Kazuki Kubo

Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38)

A novel neuropeptide with 38 residues (PACAP38) was isolated from ovine hypothalamic tissues using the pituitary adenylate cyclase activation in rat pituitary cell cultures as a parameter of the biological activity (Miyata et al, Biochem. Biophys. Res. Commun. 164, 567-574, 1989). From the side fractions obtained during the purification of PACAP38, a shorter form peptide with 27 residues corresponding to the N-terminal 27 amino acids of PACAP38 and amidated C-terminus was isolated and named as PACAP27. Synthetic PACAP27 showed a biological activity of adenylate cyclase stimulation comparable to PACAP38. Moreover PACAP27 which shows a considerable homology with vasoactive intestinal polypeptide (VIP) demonstrated a similar vasodepressor activity as VIP, but the adenylate cyclase stimulating activity was about 1000 times greater than VIP.

Pharmacology of a non‐selective ETA and ETB receptor antagonist, TAK‐044 and the inhibition of myocardial infarct size in rats

1 The aims of the present study were to characterize the pharmacological profile of a new endothelin (ET) receptor antagonist, TAK‐044 and to consider whether it limits the extension of myocardial infarct size in rats. 2 Binding of [125I]‐ET‐1 to ET receptors on rabbit ventricular and cerebellar membrane fractions was inhibited by TAK‐044 with IC50 values of 3.8 nm and 130 nm, respectively. 3 It inhibited ET‐1, ET‐2 and ET‐3‐induced vasoconstriction of porcine isolated coronary arteries in a competitive (ET‐1, ET‐2) and a non‐competitive (ET‐3) manner. 4 In the rat in vivo, the ET‐1‐induced blood pressure changes including transient hypotension followed by sustained hypertension, were inhibited by TAK‐044 (0.1–10 mg kg−1, i.v.) in a dose‐dependent manner. 5 Acute myocardial infarction induced by 1 h coronary occlusion followed by 24 h reperfusion in rats caused an infarct size of 60 ± 2% (n = 12) of the area‐at‐risk by weight. 6 Intravenous injection of TAK‐044 10 min before coronary occlusion reduced the infarct size in a dose‐dependent manner: 32% and 54% reductions at 1 and 3 mg kg−1, respectively. 7 TAK‐044 administered 10 min before or 1 h after reperfusion (1 mg kg−1, i.v.) showed similar inhibitory effects: 34% and 23% reductions, respectively. 8 We conclude that TAK‐044 is an ETA/ETB receptor antagonist which shows strong inhibitory effects on the extension of myocardial infarct size after coronary artery occlusion‐reperfusion in rats.

Effects of a new endothelin antagonist, TAK-044, on post-ischemic acute renal failure in rats

Protective effects of a new endothelin (ET) receptor antagonist, TAK-044, were studied in a model of acute renal failure (ARF) in rats. ARF was induced by clamping the left renal pedicle for 45 minutes with contralateral nephrectomy and subsequent reperfusion of the left kidney. Plasma creatinine concentration (Pcr) increased to 2.28 mg/dl 24 hours after reperfusion of the ischemic kidney. Intravenous administration of TAK-044 (1-10mg/kg) prior to renal occlusion dose-dependently but partially attenuated the increase in Pcr and the morphological damages of the kidney. ET-1 and ET-3 increased perfusion pressure in isolated kidney preparations with similar potency, indicating that the renal vasoconstriction evoked by these ET isomers is mainly via ETB receptors, and TAK-044 (10nM) shifted the ET-1 dose-response curve to the right by a factor about 10. In a rat renal membrane fraction, ET-1 showed competitive inhibition of specific [125I]ET-1 binding with an IC50 value of 0.34nM and a Hill slope of 1.10. ET-3 did so with a higher IC50 value (3.3nM) and a lower Hill slope (0.56), suggesting that rat kidney contains both ETA and another receptor subtype, probably ETB. TAK-044 inhibited ET-1 binding with an IC50 value of 6.6nM and a Hill slope of 0.41. Plasma concentrations of immunoreactive TAK-044 were maintained over 7nM for 8 hours following i.v. injection of 10mg/kg TAK-044. These results suggest that endogenous ET is involved in the pathogenesis of post-ischemic ARF, at least, in part and that TAK-044 provided protective effects against ARF by blocking ET receptors, possibly both ETA and ETB receptors in renal vasculature and parenchymal cells.

Suppression of neointimal thickening by a newly developed HMG-CoA reductase inhibitor, BAYw6228, and its inhibitory effect on vascular smooth muscle cell growth

The aim of this study was to determine whether BAYw6228 (BAYw), a newly developed 3‐hydroxy‐3‐methylglutaryl‐CoA (HMG‐CoA) reductase inhibitor, could suppress an atherogenic process such as intimal thickening by a mechanism other than lowering the level of serum cholesterol. First, we evaluated the in vitro effect of BAYw on the proliferation of vascular smooth muscle cells (SMC) from various species: Sprague‐Dawley (SD) rats, New Zealand (NZ) white rabbits, intimal cells from Watanabe hereditary hyperlipidemic (WHHL) rabbit and SMC from the new‐born human aorta. The increasing rate of total protein content of these cells was inhibited by the addition of BAYw in a dose‐dependent fashion. In the presence of 2% foetal calf serum (FCS), the value of IC50 was 1.0 μm in SD rats. 2.1 μm in NZ white rabbits, and 0.3 μm in WHHL rabbits. With human SMC, the value was 0.02 μm in the presence of 10% FCS and 0.2 μm with a mixture of growth factors. Based on these above in vitro findings, we next examined the in vivo effect of the agent to determine whether it could suppress rabbit intimal thickening induced by balloon catheterization. A balloon catheter was inserted from a peripheral branch of the left external carotid artery to the aorta to denude the endothelium of the left common carotid artery in Japanese white rabbits. After 12 days they were divided into control and BAYw groups. The former were subcutaneously injected with saline and the latter with BAYw 1 mg kg−1 day−1. Two days after the beginning of treatment, a second balloon injury was performed to the previously injured left common carotid artery in both groups. After another two weeks, the left common carotid artery was removed and variously stained. Although the total serum cholesterol in the BAYw group was significantly lower than in the control (P<0.05), the difference was not enough to affect intimal thickening. In addition, the BAYw group had a smaller intima/media ratio than the control group, decreasing to 45% of control (P<0.05). By anti‐α smooth muscle actin antibody staining, these intimal thickening areas were entirely occupied by SMCs, and their amount was attenuated by BAYw. By anti‐rabbit macrophage antibody (RAM 11) staining, the number of positive cells in the intimal thickening was markedly decreased in the BAYw group compared to control (P<0.01). These results indicate that BAYw has an inhibitory effect on intimal thickening by attenuating intimal SMC proliferation and infiltration of macrophages, suggesting that BAYw could be effective in the prevention of the progression of atherosclerotic plaque‐like restenosis after angioplasty.

Comparison of Contrast Agents for Atherosclerosis Imaging Using Cultured Macrophages: FDG Versus Ultrasmall Superparamagnetic Iron Oxide

Various noninvasive imaging methods have been developed to evaluate atherosclerotic plaques. Among them, 18F-FDG PET and MR imaging with ultrasmall superparamagnetic iron oxide particles (USPIO) have been used to quantify plaque inflammation. Both methods are based on the efficient uptake of FDG and USPIO by macrophages in atherosclerotic lesions. Differently polarized macrophages have been reported to have different characteristics that are involved in the pathologic development of atherosclerosis. M1 polarized macrophages are considered the more proatherogenic phenotype than M2 polarized macrophages. However, little is known regarding the association between macrophage polarization and FDG or USPIO accumulation. In this study, we investigated intracellular FDG and USPIO accumulation in M1 and M2 polarized macrophages. Methods: THP-1 macrophages were differentiated into M1 and M2 polarized macrophages. Under optimal glucose conditions, we investigated the 3H-labeled FDG uptake in M1 and M2 polarized macrophages. We then investigated intracellular USPIO uptake by M1 and M2 macrophages. Results: We found that M1 polarization, compared with M2 polarization, results in increased intracellular accumulation of FDG. To elucidate the mechanism by which FDG was preferentially accumulated in M1 macrophages, we examined messenger RNA expressions of glucose transporters (GLUTs) and hexokinases, which have pivotal roles in glucose uptake, and glucose-6-phosphatase (G6Pase), which catalyzes the reverse reaction of hexokinase. In M1 macrophages, GLUT-1, GLUT-3, hexokinase 1, and hexokinase 2 were upregulated and G6Pase was downregulated. In contrast to FDG, M1 polarization resulted in decreased intracellular accumulation of USPIO. We found that scavenger receptor A and CD11b, which are involved in USPIO binding and uptake, were significantly downregulated by M1 polarization. Conclusion: Compared with M2, proatherogenic M1 macrophages preferentially accumulated FDG but not USPIO, suggesting that FDG PET is a useful method for the detection of proinflammatory M1 macrophages.

Expression of endothelin-2 (ET-2) gene in a human renal adenocarcinoma cell line : purification and cDNA cloning of ET-2

It has been found that human renal adenocarcinoma ACHN cells synthesize and secrete immunoreactive endothelin (ir-ET) in the culture medium. Partial characterization of this material with reverse-phase high-performance liquid chromatography (RP-HPLC) suggested that ACHN cells synthesized only human endothelin-2 (ET-2). Isolation and characterization of this ir-ET-2 has revealed that this peptide has almost the same amino acid sequence and molecular weight as that of human ET-2 deduced from the nucleotide sequence of cloned human ET-2 gene. To delineate the precise structure of human ET-2 precursor, ET-2 cDNAs were cloned from a cDNA library constructed with mRNA derived from the ACHN cells, and the nucleotide and deduced amino acid sequences were determined. The ET-2 cDNA that has the longest open reading frame encodes prepro-ET-2 protein, consisting of 178 amino acid residues. The ET-like sequence found in the prepro-ET-1 and -ET-3 was also conserved in this prepro-ET-2. The Northern blot analysis of mRNA revealed that the transcript of the human ET-2 gene was 1.4 kb.

Isolation and identification of hemin as an endogenous Na+K+-ATPase inhibitor from porcine blood cells

A substance which is a potent inhibitor of Na+/K(+)-ATPase activity and competitively displaces [3H]ouabain binding to this enzyme was isolated from porcine blood cells. From its chemical and physiochemical properties, this activity was identified as hemin (chloroprotohemin IX). Hemin showed a dose dependent curve for Na+/K(+)-ATPase inhibitory activity similar to that of ouabain and displaced [3H]ouabain binding as potent as 1/100 of ouabain itself.

Pharmacologic Profile of EndothelinA/B Antagonist, [Thr18,γMethylLeu19]Endothelin-1

The pharmacologic profile of [Thr 18 ,γmethyl Leu 19 ]endothelin-1 (TM ET-1) was investigated in several in vitro and in vivo studies. We found that TM ET-1 inhibited 125 I-ET-1 binding in porcine cardiac membrane (ET A receptor) and in bovine brain membrane (ET B receptor), with IC 50 values of 0.7 and 0.25 nM, respectively. These values were almost comparable to those for ET-1

Effects of a new endothelin receptor antagonist, TAK-044, on myocardial stunning in dogs

The effects of a new endothelin receptor antagonist, TAK-044, (cyclo[D-alpha-aspartyl-3-[(4-phenylpiperazin-1-yl)carbonyl]L-alan yl-L-alpha aspartyl-D-2-(2-thienyl)glycyl-L-leucyl-D-tryptophyl]disodium salt, on ischemic and post-ischemic myocardial dysfunction (stunned myocardium) were studied in anesthetized open-chest dogs. A short (15 min) occlusion of the left anterior descending coronary artery followed by 5-h reperfusion significantly reduced myocardial segment shortening during and after the ischemic period in the ischemic region. Regional myocardial blood flow was also decreased significantly 10 min after the occlusion, whereas it returned almost completely to its pre-ischemic value 5 h after reperfusion TAK-044 (3 mg/kg,i.v.) administered 10 min before occlusion significantly improved the reduced myocardial segment shortening in the ischemic region during and after occlusion. Cardiovascular hemodynamics and regional myocardial blood flow in a TAK-044-treated group were identical to those in the control group. These results indicate that endogenous endothelin contributes to the cause of ischemic and post-ischemic myocardial dysfunction without changing either hemodynamics or regional myocardial blood flow.

Discovery of 3,5-Diphenyl-4-methyl-1,3-oxazolidin-2-ones as Novel, Potent, and Orally Available Δ-5 Desaturase (D5D) Inhibitors

The discovery and optimization of Δ-5 desaturase (D5D) inhibitors are described. Investigation of the 1,3-oxazolidin-2-one scaffold was inspired by a pharmacophore model constructed from the common features of several hit compounds, resulting in the identification of 3,5-diphenyl-1,3-oxazolidin-2-one 5h as a novel lead showing potent in vitro activity. Subsequent optimization focused on the modification of two metabolic sites, which provided (4S,5S)-5i, a derivative with improved metabolic stability. Moreover, adding a substituent into the upper phenyl moiety further enhanced the intrinsic activity, which led to the discovery of 5-[(4S,5S)-5-(4fluorophenyl)-4-methyl-2-oxo-1,3-oxazolidin-3-yl]benzene-1,3-dicarbonitrile (4S,5S)-5n, endowed with excellent D5D binding affinity, cellular activity, and high oral bioavailability in a mouse. It exhibited robust in vivo hepatic arachidonic acid/dihomo-γ-linolenic acid ratio reduction (a target engagement marker) in an atherosclerosis mouse model. Finally, an asymmetric synthetic procedure for this compound was established.