Kazunao Kondo

Pharmacokinetics of Voriconazole and Cytochrome p450 2C19 Genetic Status

gested and that possibly could be suitable for drugs eliminated by biliary excretion. Even though a specific classification is used for including subjects in the study, evaluation of data and dose recommendations may very well be based on relevant individual laboratory parameters (albumin levels, prothrombin time, bilirubin levels, alkaline phosphatase levels, fibrinogen levels, and so on). Sponsors and regulatory authorities also need to consider that the characteristics of the classification systems are not well known by the prescribers. In conclusion, we have presented several arguments disqualifying the Maddrey function as a general marker for hepatic function in pharmacokinetic trials. An inappropriate and routinelike classification of hepatic impairment may result in the selection of a study population that does not reflect the effects of reduced elimination capacity and a study with a low predictive value for patients at risk. Clearly, there is a need for further development with respect to what hepatic marker or classification systems to use in pharmacokinetic hepatic impairment studies. The pharmaceutical industry is encouraged to implement its knowledge of the mechanism of drug elimination into the design of the studies and to further explore hepatic markers that may be used for characterizing the pharmacokinetic profile of drugs dependent on hepatic function for their elimination.

Inhibitory effects of total flavones of Hippophae Rhamnoides L on thrombosis in mouse femoral artery and in vitro platelet aggregation.

Total flavones of Hippophae Rhamnoides L (TFH) are extracted from Sea buckthorn, a Chinese herbal medicine. Sea buckthorn has antioxidant, anti-ulcerogenic and hepato-protective actions, and its berry oil is reported to suppress platelet aggregation. Though it is frequently used for patients with thrombosis, the likely mechanism(s) and effects of TFH on thrombogenesis remain unclear. Thus, we have investigated the effect in-vivo of TFH on thrombogenesis and in vitro on platelet aggregation, comparing them to those of aspirin. We measured thrombotic occlusion time in a mouse femoral artery thrombosis model by the photochemical reaction between intravenously injected rose bengal and green light irradiation. In vitro platelet aggregation in whole blood was measured by single platelet counting. Thrombotic occlusion time was 8.5 +/- 0.6 min in the control group. TFH at a dose of 300 micro g/kg, intravenously administered 15 min before the rose bengal injection, significantly prolonged it to 11.6 +/- 1.0 min (P < 0.05), a similar effect on in-vivo thrombogenesis to that of aspirin. TFH at a concentration of 3.0 micro g/ml significantly (P < 0.01) inhibited in vitro platelet aggregation induced by collagen (2 micro g/ml) in a concentration dependent manner, in contrast TFH did not affect aggregation induced by arachidonic acid (80 micro M) and ADP (0.3 micro M). The results of the present study, in which TFH prevented in-vivo thrombogenesis, probably due to inhibition of platelet aggregation, suggest a possible clinical approach for the prevention of thrombosis.

Neutrophil elastase inhibition reduces cerebral ischemic damage in the middle cerebral artery occlusion

It has been reported that activated neutrophils are involved in the development of cerebral damage induced by ischemia. Activated neutrophils release a lot of mediators including toxic oxygen metabolites, elastase and cytokines which damage brain tissue. Therefore, we investigated roles of neutrophil elastase in the development of cerebral damage using an elastase inhibitor, ONO-5046. The rat middle cerebral artery (MCA) was occluded by a thrombus induced by photochemical reaction between green light and the photosensitizer dye, Rose Bengal. Photochemical reaction causes endothelial injury followed by formation of a platelet and fibrin-rich thrombus at the site of the irradiation. Photochemical reaction is routinely used in our laboratory to produce arterial occlusion in experimental animals. Twenty-four hours after the MCA occlusion, the size of cerebral damage was measured by histochemical technique. Water content in the brain was measured and neuronal deficits were examined 24 h after the MCA occlusion. ONO-5046 was administered at various doses as continuous infusion for 24 h, starting just after the MCA occlusion or from 3 h after. ONO-5046 at doses of 10 and 30 mg/kg/h significantly (p<0.05 and p<0.01, respectively) reduced the size of cerebral damage and water content (p<0.05, p<0.01, respectively) in different eight rats. Further, ONO-5046 at a dose of 30 mg/kg/h significantly (p=0.01) improved neuronal deficits. ONO-5046 which was administered starting from 3 h after the MCA occlusion, also reduced the size of cerebral damage. Neutropenia by anti-neutrophil antibody injection significantly (p<0. 01) reduced the size of cerebral damage. Elastase released from activated neutrophils may play a key role in the development of cerebral damage.

Dietary supplementation of fermented soybean, natto, suppresses intimal thickening and modulates the lysis of mural thrombi after endothelial injury in rat femoral artery.

We have previously demonstrated that natto-extracts containing nattokinase (NK) inactivates plasminogen activator inhibitor type 1 and then potentiates fibrinolytic activity. In the present study, we investigated the effects of dietary supplementation with natto-extracts on neointima formation and on thrombolysis at the site of endothelial injury. Endothelial damage in the rat femoral artery was induced by intravenous injection of rose bengal followed by focal irradiation by transluminal green light. Dietary natto-extracts supplementation containing NK of 50 or 100 CU/body was started 3 weeks before endothelial injury and then continued for another 3 weeks. Intimal thickening in animals given supplementation was significantly (P<0.01) suppressed compared with controls and the intima/media ratio in animals with 50 and 100 CU/body NK and control group was 0.09 +/- 0.03, 0.09 +/- 0.06 and 0.16 +/- 0.12, respectively. Although femoral arteries were reopened both in control animals and those treated with NK within 8 hours after endothelial injury, mural thrombi were histologically observed at the site of endothelial injury. In the control group, the center of vessel lumen was reopened and mural thrombi were attached on the surface of vessel walls. In contrast, in NK-treated groups, thrombi near the vessel wall showed lysis and most of them detached from the surface of vessel walls. In conclusion, dietary natto-extracts supplementation suppressed intimal thickening produced by endothelial injury in rat femoral artery. These effects may partially be attributable to NK, which showed enhanced thrombolysis near the vessel wall.

Genistein, an isoflavone included in soy, inhibits thrombotic vessel occlusion in the mouse femoral artery and in vitro platelet aggregation.

Diet can be the most important factor that influences risks for cardiovascular diseases. Genistein included in soy is one candidate that may benefit the cardiovascular system. Here, we investigated the inhibitory effects of genistein on thrombotic vessel occlusion in the mouse femoral artery using a photochemical reaction, and in vitro platelet aggregation in whole blood measured by single platelet counting. Genistein (10 mg/kg), intravenously administered 10 min before the rose bengal injection, significantly prolonged the thrombotic occlusion time from 6.1+/-0.4 to 8.4+/-0.8 min (P<0.05). Genistein at doses higher than 30 microM significantly (P<0.01) inhibited in vitro platelet aggregation induced by collagen (1 and 3 microg/ml). When 10 mg/kg genistein was intravenously administered, ex vivo platelet aggregation induced by collagen (1 and 3 microg/ml) was significantly suppressed (P<0.01). In conclusion, genistein prevented in vivo thrombogenesis and suppressed in vitro platelet aggregation. These results suggest that dietary supplementation of soy may prevent the progression of thrombosis and atherosclerosis.

Role of histamine H1 and H2 receptor antagonists in the prevention of intimal thickening

Vascular smooth muscle cell migration to the intima from the media and proliferation in the intima play key roles in atherosclerosis and restenosis after coronary angioplasty. Histamine released from adherent platelets at the injured artery and from mast cells in atheromas has stimulant actions on both smooth muscle cell migration and proliferation, and histamine receptor antagonists abolish the effect of histamine in vitro. The aim of this study was to examine the effect of histamine receptor antagonists on intimal thickening. Endothelial injury in the mouse femoral artery was induced by a photochemical reaction between localized irradiation by green light and intravenously administered rose bengal. The histamine H1 receptor antagonist, diphenhydramine, at a dose of 30 mg/kg or the histamine H2 receptor antagonist, cimetidine, at a dose of 200 mg/kg was intraperitoneally administered to mice for 21 days after endothelial injury. Twenty-one days after endothelial injury, morphometric analysis was performed to measure the cross-sectional areas of the intima and media. Diphenhydramine significantly reduced the intimal area to 1.1 +/- 0.3 (x 10(-3) mm2) compared with the value in the control group, which was 6.2 +/- 1.4 (x 10(-3) mm2), but cimetidine (5.5 +/- 1.9, x 10(-3) mm2) did not. Similarly, the ratio of intimal area to medial area in the diphenhydramine-treated group but not in the cimetidine-treated group was significantly reduced (83%). In the in vitro study, cimetidine inhibited neither proliferation nor migration of mouse vascular smooth muscle cells stimulated by platelet-derived growth factor (PDGF). In contrast, diphenhydramine significantly (P < 0.05) inhibited proliferation in a dose-dependent manner, but did not inhibit migration. These results suggest that diphenhydramine, a histamine H1 receptor antagonist, reduced the formation of intimal hyperplasia, at least in part due to inhibition of cell proliferation. However, cimetidine, a histamine H2 receptor antagonist, was ineffective. Histamine may play a key role in intimal thickening, in part via histamine H1 receptors in this model.

Neuroprotective effects depend on the model of focal ischemia following middle cerebral artery occlusion

The purpose of the present study was to compare the characteristics of the photochemical-induced thrombotic occlusion model and the thermocoagulated occlusion model of the middle cerebral artery in rats. We evaluated the neuroprotective effects of a NMDA receptor antagonist, (+)-MK-801 (dizocilpine, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptan-5,10-imine), an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist, YM90K (6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione monohydrochloride), a Ca2+ channel antagonist, S-312-d (S-(+)-methyl-4,7-dihydro-3-isobutyl-6-methyl-4-(3-nitrophenyl)-thieno[2 ,3-b]pyridine-5-carboxylate), the radical scavengers, MCI-186 (3-methyl-1-phenyl-2-pyrazolin-5-one) and EPC-K1 (L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyl-tridecyl)-2H-1-be nzopyran-6yl-hydrogen phosphate] potassium salt), and a calcineurin inhibitor, FK506 (tacrolimus, Prograf). Although all tested agents in the present study attenuated the brain damage in the photochemical-induced thrombotic occlusion model, the radical scavengers did not attenuate the brain damage in the thermocoagulated occlusion model. The time course of brain damage and brain edema formation in the two models was examined. The time course of brain damage was not different in the two models, but the time course of brain edema was quite different. Brain edema formation in the photochemical-induced thrombotic occlusion model was significantly greater (P < 0.01) than that in the thermocoagulated occlusion model at all time point studied until 24 h after occlusion of the middle cerebral artery. The present study suggests that the photochemical-induced thrombotic occlusion model has characteristics of both permanent ischemia and ischemia-reperfusion.

SM-20220, a Na(+)/H(+) exchanger inhibitor: effects on ischemic brain damage through edema and neutrophil accumulation in a rat middle cerebral artery occlusion model.

The Na(+)/H(+) exchanger (NHE) is activated during ischemia-reperfusion in an effort to restore intracellular pH to normal levels. The NHE is recognized to exist as a distinct protein in the plasma membranes of a variety of cells. We investigated the pharmacological effects of a Na(+)/H(+) exchanger inhibitor, SM-20220 (N-(aminoiminomethyl)-1-methyl-1-H-indole-2-carboxamide methanesulfonate), on ischemic brain damage, edema and neutrophil accumulation at 72 h after middle cerebral artery (MCA) occlusion in a rat MCA occlusion model. SM-20220 was intravenously administered as a bolus injection immediately after occlusion, followed by a continuous infusion over 2.5 h. At 72 h after occlusion, the infract area was measured using hematoxylin-eosin staining and, using the same slices, neutrophils in the brain were immuno-stained with anti-myeloperoxidase (n=11). In a separate study, rat behavior was scored and scaled, and brains removed for the determination of water content by the dry-weight method. SM-20220 significantly (P<0.05) attenuated cerebral infarct volume, water content, and the neutrophil accumulation at 72 h after the MCA occlusion, and ameliorated neurological deficits. SM-20220, an NHE inhibitor prevented the progress of cerebral ischemic damage and edema following MCA occlusion in rats though a possible mechanism that may be due to the inhibition of neutrophil accumulation. The NHE in neutrophils may enhance the progress of cerebral damage following cerebral ischemia-reperfusion.

Dietary supplementation with fermented soybeans suppresses intimal thickening

Although soy foods have been consumed for more than 1000 y, it is only in the past 20 y that they have made inroads into Western diets. We investigated the effect of dietary supplementation with natto extracts produced from fermented soybeans on intimal thickening of arteries after vessel endothelial denudation. Natto extracts include nattokinase, a potent fibrinolytic enzyme having four times greater fibrinolytic activity than plasmin. Intimal thickening was induced in the femoral arteries by intravenous infusion of rose bengal followed by focal irradiation with a transluminal green light. Dietary natto extract supplementation was started 3 wk before endothelial injury and continued for another 3 wk after. In ex vivo studies, euglobulin clot lysis times were measured 3 wk after the initial supplementation. Neointima formation and thickening were also initiated successfully. The intima media ratio 3 wk after endothelial injury was 0.15 +/- 0.03 in the control group. Dietary natto extract supplementation suppressed intimal thickening (0.06 +/- 0.01; P < 0.05) compared with the control group. Natto extracts shortened euglobulin clot lysis time, suggesting that their thrombolytic activities were enhanced. These findings suggest that natto extracts, because of their thrombolytic activity, suppress intimal thickening after vascular injury as a result of the inhibition of mural thrombi formation.

Hydroxyl radical generation after the third hour following ischemia contributes to brain damage

The purpose of the present study was to determine after what time period hydroxyl radical formation contributes most to ischemic brain damage in focal ischemia, using a hydroxyl radical scavenger, EPC-K1, L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyl-tridecyl)-2H-1-be nzopyran-6yl-hydrogen phosphate] potassium salt. Focal ischemia was produced by thrombotic occlusion of the left middle cerebral artery in rats. After evaluation of the pharmacokinetics of EPC-K in the brain tissue and plasma following 10 mg/kg intravenous bolus treatment of conscious rats, we investigated the neuroprotective effect of EPC-K1 in the middle cerebral artery occlusion model. A single intravenous bolus of EPC-K1 was given immediately, 3 or 6 h after ischemia, and cerebral brain damage was measured 24 h after ischemia. When EPC-K1 was injected 3 h after ischemia, a significant (P < 0.01) reduction of cerebral brain damage was observed. EPC-K1 delivered by intravenous infusion that started immediately after ischemia and lasted for 24 h, also significantly (P < 0.05) reduced brain damage, but the efficacy of the neuroprotective effect was the same as that of the 3 h after ischemia bolus treatment. These results may indicate that the period of hydroxyl radical formation most critical for ischemic brain damage is a few hours after the third hour following ischemia in this model.