Yasuhiko Ikeda

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.

Mutanolysin, Bacteriolytic Agent for Cariogenic Streptococci: Partial Purification and Properties

Mutanolysin partially purified from the culture filtrate of Streptomyces globisporus 1829 consists of two main lytic enzymes with an isoelectric point near pH 8.5 and 10, respectively, and proteolytic enzyme is associated with the latter lytic enzyme. Mutanolysin exhibited maximal lytic activity at 60 C in the pH range 6.5 to 7.0 and was stable at 50 C in the acid range. N-bromosuccinimide caused complete inhibition of lytic activity at 1 mM, whereas calcium and magnesium ions at the same concentration caused activation. Mutanolysin had lytic or bactericidal activity against the living cells of Streptococcus mutans, Streptococcus salivarius, Streptococcus sanguis, Lactobacillus acidophilus, and Actinomyces viscosus, which are considered to be etiologic agents of dental caries, but had no activity against S. aureus and all gram-negative strains tested. The lytic activity was well retained in human saliva. Digestion of the cell walls of S. mutans BHT by mutanolysin was accompanied by the liberation of free amino groups and reducing sugars. Mutanolysin may be expected to be a useful agent for dental caries control. Images

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.

Rosuvastatin, a new HMG-CoA reductase inhibitor, protects ischemic reperfused myocardium in normocholesterolemic rats

The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been shown to upregulate endothelial nitric oxide synthase in isolated endothelial cells in a manner that is independent of their lipid-lowering effects. Nitric oxide inhibits polymorphonuclear leukocyte (PMN) adherence and attenuates cardiac dysfunction caused by PMNs after ischemia/reperfusion. Therefore, the authors hypothesized that a new statin, rosuvastatin, could attenuate PMN-induced cardiac dysfunction, and examined the effects of rosuvastatin in isolated ischemic (20 min) and reperfused (45 min) rat hearts perfused with PMNs. Rosuvastatin (0.25 or 1.25 mg/kg) given 18 h before ischemia/reperfusion significantly improved left ventricular developed pressure (P < 0.01) and the maximal rate of development of left ventricular developed pressure (+dP/dtmax, P < 0.01) compared with ischemia/reperfused hearts obtained from rats given 0.9% NaCl. The time point for the improved cardiac performance caused by rosuvastatin (1.25 mg/kg) was 20 min after reperfusion. In addition, rosuvastatin significantly reduced PMN adherence to the vascular endothelium and subsequent infiltration into the postischemic myocardium (P < 0.01). The nitric oxide synthase inhibitor Nω-nitro-l-arginine methyl ester (50 &mgr;mol/l) blocked these cardioprotective effects. These results provide evidence that rosuvastatin significantly attenuates PMN-induced cardiac contractile dysfunction in the isolated perfused rat heart.

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.

Down syndrome and GATA1 mutations in transient abnormal myeloproliferative disorder: mutation classes correlate with progression to myeloid leukemia

Twenty percent to 30% of transient abnormal myelopoiesis (TAM) observed in newborns with Down syndrome (DS) develop myeloid leukemia of DS (ML-DS). Most cases of TAM carry somatic GATA1 mutations resulting in the exclusive expression of a truncated protein (GATA1s). However, there are no reports on the expression levels of GATA1s in TAM blasts, and the risk factors for the progression to ML-DS are unidentified. To test whether the spectrum of transcripts derived from the mutant GATA1 genes affects the expression levels, we classified the mutations according to the types of transcripts, and investigated the modalities of expression by in vitro transfection experiments using GATA1 expression constructs harboring mutations. We show here that the mutations affected the amount of mutant protein. Based on our estimates of GATA1s protein expression, the mutations were classified into GATA1s high and low groups. Phenotypic analyses of 66 TAM patients with GATA1 mutations revealed that GATA1s low mutations were significantly associated with a risk of progression to ML-DS (P < .001) and lower white blood cell counts (P = .004). Our study indicates that quantitative differences in mutant protein levels have significant effects on the phenotype of TAM and warrants further investigation in a prospective study.

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.

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.