Hidetada Komatsu

Effects of thromboxane synthetase inhibitors on aggregation of rabbit platelets

Thromboxane (TX) synthetase activity was selectively inhibited by (E)-3-[4-(1-imidazolylmethyl)phenyl]-2-propenoic acid hydrochloride monohydrate (OKY-046) and sodium (E)-3-[4-(3-pyridylmethyl)phenyl]-2-methyl-propenoate (OKY-1581) (OKYs). Their IC50 for the rabbit platelet enzyme were found to be 11nM and 3nM respectively. Arachidonic acid (AA) or collagen induced platelet aggregation, and generated TXA 2 and prostaglandins (PGs) in rabbit platelets. OKYs inhibited platelet aggregation and TXA2 generation without affecting PGs generation, while aspirin inhibited platelet aggregation, and TXA2 and PGs generation. There was a parallel relation between the degree of inhibition of platelet aggregation and TXA2 generation by OKYs, but the inhibitory effects of aspirin on platelet aggregation was related to that on both TXA2 and PGs generation. However, OKYs and aspirin did not inhibit ADP-induced platelet aggregation which did not involve the generation of TXA2 and PGs. These results suggested that TXA2 generation is related to platelet aggregation induced by AA or collagen, and that the inhibitory effect of OKYs on platelet aggregation is due to the inhibition of TX synthetase.

Reductions in Bone Mass, Structure, and Strength in Axial and Appendicular Skeletons Associated With Increased Turnover After Ovariectomy in Mature Cynomolgus Monkeys and Preventive Effects of Clodronate

Over 16 months, we evaluated the effects of ovariectomy (OVX) and bisphosphonate clodronate (CLO) on bone in 48 cynomolgus monkeys (9‐15 years old) fed a normal calcium diet. We established three OVX groups(oral CLO at 0 [OVX control], 12, or 60 mg/kg per day) and one sham‐operated (SHAM) group. At 16 months, the bone mineral density (BMD) values (percentage of group baseline; OVX control vs. SHAM) for lumbar bone (L3‐L5), proximal femur, midfemur, radius, and tibia were −2.6% versus 11.2%, −3.5% versus 8.9%, −3.0% versus 9.0%, −5.5% versus 15.7%, and −6.7% versus 13.9%, respectively. In OVX control (i) tibia showed significant loss of bone mineral content (BMC; vs. baseline), (ii) urinary deoxypyridinoline (DPD) and serum osteocalcin (OC) levels increased (peak = 182% and 168%, respectively, of SHAM), (iii) in lumbar bone and midfemur, ultimate load (UL) was reduced (vs. SHAM), (iv) in lumbar bone, trabecular bone‐formation rates (BFRs) were not changed significantly, but tibial endocortical and intracortical bone formation rates were significantly raised (vs. SHAM), (v) the volumetric BMD (vBMD) and geometry of the tibial cortex (measured by peripheral quantitative computed tomography [pQCT]) were significantly reduced (vs. SHAM). CLO, 60 mg/kg per day but not 12 mg/kg per day, significantly inhibited OVX‐induced changes, age‐dependent increases in bone mass, and ability to maintain structure. Thus, in OVX mature cynomolgus monkeys (possibly, a unique model of the cortical bone loss secondary to estrogen deficiency), the post‐OVX increases in systemic bone markers were slight, but stimulation of local turnover in the cortical envelope was enough to cause bone loss (more so in tibia than in lumbar trabecular bone). High‐dose CLO prevented these changes.

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.

Effects of dopamine d2 receptor agonists in a pituitary transplantation-induced hyperprolactinaemia/anovulation model in rats.

1. In the present study, we investigated the effects of hyperprolactinaemia, induced by transplantation of anterior pituitary glands under the kidney capsule in female rats, on the relationship between serum and pituitary concentrations of the gonadotropins and on the oestrous cycle.

Effect of KAD-1229, a nonsulfonylurea hypoglycemic agent, on plasma glucose and insulin in streptozotocin-induced diabetic dogs.

Hypoglycemic agents with a rapid onset and short duration of action should be useful for controlling postprandial hyperglycemia. Our aim was to establish a diabetes mellitus model in dogs, and then during an oral glucose tolerance test to compare the hypoglycemic effect and insulinotropic action of KAD-1229, a new hypoglycemic agent, with that of gliclazide, a conventional sulfonylurea. In this model, KAD-1229 reduced the increase in plasma glucose level without producing hypoglycemia. Gliclazide had a weaker effect on reduction of the glucose increase and caused hypoglycemia via a significantly raised insulin secretion in the late phase. A rapid insulinotropic action of KAD-1229 was clearly observed in the portal venous blood. The results suggest that in type 2 diabetes caused by, at least, insulin deficiency, KAD-1229 may improve impaired insulin secretion in the early phase and attenuate hyperglycemia without causing a sustained hypoglycemia.

Beneficial effect of low molecular weight heparin on the hemodialysis model in dogs

The effect of FR-860, one of low molecular weight heparins, was investigated on the hemodialysis model in dogs for comparison with that of conventional unfractionated heparin (UF-heparin). In a bolus injection model, FR-860 at 12.5-100 U/kg prolonged the dialysis time (the time until the arterial circuit pressure reaching 500 mmHg) in a dose-dependent manner and UF-heparin at 50 U/kg also prolonged it. Additionally, FR-860 (12.5-50 U/kg/hr) and UF-heparin (25 and 50 U/kg/hr) continuously inhibited the rise of the arterial circuit pressure in an infusion model. In both models, the efficacy of FR-860 was more potent than that of UF-heparin. FR-860 and UF-heparin decreased the amount of blood loss remaining in the dialyzer in the infusion model. During hemodialysis, both FR-860 and UF-heparin showed activated plasma anti-F.Xa activity, prolongation of aPTT and such of thrombin time in a dose-dependent manner in those models. However, FR-860 was higher in anti-F.Xa activity and weaker in prolongation of aPTT and thrombin time than UF-heparin. These results suggest that FR-860 is more beneficial in in efficacy and higher in safety against the bleeding risk than UF-heparin hemodialysis.

Study of anticoagulant mechanism of low molecular weight heparin

The binding ability of low molecular weight heparin (FR-860), and conventional unfractionated heparin (UF-heparin) to factor Xa (F.Xa), thrombin and AT III was investigated using FR-860- and UF-heparin-Sepharoses. FR-860 could not bind directly to F.Xa. FR-860 bound to thrombin and AT III with stronger affinity to AT III than to thrombin. On the other hand, UF-heparin bound to F.Xa, thrombin and AT III with the strongest affinity to AT III followed by thrombin and F.Xa. AT III mediated the binding between F.Xa and FR-860 and accelerated the reaction between F.Xa and UF-heparin. On the other hand, AT III did not affect the binding between thrombin and FR-860 or UF-heparin. Diisopropyl fluorophosphate-treated thrombin inhibited the binding between AT III and FR-860, but not that between AT III and UF-heparin. These results suggest that the anti-F.Xa activity of FR-860 is mediated by AT III. Furthermore, the difference of antithrombin activity between FR-860 and UF-heparin depends on the capability to form ternary complex of FR-860 or UF-heparin, AT III and thrombin.

Study of low molecular weight heparin effect on the relation between anticoagulant activity and antithrombin III affinity

Low molecular weight heparin (FR-860), and conventional unfractionated heparin (UF-heparin) were fractionated by rabbit antithrombin III (AT III)-Sepharose, and the effects of each affinity fraction on the coagulation and fibrinolytic activities were investigated. FR-860 was fractionated to no-affinity, low-affinity (LA) and high-affinity (HA) fractions, and UF-heparin to LA and HA fractions. The HA fractions showed higher activities regarding the prolongation of activated partial thromboplastin time, anti-factor Xa activity and antithrombin activity compared with those of LA. The HA and LA fractions exhibited the enhancement of heparin cofactor II (HC II) activity and fibrinolytic activity in a dose-dependent manner. These results suggest that the antithrombotic activity of FR-860 is exerted through AT III and other mechanism such as HC II-mediated system.

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．