Yoshiharu Takiguchi

A simple and reproducible cerebral thrombosis model in rats induced by a photochemical reaction and the effect of a plasminogen-plasminogen activator chimera in this model

In this study a new model of cerebral ischemia, based on a middle cerebral artery (MCA) thrombosis in rats is described. Furthermore, the effect of the novel plasminogen activator (SUN9216), a plasminogen-plasminogen activator chimera, comprising the fibrin kringle 1 domain of a plasminogen, and the two kringles, and the serine protease domains of wild-type tissue plasminogen activator (t-PA), including a modification of the mannose glycosylation site on the kringle 1 of t-PA (PK1de1FE1X), was studied in this model. In the newly described model of thrombotic cerebral ischemia, an occlusive thrombus occurred usually within 8 min in the MCA as a consequence of an endothelial injury subsequent to a photochemical reaction between a systemically administered photosensitive dye (rose bengal) and a transillumination of the MCA with a high-intensity green light with a wavelength of 540 nm. The study was quantitated by means of pathological examination of the MCA and the brain. A platelet-rich thrombus was observed in the MCA using electron microscopical analysis based on ion beam bombardment. At 24 hr after induction of the thrombus, the brain was removed from 13 control animals, nine coronal sections were stained from each brain with triphenyltetrazoliumchloride (TTC), and the ischemic area was quantitated. A constant area of infarction was observed in the cortex and the lateral part of the basal ganglia. In a second group (n = 8), at 1 or 8 weeks after induction of the thrombosis in the MCA, the coronal sections were stained with hematoxylin and eosin.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in Vascular Reactivity in Experimental Diabetic Rats: Comparison with Hypothyroid Rats

The responsiveness to vasoactive agents in the perfused mesenteric vascular bed of streptozocin-induced diabetic rats was examined and compared with that of propylthiouracil-induced hypothyroid rats. Diabetic rats at 4 and 8 weeks after the induction of diabetes showed a significant decrease in isoproterenol-induced vasodilatation. In addition, the contractile responses to norepinephrine and 5-hydroxytryptamine and the vasodilative response to acetylcholine were significantly decreased in 12-week-diabetic rats. The contractile response to nerve stimulation was markedly decreased at 8 and 12 weeks. On the other hand, hypothyroid rats showed a decreased response to isoproterenol, but they did not show any change in the response to nerve stimulation. A decrease in plasma thyroid hormone levels in diabetic rats at any time period was similar in extent to that in hypothyroid rats. The data indicate that the progressive changes in vascular reactivity in diabetic rats may be divided into two stages. In the early stage, the altered reactivity of vasculature is likely to be mediated by hypothyroidism, whereas in the later stage, it is induced by other factors, e.g. hyperglycemia and hypoinsulinemia. Adrenergic neuropathy is not caused by hypothyroidism.

Carbapenem antibiotics inhibit valproic acid transport in Caco-2 cell monolayers

The concomitant use of carbapenem antibiotics with valproic acid has been prohibited because carbapenems induced a decrease in plasma concentration of valproic acid in epileptic patients during valproic acid therapy. Our previous in vivo study in rats proposed that inhibition by carbapenem of the intestinal absorption of valproic acid might be a possible mechanism for the drug-drug interaction. To demonstrate the hypothesis, we examined the effects of imipenem and panipenem on intestinal transepithelial transport of valproic acid using Caco-2 cell monolayers. Imipenem and panipenem inhibited the transport of [14C]-valproic acid across the Caco-2 cell monolayers from apical-to-basolateral side in a concentration-dependent manner, although they had no effect on the uptake of [14C]-valproic acid by Caco-2 cells. The inhibition by the carbapenems of the valproic acid transport was found even when they were added to only the basolateral side. From these results, the carbapenems may inhibit the absorption of valproic acid at the basolateral membrane of intestinal epithelial cells, which contributes to the decrease in plasma concentration of valproic acid after oral administration.

Inhibition by carbapenem antibiotic imipenem of intestinal absorption of valproic acid in rats

The concomitant use of carbapenem antibiotics with valproic acid has been prohibited because panipenem induced a decrease in plasma concentration of valproic acid in epileptic patients during valproic acid therapy. To clarify the possible mechanism of the carbapenem‐valproic acid interaction, we investigated the effect of imipenem on the pharmacokinetic behaviour of valproic acid in rats. Co‐administration of imipenem (30 mg kg−1, i.v.) induced a decrease in the peak plasma concentration of valproic acid after oral administration. However, the imipenem‐induced decrease in plasma concentrations of valproic acid was not observed within 60 min after intravenous injection of valproic acid. By utilizing in‐situ vascular and luminal perfused small intestine, it was confirmed that absorption of valproic acid from the luminal to the vascular perfusate was decreased in the presence of imipenem (0.5 mM) in the vascular perfusate. The everted gut sac method was used to determine the effect of imipenem on active transport of valproic acid. The accumulation of valproic acid on the serosal side of the intestinal sac against the concentration gradient was reduced by lactic acid that inhibits the carrier‐mediated transport of valproic acid across the intestinal brush‐border membrane. However, imipenem did not affect the active transport of valproic acid. Therefore, the inhibition by imipenem of valproic acid absorption may be caused by a mechanism different from that of lactic acid. In conclusion, imipenem inhibits the intestinal absorption of valproic acid, which contributes to the decrease in plasma concentration of valproic acid after oral administration.

Reversal Effect of Thyroxine on Altered Vascular Reactivity in Diabetic Rats

The influence of thyroxine treatment on the altered reactivity of the isolated perfused mesenteric vasculature from streptozocin-induced diabetic rats was examined and compared with that of insulin. After 8 weeks of diabetes, the time when the decreased response to isoproterenol appeared, treatment with thyroxine reversed this decreased response to control levels. However, thyroxine replacement did not reverse the decreased responsiveness to norepinephrine, 5-hydroxytryptamine, acetylcholine, and isoproterenol after 12 weeks of diabetes. On the other hand, insulin replacement improved the vascular responsiveness to these agonists at 8 and 12 weeks. Insulin treatment also reversed the attenuated response to nerve stimulation found in diabetic rats, whereas thyroxine treatment did not improve it. Insulin treatment reversed the decreased plasma thyroid hormone levels similarly as thyroxine treatment. These results suggest that thyroid hormone deficiency is likely to be involved partly in the altered reactivity of the rat mesenteric vasculature at the early period of diabetes. On the other hand, adrenergic neuropathy is not induced by hypothyroidism.

Phase I Clinical Studies of 7432-S, a New Oral Cephalosporin: Safety and Pharmacokinetics

Phase I clinical studies of 7432‐S, a new oral cephalosporin, including a randomized placebo‐controlled trial were conducted with 40 healthy volunteers, in single‐dose studies, 7432‐S was orally administered at doses of 25, 50, 100, and 200 mg. The mean plasma levels peaked at 2.1 to 3.0 hours and reached 1.9, 3.6, 5.6, and 11.6 μg/ml, respectively. Linear correlation was observed between plasma AUC values and doses given. The half‐lives of the plasma levels were 0.88 to 2.26 hours with a mean of 1.53 ± 0.33 hours. The mean urinary recoveries were 67.5 to 75.2% of the dose within 24 hours. 7432‐S was partially metabolized to 7432‐S‐trans which was excreted in urine at 7.2 to 9.2% of the doses. Study of the meal effect showed that AUC values and peak levels were not altered although the time to the peak levels was slightly prolonged, In multiple‐dose studies, 100 mg of 7432‐S twice daily for 2 weeks and 200 mg twice daily for 1 week were administered and there was no abnormal accumulation of 7432‐S in plasma throughout the study. No significant differences were observed in plasma levels and urinary recoveries between single‐ and multiple‐dose regimens. Clinical symptoms, physical tests, laboratory parameters, and fecal levels of vitamins K1 and K2 were in normal ranges. 7432‐S was concluded to be safe and well tolerated.

Roles of platelet-activating factor, thromboxane A2, ADP and thrombin in thrombogenesis in the guinea pig

The mediators of photochemically induced thrombosis in the femoral artery of guinea pig were investigated. The femoral artery was occluded by a thrombus about 7 min after the initiation of photochemical reaction between rose bengal and green light. Pretreatment with a specific thromboxane (TX) A2 receptor antagonist, vapiprost, a platelet-activating factor (PAF) antagonist, WEB-2086, and ADP-induced platelet aggregation inhibitor, ticlopidine, prolonged the time to occlusion. Within the range of doses used, platelet aggregation in whole blood, which was induced by U-46619, PAF and ADP ex vivo, was inhibited by vapiprost, WEB-2086 and ticlopidine, respectively. In contrast, argatroban, a thrombin inhibitor, had no effect on the time to occlusion, although the dose of argatroban was sufficient to delay the prothrombin time and the activated partial thromboplastin time and to inhibit thrombin-induced platelet aggregation ex vivo. These results suggest that TXA2, PAF and ADP are involved in photochemically induced thrombosis of the guinea pig femoral artery, although the coagulation cascade does not play an important role.

Arterial thrombosis model with photochemical reaction in guinea-pig and its property.

We have already developed an arterial thrombosis model in the rat femoral artery which utilized photochemical reaction between systemically injected rose bengal and transillumination of a green light with 540 nm wave length from the outside of the vessel. In the present study, we applied this model to guinea-pigs in order to produce a more suitable thrombus model for evaluation of antithrombotic drugs which act on the prostaglandin cascade. In the guinea-pigs, the irradiated femoral artery was completely occluded in 7 min after the injection of rose bengal (10 mg/kg) in a similar manner to the rats. The processes of primary endothelial injury and the subsequent formation of thrombus during this manipulation were observed by the electron microscopy. Pretreatment with aspirin and Y-20811, a thromboxane synthetase inhibitor, significantly prolonged the time required for occlusion in the guinea-pigs, while these drugs were ineffective in the rats. The antithrombotic effect of vapiprost, a thromboxane A2 receptor antagonist, was more pronounced in the guinea-pigs than the rats. In conclusion, this model in guinea-pigs is more suitable for evaluating antithrombotic drugs, particularly, the action of which is exerted involving the prostaglandin cascade.

Early administration of YT-146, an adenosine A2 receptor agonist, inhibits neointimal thickening after rat femoral artery endothelium injury

Adenosine is known to inhibit vascular smooth muscle cell proliferation in vitro via adenosine A2 receptor activation. We tested the inhibitory effect of an adenosine A2 receptor agonist, 2-octynyladenosine (YT-146), on in vivo intimal thickening following a photochemically induced injury of the endothelium of rat femoral artery. YT-146 (1 mg/kg/day) was administered s.c. for the first 3, 7 or 28 days after the injury. YT-146 significantly decreased neointimal area and the ratio of intima to medial area measured 28 days after the injury, regardless of the duration of administration. These results suggest that YT-146 may inhibit the early events of neointimal formation. The effect is long lasting and is not reversed even if YT-146 is stopped after a short course of administration.

Effects of vapiprost, a novel thromboxane receptor antagonist, on thrombus formation and vascular patency after thrombolysis by tissue-type plasminogen activator

1 A thrombus was induced in the rat femoral artery by endothelial damage due to the photochemical reaction between systemically‐injected Rose Bengal and transillumination with green light (wavelength: 540 nm). The artery of the control rat was completely occluded in 302.8 ± 27.0 s after the initiation of the reaction. 2 Pretreatment with vapiprost (0.1, 0.3 and 1.0 mg kg−1, i.v., 5 min before the reaction) prolonged the time required to occlude the femoral artery in a dose‐dependent manner. The efficacy of vapiprost on the time required for occlusion was over 10 times higher than that of aspirin which was administered 30 min before the reaction. 3 The thrombolytic effects of tissue‐type plasminogen activator (tPA) on the established arterial thrombus in the presence and absence of vapiprost were also studied in the same model. When vapiprost (0.3 mg kg−1, i.v.) was administered just before tPA infusion (100 μg kg−1 min−1 for 30 min), the time required to reperfuse the occluded artery was reduced, the incidence of the reperfusion was increased and the arterial blood flow after reperfusion was improved. 4 When vapiprost (1.0 mg kg−1 daily p.o.) was administered for 1 week after the establishment of reperfusion by tPA combined with vapiprost, the patency of the reperfused artery was improved and the femoral arterial blood flow was better preserved than after treatment with only tPA. 5 These findings suggest that this thromboxane receptor antagonist may be a useful adjunct to anti‐thrombotic therapy. The combination therapy with tPA may be more effective than treatment with tPA alone and provides greater protection against reocculsion after reperfusion.