, 9.7–15.6% of the wild-type control). In conclusion, Gstm1-null mice showed markedly low ability for glutathione conjugation to DCNB in the cytosol and in vivo and would be useful as a deficient model of GSTM1 for absorption, distribution, metabolism, and excretion/toxicology studies.

In vitro cytotoxicity assay to evaluate the toxicity of an electrophilic reactive metabolite using glutathione-depleted rat primary cultured hepatocytes.

Glutathione plays an important role as not only a scavenger of reactive oxygen species but also in the conjugation or detoxification of electrophilic reactive metabolites, which has been thought to be one of the causes for idiosyncratic drug toxicity (IDT). Therefore, toxic responses to the reactive metabolites have been expected to be expressed more strongly in a glutathione-depleted condition. In the present study, we attempted to establish an in vitro cytotoxicity assay method to evaluate the toxicity of the reactive metabolite using rat primary cultured hepatocytes with cellular glutathione depletion by l-buthionine-S,R-sulfoximine. Also, we investigated whether the IDT risk is predictable by comparing the cytotoxic sensitivity between glutathione-depleted hepatocytes and untreated hepatocytes. Consequently, 10 drugs of 42 approved drugs, which were classified into 4 IDT categories (Withdrawn, Black box warning, Warning, and Safe), demonstrated higher cytotoxic sensitivity in the glutathione-depleted hepatocytes. Furthermore, a correlation was observed between the incidence of drugs with higher cytotoxic sensitivity in the glutathione-depleted hepatocytes and the IDT risk. The incidence was 50% in the Withdrawn category, 38% in the Black box warning category, 22% in the Warning category, and 8% in the Safe category. These results suggest that the IDT risk of some drugs may be predicted by comparing the cytotoxic sensitivity between them. Additionally, this method may be useful as a screening in the early stage of drug development where leads/candidates are optimized.

Genetic mapping of a neurological mutation cerebellar calcification in the rat

The rat genetic map has been markedly improved with application of PCR-analyzed microsatellite markers, particularly gene-derived and anonymous SSLP markers, in combination with cytogenetical methods for clarification of coverage, orientation, and centromere position (Rat Genome Database 1999; Rat Genome Data 1999). With this progress, comparative mapping studies between the rat, mouse, and human have become possible in almost the whole genome through identification of conserved genomic regions containing homologous genes common to these species (Mouse Genome Database 2000). Such a situation has not only promoted genetic mapping of rat mutations, but also made it possible to select out the candidate genes for particular diseases by referring to the syntenic regions of the mouse and human chromosomes (Kren et al. 1997; Yokoi et al. 2000). A new neurological mutant was found in the inbred F344 strain of rats maintained in the Medicinal Safety Research Laboratories, Sankyo Co., Ltd. in Japan (Ando et al. 1999). They developed a hesitant and wobbly gait with asynergic movement of limbs and a slight tremor by 2–3 weeks of age. The mutation was inherited as an autosomal recessive trait (Ando et al. 1999) and revealed pathologically slight cerebellar atrophy and distinct symmetrical calcium deposition in the Purkinje cells and dendrites, followed by their degeneration in the cerebellar cortex. Moreover, periodic acid-Schiff (PAS)-positive substances accumulated markedly in the lesioned sites before the Purkinje cells started to degenerate. Thus, we named this mutant cerebellar calcification (CC) rat with the gene symbol cc. Severe Purkinje cell degeneration has been recognized in cerebellar ataxic mutants such as the pcd mouse (Mullen et al. 1976) and the shaker rat (Tolbert et al. 1995). However, no mutations exhibiting similar degenerative lesions as observed in the cerebellum of the CC rat have been reported so far. In some cases of human hereditary ataxic diseases, Purkinje cell degeneration and/or calcification is observed in the brain (Rowlatt 1969; Kobayashi et al. 1987; Adams and Duchen 1992). Therefore, the CC rat may be useful as an animal model for studying the mechanisms of Purkinje cell degeneration and intracranial calcification in the human neurological disorders. In the present study, we performed genetic mapping of the causative gene responsible for cerebellar calcification in the CC rat, and comparative mapping of the cc region in the rat, mouse, and human as an approach to clarification of genetic bases leading to the pathogenesis of the neurological disease in the CC rat.

Establishment of an in vitro cytotoxicity assay platform using primary monkey cardiomyocytes

To establish an in vitro cytotoxicity assay platform using monkey cardiomyocytes, we isolated primary cardiomyocytes from fetal cynomolgus monkeys at different gestation days (from day 39 to 90) using the trypsin and collagenase digestion method, which was identical to the standard procedure for rat cardiomyocytes. Under these conditions, the primary cells obtained from monkeys at gestation day 63 or earlier showed spontaneous beating, with >80% cells being viable from all fetuses. Transcriptome analysis of the monkey cardiomyocytes indicated that the cells have essential components of cardiac functions, such as myosins, α-actin, cardiac troponins, and calcium-related molecules. The susceptibility to doxorubicin-induced cytotoxicity in monkey cardiomyocytes was comparable to that in rat cardiomyocytes, as evaluated based on intracellular ATP levels. Microarray analysis with Ingenuity Pathway Analysis revealed that doxorubicin predominantly increased the expression of several key genes involved in the endoplasmic reticulum stress pathway in monkey cardiomyocytes than in rat cardiomyocytes. In conclusion, we isolated primary monkey cardiomyocytes that showed similar sensitivity to doxorubicin as compared with rat cardiomyocytes. This in vitro monkey cardiomyocyte assay platform would serve as a powerful tool for the investigation of the interspecies differences in drug-induced cardiotoxicity and its underlying mechanism.

Discovery of DS-6930, a potent selective PPARγ modulator. Part II: Lead optimization

Attempts were made to reduce the lipophilicity of previously synthesized compound (II) for the avoidance of hepatotoxicity. The replacement of the left-hand side benzene with 2-pyridine resulted in the substantial loss of potency. Because poor membrane permeability was responsible for poor potency in vitro, the adjustment of lipophilicity was examined, which resulted in the discovery of dimethyl pyridine derivative (I, DS-6930). In preclinical studies, DS-6930 demonstrated high PPARγ agonist potency with robust plasma glucose reduction. DS-6930 maintained diminished PPARγ-related adverse effects upon toxicological evaluation in vivo, and demonstrated no hepatotoxicity. Cofactor recruitment assay showed that several cofactors, such as RIP140 and PGC1, were significantly recruited, whereas several canonical factors was not affected. This selective cofactor recruitment was caused due to the distinct binding mode of DS-6930. The calcium salt, DS-6930b, which is expected to be an effective inducer of insulin sensitization without edema, could be evaluated clinically in T2DM patients.