Shota Ikeda

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.

Discovery, synthesis and biological evaluation of isoquinolones as novel and highly selective JNK inhibitors (2)

A novel series of 4-phenylisoquinolones were synthesized and evaluated as c-Jun N-terminal kinase (JNK) inhibitors. Initial modification at the 2- and 3-positions of the isoquinolone ring of hit compound 4, identified from high-throughput screening, led to the lead compound 6b. The optimization was carried out using a JNK1-binding model of 6b and several compounds exhibited potent JNK inhibition. Among them, 11g significantly inhibited cardiac hypertrophy in rat pressure-overload models without affecting blood pressure and the concept of JNK inhibitors as novel therapeutic agents for heart failure was confirmed.

Inhibitory effects of TAK‐044 on endothelin induced vasoconstriction in various canine arteries and porcine coronary arteries: a comparison with selective ETA and ETB receptor antagonists

1 The inhibitory effects of the endothelin (ET) receptor antagonist, TAK‐044, on ET‐induced vasoconstriction in various canine arteries and porcine coronary arteries were studied and were compared to those of selective ETA and ETB receptor antagonists. 2 ET‐1 (0.1 nM‐0.3 μm) caused vasoconstriction in canine coronary, femoral, renal, mesenteric and basilar arteries, and the strongest responses were obtained in coronary and basilar arteries. TAK‐044 (10 nM, 100 nM) inhibited this ET‐1‐induced vasoconstriction except in the case of mesenteric arteries. The strongest inhibitory effects were obtained in coronary arteries; an EC50 value for ET‐1 was 5.2 ± 0.77 nM (n = 12) in the control and 24 ± 3.8 nM (n = 4) in the presence of TAK‐044 at 10 nM. BQ‐123 (1 μm) inhibited the vasoconstriction in coronary and femoral arteries but did not in renal, mesenteric or basilar arteries. 3 TAK‐044 (10–100 nM) inhibited the ET‐1‐induced vasoconstriction in porcine coronary arteries to a degree similar to that in canine coronary arteries. In contrast, BQ‐123 (10 μm) did not inhibit the contraction completely, and a BQ‐123‐insensitive component was identified. Although BQ‐788 (1 μm) did not modify the concentration‐response curve at all, it abolished the BQ‐123‐insensitive component when applied together with BQ‐123 (10 μM). 4 Sarafotoxin S6c (10 pM‐30 nM) caused vasoconstriction in porcine coronary arteries with the maximum amplitude of the contraction being 39% of that with ET‐1. Both TAK‐044 (10 nM, 100 nM) and BQ‐788 (1 μM) inhibited this vasoconstriction, while BQ‐123 (3 μM, 10 μm) did not. 5 Vasoconstriction induced by ET‐3 (0.1 nM‐0.3 μm) in porcine coronary arteries showed a concentration‐response curve with two distinct phases in contrast to that seen with sarafotoxin S6c. TAK‐044 (0.3 nM‐10 nM) inhibited both phases in a concentration‐dependent manner. BQ‐123 (1 μM, 3 μm) inhibited only the second phase, while BQ‐788 (1 μm) inhibited the first phase. 6 We concluded that the inhibitory effects of TAK‐044 on ET‐1‐induced vasoconstriction were the strongest in coronary arteries among the canine arteries examined. In addition, we showed that both ETA and ETB receptors mediate vasoconstriction in porcine coronary arteries and TAK‐044 inhibits the vasoconstriction mediated by both of these receptors.

In vitro and in vivo pharmacology of a structurally novel Na+-H+ exchange inhibitor, T-162559

We investigated the inhibitory effects of a non‐acylguanidine Na+‐H+ exchange (NHE) inhibitor, T‐162559 ((5E,7S)‐[7‐(5‐fluoro‐2‐methylphenyl)‐4‐methyl‐7,8‐dihydro‐5(6H)‐quinolinylideneamino] guanidine dimethanesulphonate), on NHE‐1, and its cardioprotective effect against ischaemia and reperfusion injury in rats and rabbits. T‐162559 inhibited human platelet NHE‐1 in a concentration‐dependent manner, with an IC50 value of 13±3 nmol l−1, making it 16 and three times more potent than cariporide IC50: 209±75 nmol l−1, P<0.01) and eniporide (IC50: 40±11 nmol l−1, P=0.066), respectively. T‐162559 also inhibited rat NHE‐1 with an IC50 value of 14±2 nmol l−1, which was five and three times lower than that of cariporide (IC50: 75±7 nmol l−1, P<0.01) and eniporide (IC50: 44±2 nmol l−1, P<0.01), respectively. T‐162559 inhibited, in a concentration‐dependent manner, the reduction in cardiac contractility, progression of cardiac contracture, and increase in lactate dehydrogenase release after global ischaemia and reperfusion in perfused rat hearts. The inhibitory effects of T‐162559 were observed at a lower concentration range (10 – 100 nmol l−1) than with cariporide and eniporide. T‐162559 did not alter basal cardiac contractility or coronary flow after reperfusion, suggesting that it exerts direct cardioprotective effects on the heart. Intravenous administration of T‐162559 (0.03 and 0.1 mg kg−1) significantly inhibited the progression of myocardial infarction induced by left coronary artery occlusion and reperfusion in rabbits; the infarct size normalized by area at risk was 74±6% in the vehicle group, and 47±5% and 51±7% in the T‐162559‐0.03 mg kg−1 and T‐162559‐0.1 mg kg−1 groups (both P<0.05), respectively. These results indicate that the new structural NHE‐1 inhibitor T‐162559 is more potent than cariporide and eniporide and possesses a cardioprotective effect against ischaemia and reperfusion injury in rat and rabbit models.

Effects of troglitazone and pioglitazone on the action potentials and membrane currents of rabbit ventricular myocytes

The effects of the antidiabetic thiazolidinediones troglitazone and pioglitazone on action potentials and membrane currents were studied in rabbit ventricular myocytes. Troglitazone (10 microM) reversibly reduced excitability of the myocytes and modified their action potential configuration. It significantly increased the stimulation threshold required to elicit action potentials and decreased action potential amplitude and the maximum upstroke velocity of the action potentials. The Inhibition of the maximum upstroke velocity by troglitazone was also significant at 1 microM. Voltage-clamp experiments revealed that troglitazone (10 microM) reversibly inhibited both the slow inward Ca2+ current and the steady-state K+ current. In contrast to troglitazone, pioglitazone (1-10 microM) had no significant effect on the excitability, action potential configuration, or membrane currents of myocytes. These results suggest that troglitazone, but not pioglitazone, modulates Na+, Ca2+ and K+ currents, leading to the changes in excitability and action potential configuration of ventricular myocytes.

A Novel Selective Inhibitor of Delta-5 Desaturase Lowers Insulin Resistance and Reduces Body Weight in Diet-Induced Obese C57BL/6J Mice

Obesity is now recognized as a state of chronic low-grade inflammation and is called as metabolic inflammation. Delta-5 desaturase (D5D) is an enzyme that metabolizes dihomo-γ-linolenic acid (DGLA) to arachidonic acid (AA). Thus, D5D inhibition increases DGLA (precursor to anti-inflammatory eicosanoids) while decreasing AA (precursor to pro-inflammatory eicosanoids), and could result in synergistic improvement in the low-grade inflammatory state. Here, we demonstrate reduced insulin resistance and the anti-obesity effect of a D5D selective inhibitor (compound-326), an orally active small-molecule, in a high-fat diet-induced obese (DIO) mouse model. In vivo D5D inhibition was confirmed by determining changes in blood AA/DGLA profiles. In DIO mice, chronic treatment with compound-326 lowered insulin resistance and caused body weight loss without significant impact on cumulative calorie intake. Decreased macrophage infiltration into adipose tissue was expected from mRNA analysis. Increased daily energy expenditure was also observed following administration of compound-326, in line with sustained body weight loss. These data indicate that the novel D5D selective inhibitor, compound-326, will be a new class of drug for the treatment of obese and diabetic patients.

Pharmacological characterization of cardiovascular responses induced by endothelin-1 in the perfused rat heart

The effects of the endothelin receptor antagonist TAK-044 (cyclo[D-alpha-aspartyl-3-[(4-phenylpiperazin-1-yl)carbonyl]-L-ala nyl-L-alpha-aspartyl-D-2-(2-thienyl)glycyl-L-leucyl-D-tryptophyl]+ ++disodiu m salt) and BQ-123 (cyclo[D-Asp-Pro-D-Val-Leu-D-Trp]) were studied in the rat heart to characterize the receptor subtypes responsible for the cardiovascular actions of endothelin-1. Endothelin-1 induced a transient decrease and subsequent increase in perfusion pressure in perfused rat hearts, and increased left ventricular developed pressure. TAK-044 diminished these endothelin-1-induced responses (100 pmol/heart) with IC50 values of 140, 57 and 1.3 nM, respectively. BQ-123 (1-30 mu M) partially inhibited the endothelin-1-induced hypertension (30-40%) in the rat heart, and failed to inhibit the hypotension. The positive inotropic effect of endothelin-1 was abolished by BQ-123. Neither indomethacin (10 mu M) nor Nomega-nitro-L-arginine methyl ester (100 mu M) attenuated the endothelin-1-induced hypotension. TAK-044 and BQ-123 attenuated the positive inotropic effect of endothelin-1 in rat papillary muscles. In rat cardiac membrane fractions, TAK-044 and BQ-123 inhibited [125I]endothelin-1 binding to endothelin ET(A) receptors with IC50 values of 0.39 +/- 0.6 and 36 +/- 9 nM, respectively, whereas only TAK-044 potently blocked the endothelin ET(B) receptor subtype (IC50 value: 370 +/- 180 nM). These results suggest that endothelin-1 modulates cardiovascular functions in the rat heart by activating both endothelin ET(A) and endothelin ET(B) receptors, all of which are sensitive to TAK-044.

Design, Synthesis, and Evaluation of the Highly Selective and Potent G-Protein-Coupled Receptor Kinase 2 (GRK2) Inhibitor for the Potential Treatment of Heart Failure

A novel class of therapeutic drug candidates for heart failure, highly potent and selective GRK2 inhibitors, exhibit potentiation of β-adrenergic signaling in vitro studies. Hydrazone derivative 5 and 1,2,4-triazole derivative 24a were identified as hit compounds by HTS. New scaffold generation and SAR studies of all parts resulted in a 4-methyl-1,2,4-triazole derivative with an N-benzylcarboxamide moiety with highly potent activity toward GRK2 and selectivity over other kinases. In terms of subtype selectivity, these compounds showed enough selectivity against GRK1, 5, 6, and 7 with almost equipotent inhibition to GRK3. Our medicinal chemistry efforts led to the discovery of 115h (GRK2 IC50 = 18 nM), which was obtained the cocrystal structure with human GRK2 and an inhibitor of GRK2 that potentiates β-adrenergic receptor (βAR)-mediated cAMP accumulation and prevents internalization of βARs in β2AR-expressing HEK293 cells treated with isoproterenol. Therefore, 115h appears to be a novel class of therapeutic for heart failure treatment.

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.

Zooxanthellal genetic varieties in giant clams are partially determined by species-intrinsic and growth-related characteristics

Giant clams (tridacnine shellfishes) are large bivalves that inhabit tropical and subtropical waters and harbor the symbiotic microalgae zooxanthellae, which consist of diverse phylotypes (clades). Each clade exhibits unique physiological characteristics, and the cladal composition may influence the hosts survival and its ability to tolerate environmental changes. Using quantitative PCR (qPCR) assays, we investigated the zooxanthellal genetic clades in Tridacna crocea (n = 93) and Tridacna squamosa (n = 93). These two clam species were artificially bred and maintained for an extended time period under an equivalent environment in an outdoor pond. Results showed that T. crocea had a simpler cladal composition and with an apparent dominance of clade A, whereas multiple clades were present in T. squamosa. The zooxanthellae clade A is known to occur in other zooxanthellae-bearing animals that inhabit shallow waters, which is consistent to the shallow water habitat preference of T. crocea. Interestingly, in larger individuals of T. squamosa, the main zooxanthellal clade was C rather than A. The mechanism underlying the dominance of clade C in the larger T. squamosa has not yet been clarified. However, the additional photosynthates supplied by clade C may be preferable for growing clams, as is observed in corals. The cladal composition of giant clams has previously been reported to be primarily controlled by environmental factors. However, our experiments subjected different clam species to the same environmental conditions, and our results suggested that species-intrinsic and/or growth-related processes may also influence the cladal composition.