Hiroshi Kusama

The effects of mitiglinide (KAD-1229), a new anti-diabetic drug, on ATP-sensitive K+ channels and insulin secretion: comparison with the sulfonylureas and nateglinide

Mitiglinide (KAD-1229), a new anti-diabetic drug, is thought to stimulate insulin secretion by closing the ATP-sensitive K+ (K(ATP)) channels in pancreatic beta-cells. However, its selectivity for the various K(ATP) channels is not known. In this study, we examined the effects of mitiglinide on various cloned K(ATP) channels (Kir6.2/SUR1, Kir6.2/SUR2A, and Kir6.2/SUR2B) reconstituted in COS-1 cells, and compared them to another meglitinide-related compound, nateglinide. Patch-clamp analysis using inside-out recording configuration showed that mitiglinide inhibits the Kir6.2/SUR1 channel currents in a dose-dependent manner (IC50 value, 100 nM) but does not significantly inhibit either Kir6.2/SUR2A or Kir6.2/SUR2B channel currents even at high doses (more than 10 microM). Nateglinide inhibits Kir6.2/SUR1 and Kir6.2/SUR2B channels at 100 nM, and inhibits Kir6.2/SUR2A channels at high concentrations (1 microM). Binding experiments on mitiglinide, nateglinide, and repaglinide to SUR1 expressed in COS-1 cells revealed that they inhibit the binding of [3H]glibenclamide to SUR1 (IC50 values: mitiglinide, 280 nM; nateglinide, 8 microM; repaglinide, 1.6 microM), suggesting that they all share a glibenclamide binding site. The insulin responses to glucose, mitiglinide, tolbutamide, and glibenclamide in MIN6 cells after chronic mitiglinide, nateglinide, or repaglinide treatment were comparable to those after chronic tolbutamide and glibenclamide treatment. These results indicate that, similar to the sulfonylureas, mitiglinide is highly specific to the Kir6.2/SUR1 complex, i.e., the pancreatic beta-cell K(ATP) channel, and suggest that mitiglinide may be a clinically useful anti-diabetic drug.

Bezafibrate stimulates canalicular localization of NBD-labeled PC in HepG2 cells by PPARα-mediated redistribution of ABCB4

Fibrates, including bezafibrate (BF), upregulate the expression of ATP binding cassette protein B4 (ABCB4) through gene transcription in mice. To determine the effects of BF on the expression levels of ABCB4 and on the stimulation of biliary phosphatidylcholine (PC) transport in human HepG2 hepatoblastoma cells, mRNA and protein levels as well as subcellular localization were investigated in the cells treated with BF. The canalicular accumulation of a fluorescent PC was assessed by confocal laser scanning microscopy. Treatment with 300 μmol/l BF for 24 h increased levels of ABCB4 mRNA but not protein by up to 151%. BF caused redistribution of ABCB4 into pseudocanaliculi formed between cells. In association with this redistribution, BF accelerated the accumulation of fluorescent PC in bile canaliculi (up to 163% of that in nontreated cells). Suppression of peroxisome proliferator-activated receptor α (PPARα) expression by either a small interfering RNA duplex or morpholino antisense oligonucleotide attenuated the BF-induced redistribution of ABCB4. These findings suggest that BF may enhance the capacity of human hepatocytes to direct PC into bile canaliculi via PPARα-mediated redistribution of ABCB4 to the canalicular membrane. This provides a rationale for the use of BF to improve cholestasis and/or cholangitis that is attributable to hypofunction of ABCB4.

Bezafibrate prevents hepatic stellate cell activation and fibrogenesis in a murine steatohepatitis model, and suppresses fibrogenic response induced by transforming growth factor‐β1 in a cultured stellate cell line

Aim:  The aim of this study was to investigate the preventive actions of bezafibrate against non‐alcoholic steatohepatitis (NASH), the activation of hepatic stellate cells (HSC), and fibrogenesis by using a model of NASH and an in vitro model.

Effect of a thromboxane A2 synthetase inhibitor (OKY-046.HCl) on airway hyperresponsiveness in guinea pigs.

We studied the effect of (E)-3-[p-(1H-imidazol-1-ylmethyl)phenyl]-2-propenoic acid hydrochloride monohydrate (OKY-046.HCl), a specific thromboxane (TX) A2 synthetase inhibitor, on airway hyperresponsiveness of guinea pigs. OKY-046.HCl (30-100 mg/kg, intraduodenally (i.d.) or orally (p.o.)) suppressed dose dependently the airway hyperresponsiveness to acetylcholine (ACh) induced by formyl-methionyl-leucyl-phenylalanine (FMLP), platelet activating factor (PAF) and repetitive antigen. OKY-046.HCl (100 mg/kg) also inhibited the increase in TXB2 in bronchoalveolar lavage fluid (BALF) induced by FMLP, PAF and antigen. Aspirin 10 or 30 mg/kg i.d. or p.o.) suppressed the airway hyperresponsiveness induced by FMLP and PAF but not by antigen. Azelastine (10 mg/kg i.d.) was ineffective on PAF- and antigen-induced airway hyperresponsiveness. TXA2 mimetic drugs caused airway hyperresponsiveness that was not inhibited by OKY-046.HCl (30 mg/kg i.v.). Furthermore, OKY-046.HCl showed no effect on propranolol- and physostigmine-induced airway hyperresponsiveness which did not accompany TXB2 generation in BALF. The number of eosinophils in BALF increased after FMLP exposure, an effect which was not inhibited by OKY-046.HCl. These results suggest that OKY-046.HCl inhibits airway hyperresponsiveness by suppressing TXA2 generation. We suggest that OKY-046.HCl will be a new antiasthmatic drug.

Salivation triggered by pilocarpine involves aquaporin-5 in normal rats but not in irradiated rats.

1 Using rats, we examined the muscarinic receptor subtype mediating pilocarpine‐induced parotid salivary secretion and the contributions of ion transporter systems (effluxes of K+ and Cl‐) and aquaporin‐5 (AQP5) translocation to this response in parotid glands in irradiated‐induced xerostomia. 2 Salivary secretion was significantly lower in irradiated compared with sham‐irradiated (normal) rats. In xerostomia rats, 0.4 and 0.8 mg/kg pilocarpine significantly increased parotid salivary secretion, although the salivary volume was still significantly less than in normal rats after the same dose of pilocarpine. 3 Pirenzepine (1 × 10−6 to 1 × 10−1 mol/L), AF‐DX 116 (3 × 10−6 to 3 × 10−2 mol/L) and N‐2‐chloroethyl‐4‐piperidinyl diphenylacetate (4‐DAMP; 1 × 10−8 to 1 × 10−2 mol/L) dose‐dependently displaced radioligand binding to M1, M2 and M3 receptors, respectively, in parotid membranes from both normal and irradiated rats. In each group of rats, 4‐DAMP had the highest binding affinity. Pretreatment with 4‐DAMP or pirenzepine dose‐dependently inhibited pilocarpine‐induced parotid secretion in both normal and irradiated rats, with 4‐DAMP being markedly more potent than pirenzepine. 4 Normal and irradiated‐rat parotid cells did not differ significantly in terms of pilocarpine‐induced changes in [Ca2+]i, [K+]i and [Cl‐]i. Pilocarpine markedly increased the amount of AQP5 in the apical plasma membrane of parotid cells isolated from normal but not irradiated rats. 5 Thus, pilocarpine induces parotid salivary secretion mainly via the M3 receptor subtype in both irradiated and normal rats. The reduction in this pilocarpine‐induced secretion seen in irradiated rats is due not to disturbances of intracellular Ca2+ mobilization or ion transporter systems, but rather to a disturbance of AQP5 translocation, which may be involved in the pathogenesis of X‐ray irradiation‐induced xerostomia.

Effect Of Kad-1229, A Novel Hypoglycaemic Agent, On Plasma Glucose Levels After Meal Load In Type 2 Diabetic Rats

1. The effects of KAD‐1229 (a novel non‐sulphonylurea agent), voglibose (an α‐glucosidase inhibitor) and nateglinide (a non‐sulphonylurea antihyperglycaemic agent) on hyperglycaemia induced by a meal load were assessed in diabetic rats.

Mechanical Function and Gene Expression of α1-Adrenoceptor Subtypes in Dog Intravesical Ureter

OBJECTIVES To characterize the contractile functions and gene expression of the alpha(1)-adrenoceptor (AR) subtypes present in the dog intravesical ureter. METHODS In a functional study, alpha(1)-AR antagonists were evaluated against phenylephrine (alpha(1)-AR agonist)-induced contractions in dog isolated intravesical ureteral preparations. The quantitative expression of alpha(1)-AR subtype mRNA in this tissue was determined using real-time quantitative reverse transcriptase-polymerase chain reaction. RESULTS In the isolated intravesical ureter, prazosin (nonselective alpha(1)-AR antagonist), silodosin (selective alpha(1A)-AR antagonist), naftopidil (selective alpha(1D)-AR antagonist), and BMY-7378 (selective alpha(1D)-AR antagonist) all shifted the concentration-contractile response curve for phenylephrine to the right. The rank order of potencies (pK(B) value) was silodosin (9.45 +/- 0.14), prazosin (8.16 +/- 0.08), naftopidil (7.39 +/- 0.19), and BMY-7378 (6.78 +/- 0.20). The alpha(1A)-AR antagonist silodosin was much more potent than the 2 alpha(1D)-AR antagonists. The rank order of mRNA expression levels among the alpha(1)-AR subtypes was alpha(1d) (72.68%), alpha(1a) (24.14%), and alpha(1b) (3.18%). CONCLUSIONS In the dog intravesical ureter, alpha(1A)-AR plays a major role in contraction, despite the prevalence of alpha(1D)-AR.

Absence of exacerbation of myocardial stunning in anesthetized dogs treated with KAD-1229, a novel hypoglycemic agent

The effect of (+)-momocalcium bis[(2S,3a,7a-cis)-alpha-benzylhexahydro-gamma-oxo-2-isoindolinebutyrate]dihydrate (KAD-1229), a novel hypoglycemic agent with a chemical structure different from that of the sulfonylureas, on myocardial stunning was assessed in anesthetized dogs by comparison with that of glibenclamide, a sulfonylurea. Even though their hypoglycemic effects were of similar magnitude, glibenclamide (1 mg/kg, i.v.), but not KAD-1229, exacerbated the myocardial stunning induced by occlusion/reperfusion of the descending coronary artery. In a receptor-binding experiment, unlabeled glibenclamide completely inhibited [(3)H]glibenclamide binding to the myocardium, but KAD-1229 did not. These results suggest that the difference in binding properties of KAD-1229 and glibenclamide toward cardiac sulfonylurea receptors is one of the causes of their different effects on myocardial stunning. It is likely that KAD-1229 is highly specific for pancreatic sulfonylurea receptors and is speculated to be a safer hypoglycemic agent than, at least, glibenclamide.

Platelet-Activating-Factor(PAF)吸入誘発モルモット気道過敏性に対する特異的Thromboxane A2合成酵素阻害剤(E)-3-〔p-(1H-imidazol-1-ylmethyl)phenyl〕-2-propenoic acid hydrochloride monohydrate (OKY-C)46・HCl)の抑制作用

We studied the inhibitory effects of OKY-046.HCl on PAF-induced airway hyperresponsiveness (AHR) in guinea pigs. 1) Inhalation of PAF (1 or 10 micrograms/ml) caused AHR to acetylcholine (ACh) aerosol and increased TXB2 generation in broncho-alveolar lavage fluid (BALF) at 30 min and 60 min, but the AHR and the TXB2 generation disappeared at 2 hr. OKY-046.HCl (100 mg/kg, intraduodenally) inhibited the AHR, which was accompanied by its inhibition of the TXB2 generation. However, no changes of 6-keto-PGF1 alpha in BALF were found. 2) There were no changes in the number of leukocytes; the activities of alkaline phosphatase, N-acetyl-beta-D-glucosaminidase, and lactate dehydrogenase; and the LTC4/D4/E4 in BALF. 3) In bronchus-lung preparations, PAF (1 microgram/min) also caused the AHR and increased TXB2 generation. OKY-046.HCl (100 micrograms/min) inhibited the AHR and TXB2 generation. 4) PAF (1 microgram/ml) evoked TXB2 generation in BALF from normal guinea pigs. OKY-046.HCl (10(-4)M) inhibited its increase. 5) Stable TXA2 (STA2, 1 ng/ml) inhalation also caused AHR to ACh at 30 min. STA2 (0.45 ng/min) also caused the AHR in bronchus-lung preparations. These results suggest that OKY-046.HCl can inhibit PAF-induced AHR by suppressing the generation of TXA2. We also supposed that TXA2 is released from lung parenchyma, airway epithelium and cell components in BALF.モルモットのPAF吸入誘発気道過敏性モデルにおけるthromboxane(TX)A2合成酵素阻害剤OKY-046・HClの抑制作用について検討した．その結果(1)PAF(1および10μg/ml)吸入30および60分後にacetylcholine(ACh)に対する気道反応は有意に充進し，気管支肺胞洗浄液(BALF)中のTXB2濃度も有意に上昇した，OKY-046・HCl(100mg/kg，十二指腸内投与)は気道反応の亢進とTXB2の産生を有意に抑制した．アスピリン(10mg/kg，十二指腸内投与)も気道反応の亢進を抑制した。BALF中の6-keto-prostaglandin(PG)F1αの濃度はPAF吸入により有意な影響を受けなかった．(2)PAF(10μg/ml)吸入15分後に末梢血中血小板数の減少が認められたが，血中TXB2濃度には変化が認められなかった．また吸入15分後にBALF中タンパク濃度および好中球数比の上昇が認められたが，末梢血中白血球数さらにBALF中の白血球数，細胞分画比，leukotriene(LT)C4/D4/E4濃度，alkaline phosphatase活性，N-acetyl-β-D-glucosaminidase活性およびlactate dehydrogenase活性にはPAF吸入60分後まで変化は認められなかった．(3)摘出気管支-肺灌流標本においてPAF(1μg/min)によりAChに対する気道反応の充進と灌流液中のTXB2産生の増加が有意に認められ，OKY-046・HCl(100μg/min)はこれらの反応を著明に抑制した．(4)正常モルモットのBALF中にPAF(1μg/ml)を添加するとTXB2産生は著明に増加し，OKY-046・HClは10-4Mでその増加を有意に抑制した．(5)stable TXA2(STA2，1ng/ml)吸入30分後にAChに対する気道反応の著明な充進を認めたが，2時間後には消失した．また摘出気管支-肺灌流標本においてもSTA2(0.45ng/min)によりAChに対する気道反応は充進した．以上より，PAF吸入誘発気道過敏性は，主として肺実質，上皮細胞および気管支-肺胞内細胞成分から産生されるTXA2によって発症し，OKY-046・HClはこのTXA2の産生を抑制することにより，この気道過敏性の発症を防ぐものと考えられ，OKY-046・HClは抗喘息薬として期待できる．