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6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) is an essential cofactor for aromatic L-amino acid hydroxylases, including tyrosine hydroxylase (TH), alkylglycerol monooxygenase, and three types of nitric oxide (NO) synthases (NOS). Sepiapterin reductase (SPR) catalyzes the third step of BH4 biosynthesis. SPR gene-disrupted (Spr

Increasing evidence suggests the involvement of peripheral amino acid metabolism in the pathophysiology of neuropsychiatric disorders, whereas the molecular mechanisms are largely unknown. Tetrahydrobiopterin (BH4) is a cofactor for enzymes that catalyze phenylalanine metabolism, monoamine synthesis, nitric oxide production, and lipid metabolism. BH4 is synthesized from guanosine triphosphate and regenerated by quinonoid dihydropteridine reductase (QDPR), which catalyzes the reduction of quinonoid dihydrobiopterin. We analyzed Qdpr

Neopterin (NP), biopterin (BP) and monapterin (MP) exist in saliva. The physiological role of salivary NP as well as the pathophysiological role of increased NP in the immune-activated state has been unclear. Saliva is a characteristic specimen different from other body fluids. In this study, we analysed salivary NP and related pterin compounds, BP and MP and revealed some of its feature. High-performance liquid chromatography (HPLC) analysis of saliva and plasma obtained from 26 volunteers revealed that salivary NP existed mostly in its fully oxidized form. The results suggested that salivary NP as well as BP would mostly originate from the oral cavity, perhaps the salivary glands, and that salivary NP levels might not reflect those in the plasma. We also found that a gender difference existed in correlations between concentrations of salivary total concentrations of NP (tNP) and BP (tBP). HPLC analysis of saliva obtained from 5 volunteers revealed that the concentrations of salivary tNP as well as tBP fluctuated in an irregular fashion in various individuals. MP, a diastereomer of NP, might have come from oral cavity NP itself or its precursor. These results indicated that the nature of salivary NP might be different from that of NP in the blood or urine.

We evaluated the pharmacokinetics and pharmacodynamics of prasugrel used in combination with aspirin in healthy Japanese subjects. All subjects received aspirin 100 mg/day. Subsequently, in the single-administration study, 23 subjects also received prasugrel 20 mg or 30 mg; and in the multiple-administration study, 20 subjects received a loading dose of prasugrel 20 mg or 30 mg on day 1, followed by a maintenance dose of prasugrel 5 mg/day or 7.5 mg/day, respectively, on days 2–5. In both studies, the plasma concentration of the active metabolite of prasugrel, R-138727, reached a maximum at 0.5 h after administration, and rapidly decreased within 4 h. In the single-administration study, the inhibitory effect on adenosine diphosphate–induced platelet aggregation was significantly higher in the prasugrel 20 mg and 30 mg groups than in the placebo group at all time points (1–144 h) after administration. In the multiple-administration study, a similar antiplatelet effect was found after both the loading dose and the maintenance dose, and was maintained for 3–6 days after the last administration. There were study drug–related adverse events; however, all were mild, and none were clinically significant. This article is protected by copyright. All rights reserved

Objectives: According to our previous work, aripiprazole exerted a protective effect on hydrogen peroxide (H2O2)-treated PC12 cells; haloperidol did not. Because aripiprazole has distinct affinities to a set of neurotransmitter receptor subtypes, this study aimed to clarify which subtype is responsible for rescuing cells from 0.25 mM H2O2 exposure. Methods: A set of compounds, which are more specific to each subset of G-protein coupled receptors, were examined for their ability to mimic the pharmacological effects of aripiprazole or haloperidol, including their Ki values. The viability of PC12 cells cultured with test compounds with or without H2O2 was assessed using WST-8 reagent. Results: Results from in vitro studies using PC12 cells showed that agonism at serotonin 5-HT2C-receptors based on the antagonism against 5-HT2B-receptors played a significant role in resistingH2O2-induced cell death. However, the use of a specific 5-HT2B-receptor agonist instead of a 5-HT2B-receptor antagonist completely negated the effect of a specific 5-HT2C-receptor agonist. Furthermore, unlike the dopamine D1-receptor specific antagonist, none of the agonists of dopamine D2-, D3-, and D4-receptors ameliorated the cytopathic effects of H2O2. Conclusion: Antagonism at 5-HT2B-receptors is fundamental for the protection of PC12 cells against the cytopathic effects caused by 0.25 mM H2O2. However, the role of negatively regulated cyclic adenosine monophosphate in this phenomenon requires further investigation.

We previously showed that aripiprazole increases intracellular NADPH and glucose-6-phosphate dehydrogenase mRNA in PC12 cells. Aripiprazole presumably activates a system that concurrently detoxifies reactive oxygen species and replenishes NADPH. Nrf2, a master transcriptional regulator of redox homeostasis genes, also activates the pentose phosphate pathway, including NADPH production. Therefore, our aim was to determine whether aripiprazole activates Nrf2 in PC12 cells. Aripiprazole increased mRNA expression of Nrf2-dependent genes (NAD(P)H-quinone oxidoreductase-1, Nqo1; heme oxygenase-1, HO1; and glutamate-cysteine ligase catalytic subunit) and protein expression of Nqo1 and HO1 in these cells (p < 0.05). To maintain increased Nrf2 activity, it is necessary to inhibit Nrf2 degradation; this is done by causing Nrf2 to dissociate from Keap1 or β-TrCP. However, in aripiprazole-treated cells, the relative amount of Nrf2 anchored to Keap1 or β-TrCP was unaffected and Nrf2 in the nuclear fraction decreased (p < 0.05). Aripiprazole did not affect phosphorylation of Nrf2 at Ser40 and decreased the relative amount of acetylated Nrf2 (p < 0.05). The increase in Nqo1 and HO1 in aripiprazole-treated cells cannot be explained by the canonical Nrf2-degrading pathways. Further experiments are needed to determine the biochemical mechanisms underlying the aripiprazole-induced increase in these enzymes.

Von Willebrand factor (VWF) is one of the plasma protein carrying ABO(H) blood group antigens, but the combining process of these antigens is not clear. In the present study, we examined whether plasma glycosyltransferase affects the blood group antigens on VWF. VWF expressing H-antigen (H-VWF) from blood group O and bovine serum albumin conjugated with H-antigen (H-BSA) were incubated with recombinant α1-3-N-acetylgalactosaminyltransferase (rA-transferase) and A-plasma with or without an additional UDP-GalNAc. Transformed antigens were detected by western blotting and ELISA, using an anti-A antibody. Both H-VWF and H-BSA acquired the A-antigen after incubation with rA-transferase and UDP-GalNAc. Incubation with A-plasma very weakly converted the H-antigen on BSA and VWF to A-antigen only in the presence of supplemented UDP-GalNAc. This conversion was enhanced on desialylation of H-VWF. These results indicate that sugar chains of plasma VWF can be modified by the external glycosyltransferase, but that plasma glycosyltransferase has no effect on the blood group antigens of VWF due to its low activity and the lack of donor sugars. Further, sialic acid residues of VWF may exert a protective effect against post-translational glycosylation. Our results clearly exclude the possibility that blood group antigens of VWF are constructed extracellularly in plasma.

(6 R )‐l‐ erythro ‐5,6,7,8‐Tetrahydrobiopterin (BH4) is an essential cofactor for monoamine and nitric oxide (NO) production. Sepiapterin reductase (SPR) catalyzes the final step in BH4 biosynthesis. We analyzed the cardiovascular function of adult Spr gene‐disrupted ( Spr −/− ) mice for the first time. After weaning, Spr −/− mice suffered from hypertension with fluctuation and bradycardia, while the monoamine contents in these mice were less than 10% of those in the wild‐type mice as a result of BH4 depletion. Heart rate variability analysis indicated the sympathetic dominant state in Spr −/− mice. The endothelium‐dependent vascular relaxation in response to acetylcholine was significantly impaired in Spr −/− mice after sexual maturation (above 4 months old). Protein amounts of α 1 adrenergic receptor and eNOS in the aorta were not altered. Spr −/− mice exhibited hypoglycemia and elevation of plasma renin activity. Our results suggest that the hypertension with fluctuation and bradycardia of Spr −/− mice would be caused by an imbalance of sympathetic and parasympathetic input and impaired nitric oxide production in endothelial cells. We suggest an important role of BH4 and SPR in age‐related hypertension and a possible relationship with the cardiovascular instabilities in autonomic diseases, including Parkinson9s disease and spinal cord injury.

Aripiprazole is the only atypical antipsychotic drug known to cause the phosphorylation of AMP-activated protein kinase (AMPK) in PC12 cells. However, the molecular mechanisms underlying this phosphorylation in aripiprazole-treated PC12 cells have not yet been clarified. Here, using PC12 cells, we show that these cells incubated for 24 h with aripiprazole at 50 μM and 25 mM glucose underwent a decrease in their NAD⁺/NADH ratio. Aripiprazole suppressed cytochrome c oxidase (COX) activity but enhanced the activities of pyruvate dehydrogenase (PDH), citrate synthase and Complex I. The changes in enzyme activities coincided well with those in NADH, NAD⁺, and NAD⁺/NADH ratio. However, the bioenergetic peril judged by the lowered COX activity might not be accompanied by excessive occurrence of apoptotic cell death in aripiprazole-treated cells, because the mitochondrial membrane potential was not decreased, but rather increased. On the other hand, when PC12 cells were incubated for 24 h with clozapine at 50 μM and 25 mM glucose, the NAD⁺/NADH ratio did not change. Also, the COX activity was decreased; and the PDH activity was enhanced. These results suggest that aripiprazole-treated PC12 cells responded to the bioenergetic peril more effectively than the clozapine-treated ones to return the ATP biosynthesis back toward its ordinary level. This finding might be related to the fact that aripiprazole alone causes phosphorylation of AMPK in PC12 cells.

Aims: Cilostazol, a type III phosphodiesterase inhibitor, is utilized for the treatment of intermittent claudication and is considered to have the beneficial effects against the atherogenic process. In the present study, we examined the effects of cilostazol on BH4 biosynthesis in HUVEC treated with a mixture of the pro-inflammatory cytokines IFN-γ and TNF-α.Methods: Isolated HUVECs were grown to confluence and treated with IFN-γ (300 units/mL) and TNF-α (300 units/mL) for 16 h in order to stimulate BH4 biosynthesis. The BH4 levels were mea-sured by HPLC. The mRNA expression of GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme of BH4 biosynthesis, and GTPCH feedback regulatory protein (GFRP) were quantified by real-time PCR. The GTPCH protein expression was assessed by western blot analysis.Results: Cilostazol significantly reduced the BH4 levels in cytokine-stimulated HUVEC. Cilostazol produced a concomitant increase in the cAMP levels in HUVEC. Cilostazol decreased the GTPCH activity as well as the expression of GTPCH mRNA and protein. 8-bromo-cAMP (8Br-cAMP), a cell-permeable cAMP analogue, did not reproduce the effects of cilostazol. Cilostazol did not affect the cytokine-induced inhibition of GFRP mRNA expression.Conclusions: We conclude that cilostazol inhibited cytokine-stimulated BH4 biosynthesis via a cAMP-independent mechanism in HUVEC. Our data indicate that cilostazol reduced GTPCH activity and did so by suppressing the GTPCH protein levels.

Postnatal development of dopaminergic system is closely related to the development of psychomotor function. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of dopamine and requires tetrahydrobiopterin (BH4) as a cofactor. To clarify the effect of partial BH4 deficiency on postnatal development of the dopaminergic system, we examined two lines of mutant mice lacking a BH4-biosynthesizing enzyme, including sepiapterin reductase knock-out (Spr(-/-)) mice and genetically rescued 6-pyruvoyltetrahydropterin synthase knock-out (DPS-Pts(-/-)) mice. We found that biopterin contents in the brains of these knock-out mice were moderately decreased from postnatal day 0 (P0) and remained constant up to P21. In contrast, the effects of BH4 deficiency on dopamine and TH protein levels were more manifested during the postnatal development. Both of dopamine and TH protein levels were greatly increased from P0 to P21 in wild-type mice but not in those mutant mice. Serotonin levels in those mutant mice were also severely suppressed after P7. Moreover, striatal TH immunoreactivity in Spr(-/-) mice showed a drop in the late developmental stage, when those mice exhibited hind-limb clasping behavior, a type of motor dysfunction. Our results demonstrate a critical role of biopterin in the augmentation of TH protein in the postnatal period. The developmental manifestation of psychomotor symptoms in BH4 deficiency might be attributable at least partially to high dependence of dopaminergic development on BH4 availability.

Inflammatory and/or autoimmune diseases like ulcerative colitis (UC) or Crohn's disease (CD) are debilitating chronic disorders that poorly respond to pharmacological interventions. Further, drug therapy has adverse effects that add to disease complications. The current thinking is that disorders like inflammatory bowel disease (IBD) reflect an over exuberant immune activation driven by cytokines including TNF-alpha. Major sources of cytokines include myeloid leukocytes (granulocytes, monocytes/macrophages), which in IBD are elevated with activation behavior and are found in vast numbers within the inflamed intestinal mucosa. Accordingly, myeloid cells should be the targets of therapy. Adacolumn is filled with cellulose acetate beads that selectively adsorb and deplete myeloid cells and a small fraction of lymphocytes (FcgammaR and complement receptors bearing cells). In one study, 20 steroid naive patients with moderate (n = 14) or severe (n = 6) UC according to Rachmilewitz despite 1.5-2.25 g/day of 5-aminosalicylic acid received 6 to 10 Adacolumn sessions at 2 sessions/week. Efficacy was assessed 1 week after the last session. The majority of patients responded to 6 sessions, 17 (85%) achieved remission. In 2 of the 3 non-responders, CAI was 8 and 12 in 1; all 3 had deep colonic ulcers at study initiation. Decreases were seen in total leukocytes (P = 0.003), % neutrophils (P = 0.003), % monocytes (P = 0.004), an increase in lymphocytes (P = 0.001), decreases in C-reactive protein (P = 0.0002), and rises in blood levels of soluble TNF-alpha receptors I (P = 0.0007), II (P = 0.0045). In a separate study, a case with very severe steroid refractory UC who received up to 11 sessions responded well and avoided colectomy. Further, myeloid cell purging with Adacolumn has been associated with the release of IL-1 receptor antagonist, suppression of TNF-alpha, IL-1beta, IL-6, IL-8, down-modulation of L-selectin and the chemokine receptor CXCR3. In conclusion, selective depletion of myeloid cells appears to induce anti-inflammatory effects and represents a non-pharmacological treatment for patients with active IBD. The treatment has a clear drug-sparing role. Changes in blood levels of inflammatory and anti-inflammatory factors are thought to contribute to the efficacy of this procedure.

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

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.

Aims The pharmacokinetics and safety profile of JTE-522, 4-(4-cyclohexyl-2 methyloxazol-5-yl)-2-fluorobenzensulphonamide, a novel selective cyclooxygenase-2 inhibitor were investigated in healthy male volunteers. Methods Initially, as a pilot study, five groups of two subjects were given oral doses of 3–100 mg of JTE-522. After safety assessment, subjects were given 150 and 200 mg of JTE-522. The effect of food-intake on the pharmacokinetics of JTE-522 at a dose of 150 mg was examined. In the multiple-dose study, subjects were given 150 mg of JTE-522 once a day for 7 days. Concentrations of unchanged JTE-522 in plasma, blood and urine were determined by high performance liquid chromatography (h.p.l.c.). Concentrations of metabolites were estimated with h.p.l.c. chromatograms and calibration curves for quantification of unchanged JTE-522. Results In the course of this study, no serious abnormality attributable to the test drug was observed, suggesting that JTE-522 was well tolerated in healthy subjects. In a single-dose study, the concentrations of JTE-522 in blood were much higher than the corresponding concentrations in plasma. JTE-522 was readily distributed to blood cells and percentage distribution into blood cells was more than 99.0%. However, the values of Cmax in blood at doses of 100, 150, 200 mg JTE-522 were 15241, 20445 ± 3918 (16333–24556), 20965 ± 3260 (17544–24386) ng ml−1, respectively. These findings suggest that JTE-522 has a high affinity for blood cells and the distribution into blood cells is limited at the higher doses of over 100 mg. In a multiple dose study, pharmacokinetic parameters including t1/2 and AUC after the fourth administration were comparable with that of the seventh administration. Thus, these findings suggest the absence of accumulation on the multiple-dosing of JTE-522. Conclusions These results indicate that JTE-522 has an acceptable pharmacokinetic profile for clinical use without any serious adverse events as we verified in healthy young male volunteers.

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

Intracerebral hemorrhage is the major complication associated with antithrombotic and thrombolytic therapy. Despite efforts directed toward achieving hemorrhagic infarction, an ideal animal model of cerebral hemorrhage has not yet to be established. Using the photothrombotic technique in rabbits, we developed a model of cerebral hemorrhage by inducing cyclic flow reductions in the middle cerebral artery (MCA). Furthermore, the hemorrhage increased 4-fold after infusion of heparin at a dose prolonging activated partial thromboplastin time by about three times that of control animals. The photothrombotic occlusion of the MCA is based on a thrombosis induced by endothelial injury through singlet oxygen produced by Rose Bengal injection and green light irradiation (Acta Neuropathol. 72 (1987) 315; Acta Neuropathol. 72 (1987) 326; J. Pharmacol. Toxicol. Methods. 29 (1993) 165). Using a pulse Doppler flowmeter, spontaneous reperfusion of the MCA after the thrombotic occlusion following cyclic flow reductions was observed within 2 h in the majority of animals. This model is unusual with respect to the development of clinical stroke, because of the MCA cyclic flow reductions. Thus it is different from permanent or ischemia/reperfusion MCA occlusion in rodents and may be suitable for studying hemorrhagic risks associated with the use of antithrombotic agents.

Tirofiban is a nonpeptide tyrosine derivative that antagonises platelet glycoprotein IIb/IIIa (GP IIb/IIIa) receptors. It is one of three GP IIb/IIIa antagonists approved by the US Food and Drug Administration for the treatment of patients with acute coronary syndromes. The clinical effect of tirofiban has been shown in large studies such as PRISM (Platelet Receptor Inhibition for Ischemic Syndrome Management), PRISM-PLUS (PRISM - Patients Limited by Unstable Signs and Symptoms) and RESTORE (Randomised Efficacy Study of Tirofiban for Outcomes and Restenosis). Tirofiban is administered as an intravenous infusion. Volume of distribution ranges from 21 to 87 L, and binding to human plasma proteins is modest at 64%. Metabolism in humans is negligible, and most drug is excreted renally with systemic clearance ranging from 4.8 to 25.8 L/h. Renal function may influence the excretion of tirofiban, but concurrent disease or other drugs generally used in patients with ischaemia seem not to do so. This review updates what is known about the pharmacokinetics of tirofiban in humans, especially in comparison with the monoclonal antibody against the IIb/IIIa receptor, abciximab.

Background: Loss of adenomatous polyposis coli (APC) gene function results in constitutive activation of the canonical Wnt pathway and represents the main initiating and rate-limiting event in colorectal tumorigenesis. APC is likely to participate in a wide spectrum of biological functions via its different functional domains and is abundantly expressed in the brain as well as in peripheral tissues. However, the neuronal function of APC is poorly understood. To investigate the functional role of Apc in the central nervous system, we analyzed the neurological phenotypes of Apc(1638T/1638T) mice, which carry a targeted deletion of the 3' terminal third of Apc that does not affect Wnt signaling. Results: A series of behavioral tests revealed a working memory deficit, increased locomotor activity, reduced anxiety-related behavior, and mildly decreased social interaction in Apc(1638T/1638T) mice. Apc(1638T/1638T) mice showed abnormal morphology of the dendritic spines and impaired long-term potentiation of synaptic transmission in the hippocampal CA1 region. Moreover, Apc(1638T/1638T) mice showed abnormal dopamine and serotonin distribution in the brain. Some of these behavioral and neuronal phenotypes are related to symptoms and endophenotypes of schizophrenia. Conclusions: Our results demonstrate that the C-terminus of the Apc tumor suppressor plays a critical role in cognitive and neuropsychiatric functioning. This finding suggests a potential functional link between the C-terminus of APC and pathologies of the central nervous system.

Geniposide is one of the constituents of Gardenia fruit (Gardenia jasminoides Ellis, Rubiaceae), which has been used in traditional medicine. Although its anti-inflammatory and antithrombotic effects have been reported, the way it acts is still unclear. We have investigated the effects of geniposide and its metabolite genipin on thrombogenesis and platelet aggregation. In an in vivo model, geniposide and genipin significantly (P < 0.05) prolonged the time required for thrombotic occlusion induced by photochemical reaction in the mouse femoral artery. In an in vitro study, both geniposide and genipin inhibited collagen-induced, but did not inhibit arachidonate-induced, mouse platelet aggregation. However aspirin, a cyclooxygenase inhibitor, inhibited arachidonate-induced platelet aggregation but only partially inhibited the collagen-induced one. We also showed, by measuring PLA 2 -catalyzed arachidonic acid release, that geniposide inhibited phospholipase A 2 (PLA 2 ) activity. We conclude that geniposide showed an antithrombotic effect in vivo due to the suppression of platelet aggregation. PLA 2 inhibition by geniposide is one possible anti-platelet mechanism.

Background and Purpose — We sought to investigate the effects of EPC-K1, a free radical scavenger, on reducing heparin-produced cerebral hemorrhage in a rabbit model of middle cerebral artery (MCA) photothrombosis and to investigate whether the combination of EPC-K1 and heparin enhances neuroprotection from cerebral ischemic damage. Methods — In the heparin-alone group (n=8), heparin was administered intravenously for 24 hours, starting from 3 hours after MCA occlusion. In the EPC-K1-alone group (n=8), EPC-K1 was administered as a bolus injection (10 mg/kg) twice at 3 and 6 hours after MCA occlusion. In the combination group (n=8), EPC-K1 and heparin both were administered as in the single-drug procedures. In the vehicle group (n=10), saline were infused for 24 hours. Results — Heparin prolonged activated partial thromboplastin time by ≈3 times that of control animals. In the heparin-treated animals, the hemorrhage size was significantly increased ( P P P P P Conclusions — These data indicate that free radical formation may play a key role in intracerebral hemorrhage exacerbated by heparin treatment and that the combination of a free radical scavenger and heparin augmented neuroprotection from acute brain ischemia. The results of the present study may suggest a potential clinical approach for the treatment of acute stroke.

The advantage of platelet integrin GPIIb-IIIa receptor antagonists in the prevention of thrombotic occlusion was clearly proven in patients who underwent interventional treatment of the coronary artery, but its value in cerebral ischemia is still under investigation. The expectation of intracranial hemorrhage on strong inhibition of platelet function restricts its application in cerebral ischemia. To minimize bleeding while keeping antithrombotic activity, we have tried to find an appropriate approach using a combination of platelet integrin GPIIb-IIIa receptor antagonist and some other antithrombotic agents. The time to thrombotic occlusion was measured using a photothrombotic occlusion model of guinea pig middle cerebral artery. A platelet integrin GPIIb-IIIa receptor antagonist, ME3277 (sodium hydrogen [4-[(4,5,6,7-tetrahydrothieno [3,2-c] pyridin-2-yl) carbonylamino] acetyl-o-phenylene] dioxydiacetate), delayed occlusion time from 7.3 min in vehicle to 15.0, 20.6 and 25.9 min (P0.05) at 0.1, 0.3 and 1 mg/kg, respectively. ME3277 profoundly inhibited ex vivo platelet aggregation and the highest dose of ME3277 prolonged (3.5 folds, P0.01) the bleeding time measured in the hind paw. A thromboxane A(2) synthase inhibitor, sodium ozagrel, significantly delayed occlusion time to 19.5 min at 30 mg/kg (P0.05) while it did not affect bleeding time or platelet aggregation. ME3277 (0.1 mg/kg) in combination with 10 mg/kg sodium ozagrel synergistically delayed occlusion time (sodium ozagrel alone; 7.9 min, combination; 26.1 min, P0.05 vs. ME3277 alone). Sodium ozagrel did not affect ex vivo platelet aggregation or bleeding time when combined with 0.1 mg/kg of ME3277. This synergy was cancelled by combination with 30 mg/kg aspirin (14.7 min). A thromboxane A(2) receptor antagonist, vapiprost (0.1 mg/kg), did not enhance the antithrombotic efficacy of ME3277. These results imply that local prostacyclin production enhances the in vivo antithrombotic effect of the platelet integrin GPIIb-IIIa receptor antagonist. Therefore, the thromboxane A(2) synthase inhibitor allowed a reduction in the dose level of the platelet integrin GPIIb-IIIa receptor antagonist for cerebral thrombosis, which resulted in a reduced risk of bleeding.

We have previously reported that tranilast, an anti-allergic drug, prevented the experimental intimal thickening in the rat and mouse femoral arteries and its effect may be exerted through the inhibition of vascular smooth muscle cell proliferation. However, its inhibitory mechanism has yet to be understood. In this study, we investigated the inhibitory effect of tranilast on platelet-derived growth factor BB-homodimer (PDGF-BB) mediated signal transduction pathways in cultured human coronary artery smooth muscle cells (CASMCs). Growth responses to PDGF-BB were measured by [3H]-thymidine incorporation or cell counting. Activation of DNA synthesis and augmentation of cell proliferation stimulated by PDGF-BB in quiescent cultures of CASMCs were inhibited by tranilast in a concentration-dependent manner. Western blot analysis of lysates from CASMCs with an anti-activated mitogen-activated protein (MAP) kinase antibody revealed that tranilast (10–300 μM) inhibited MAP kinase activation by PDGF-BB in a concentration-dependent manner. Tranilast also reduced PDGF-BB-stimulated tyrosine phosphorylation of a 180 kDa band, corresponding in mass to the PDGF β-receptor, as shown by immunoblots using an anti-phosphotyrosine antibody. Receptor-binding study with [125I]-PDGF-BB on CASMCs showed that tranilast (10–1000 μM) inhibited the specific binding of PDGF-BB to cell surface receptors in a concentration-dependent manner. Scatchard analysis revealed that pretreatment with 300 μM tranilast decreased the maximum binding capacity (Bmax) from 27.6 to 18.0 fmol 106 cells−1 without affecting binding affinity (Kd∼0.15 nM), indicating a non-competitive inhibition of the receptor binding. These results suggest that the suppression of human CASMC growth by tranilast might be at least partly due to blockade of PDGF-BB-receptor binding. British Journal of Pharmacology (2000) 130, 307–314; doi:10.1038/sj.bjp.0703285

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

Glycoprotein IIb/IIIa receptors play an important role in platelet aggregation by binding fibrinogen. Drugs that antagonize this binding have been developed for the treatment of thrombotic diseases including acute coronary syndrome. These drugs are of four main types: [1] monoclonal antibody, [2] synthetized peptide, [3] non-peptide, and [4] oral antagonist. Since these drugs inhibit the final step in platelet aggregation, i.e., binding of fibrinogen to the platelet membrane, they were expected to 1) have high specificity for inhibiting platelet aggregation, 2) inhibit any kind of aggregation regardless of stimulation, 3) have high potency in aggregation suppression. As a result of large clinical trials, however, monoclonal antibody alone was revealed to be effective, but non-peptide and oral antagonists were ineffective. The possible mechanisms that lead to such inefficiency are: 1) difference in binding duration to platelets, 2) concomitant suppression of other adhesion molecules beside IIb/IIIa, 3) difference in binding site in the receptor and presence of a partial agonist effect, 4) aggregation promotion or induction of apoptosis via caspase-3 activation, 5) modified aggregation by gene polymorphism. New findings on platelet physiology through studying ways to overcome the above problems will lead to the development of new drugs and treatment.

We have developed a photochemical model of thrombotic middle cerebral artery (MCA) occlusion in the guinea pig for investigating factors contributing to the development of cerebral infarction. In this model, cyclic flow reductions (CFRs) after recanalization of the MCA are a common observation and might contribute to the development of cerebral infarction. Therefore, we sought to measure the time course of recanalization of the guinea pig MCA after the artery had been occluded by a thrombus. Thrombotic occlusion of the MCA was induced by photochemical reaction between intravenously administered rose bengal and transluminal green light for 10, 15, 20, or 30 min. After the thrombotic occlusion of MCA and subsequent spontaneous thrombolysis, blood flow in the MCA gradually recovered to preocclusion level but with frequent CFRs. The recovery of MCA blood flow or duration of CFRs was dependent on the duration of photochemical reaction (extent of endothelial injury); thus, for a 30-min photochemical reaction, CFRs were still observed 24 h after photochemical reaction. In separate experiments, we also investigated the effect of permanent occlusion of the MCA, which was induced by electrocoagulation in the vessel on cerebral infarction. The infarct volume in the permanent occlusion model was smaller than the maximum value in the thrombotic occlusion model (12.5 vs. 17.4%; P < 0.05, n = 6). CFRs may constitute an important factor contributing to the extent of cerebral infarction.

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 (P0.05) 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.

Abstract —We have established a mouse model of intimal thickening and assessed its suitability for experimental studies of intimal thickening. Neointimal formation was observed after endothelial injury by photochemical reaction between transluminal green light and systemically administered rose Bengal, which represents a nonmechanical approach to vessel wall denudation. Intimal thickening began 7 days after endothelial injury, reached a maximum after 21 days, and then remained unchanged for as long as 42 days. Furthermore, as a consequence of neointimal proliferation, the luminal area gradually decreased. The cells in the neointimal layer were identified as smooth muscle cells by immunohistochemical staining with an α-actin–specific antibody. Extracellular matrix deposition in the neointima was markedly increased beyond 14 days after injury. Smooth muscle cell proliferation, as measured by pulse labeling of 5-bromo-2′-deoxyuridine, was identified initially in the media 2 days after vessel wall denudation, with the proliferative activity’s shifting almost exclusively to the neointima within 7 days. Endothelial regeneration, as indicated by Evans blue staining, was complete within 21 days after injury. To assess the suitability of this model for experimental studies on intimal thickening, the effect of tranilast, an antiallergy drug with a broad spectrum of pharmacological actions on intimal thickening, was investigated. Tranilast (100 mg · kg −1 · d −1 PO) significantly ( P

SummaryIntimal thickening is a major complication following percutaneous transluminal coronary angioplasty, which leads to restenosis and requires reoperation. We have investigated the effect of a 5-lipoxygenase inhibitor, MK-886, a leukotriene B4 (LTB4) receptor antagonist, ONO-4057 or a LTC4 and LTD4 receptor antagonist, ONO-1078, on intimal thickening. Photochemical reaction between green light and systemically administered Rose Bengal produced intimal thickening in the rat femoral artery. Each drug was administered orally, once a day for 7 days, starting just after the endothelial injury. Both MK-886 administration, 10 mg/kg, and ONO-4057 administration, 100 mg/kg, suppressed intimal thickening level examined three weeks after endothelial injury, while similarly administered ONO-1078 did not. In cultured rat-derived smooth muscle cells, LTB4, an active metabolite of 5-lipoxygenase whose biosynthesis in air pouch exudate was suppressed by MK-886, stimulated cell migration. Based on these observations, the 5-lipoxygenase may have a key role in intimal thickening via its metabolites such as LTB4.

YM90K is a novel, selective and competitive alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor antagonist with neuroprotective properties, and it is currently under development for the intravenous treatment of stroke and other conditions of acute neuronal degeneration. The safety and pharmacokinetics of YM90K in healthy men was investigated after single doses up to 36 mg and repeated doses of 24 mg given by intravenous infusion over 3 hours. YM90K was well tolerated in healthy men and induced only mild changes in kidney function markers. Unchanged plasma drug concentration reached a near steady state during 3-hour infusion and rapidly decreased in a biphasic manner after the completion of infusion. YM90K showed linear pharmacokinetics. In the repeated-dose study, no significant differences were observed in pharmacokinetics of YM90K between the first and fifth dose. Unchanged urinary drug excretion was as high as 63.2% to 78.3% of the dose, most of which was excreted within 1 hour after the completion of infusion. YM90K is thought to be excreted mainly by renal tubular secretion. YM90K showed neither significant adverse reactions nor severe abnormalities in physical and laboratory examinations of the study participants and demonstrated safety and pharmacokinetic profiles compatible with clinical use.

Aims To investigate the pharmacokinetics and safety profile of JTP-4819, (-)-(2S)-1-benzylaminocarbonyl-[ (2S)-2-glycoloylpyrrolidinyl]-2-pyrrolidinecarboxamide, a novel specific orally active prolyl endopeptidase (PEP) inhibitor. Methods JTP-4819 was given orally to 28 healthy male volunteers at single doses of 30 mg (n=6), 60 mg (n=6), 120 mg (n=6) and placebo (n=3) and multiple doses of 60 mg three times daily (n=5) and placebo (n=2) for 7 days to investigate its safety and pharmacokinetics following a preliminary safety evaluation of 3, 10 and 30 mg doses in six healthy volunteers. With the single dose of 60 mg, a cross-over study was conducted to examine the effect of food on the bioavailability of the drug. The concentrations of JTP-4819 in plasma and urine were determined by electrospray ionization-liquid chromatography/mass spectrometry (ESI-LC/MS) method. Results In the multiple–dose study, the cholinesterase activity was gradually increased and reached above the normal range on days 4 to 8 in all five subjects given JTP-4819 and gradually returned to normal range after completion of dosing. The elevation of plasma cholinesterase activity was considered to be an action of JTP-4819, but this remains to be verified. There were no other abnormal findings in objective symptoms and laboratory findings including blood pressure, heart rate, electrocardiogram, body temperature, haematology, blood chemistry and urinalysis. The Cmax of JTP-4819 at 30, 60 and 120 mg in fasting state were 474, 887 and 1,649 ng ml−1, respectively, at 1 h after administration, and the t1/2 was about 2 h. AUC increased in proportion to the given doses. The cumulative urinary recoveries within 24 h were approximately 66%. Cmax, AUC, t1/2 and urinary recovery were not affected by food intake. In the multiple-dose study, there was no drug accumulation trend in plasma. Conclusions These results indicate that JTP-4819 has acceptable pharmacodynamic and pharmacokinetics profiles for clinical use without any serious adverse events as we verified in healthy young male volunteers.

The inhibitory effect of prostaglandin E1, which has an anti-platelet action and a vasodilating action via intracellular cyclic AMP elevation, was studied on intimal thickening in the rat femoral artery. A segment of the femoral artery was occluded by a platelet and fibrin-rich thrombus due to photochemical reaction between systemically administered Rose Bengal and transluminal green light which causes endothelial injury followed by platelet adhesion and aggregation at the site of photochemical reaction. Three weeks after endothelial injury, intimal thickening occurred at the irradiated site. Prostaglandin E1 (0.3 microgram/kg per min), administered as a continuous infusion 10 min before photochemical reaction significantly (P < 0.05) prolonged the time to occlusion of the femoral artery. In a separate experiment, prostaglandin E1 (0.3 microgram/kg per min) administered as a continuous infusion for 7 days just after endothelial injury significantly (P < 0.05) inhibited intimal thickening compared with a control group. In cultured rat-derived vascular smooth muscle cells, prostaglandin E1 produced concentration-dependent inhibition of migration and proliferation, stimulated by platelet-derived growth factor. These results suggest that prostaglandin E1 may be effective in preventing vascular restenosis after vascular surgery and angioplasty.

Tetrahydrobiopterin (BH4) is essential for the biosynthesis of dopamine, noradrenaline, and serotonin, which serve as cofactors for tyrosine hydroxylase (TH) and tryptophan hydroxylase. GTP cyclohydrolase (GCH) is the first and rate-limiting enzyme for BH4 biosynthesis. Genetic defects in an allele of the GCH gene can result in dopa-responsive dystonia due to partial BH4 deficiency. To explore the transcriptional control of the GCH gene, we analyzed the signaling pathway. Bacterial lipopolysaccharide (LPS) greatly enhanced the expression of GCH in RAW264 cells, and the induction of GCH by LPS was suppressed by treatment with either a MEK1/2 inhibitor or an inhibitor for the NF-κB pathway. Next, we analyzed two types of biopterin-deficient transgenic mice. We found that both mice exhibited motor disorders with slight differences. Dopamine and TH protein levels were markedly and concurrently increased from birth (P0) to P21 in wild-type mice, and these increases were abolished in both types of biopterin-deficient mice. Our results suggest that the developmental manifestation of psychomotor symptoms in BH4 deficiency might be attributable at least partially to the high dependence of dopaminergic development on the availability of BH4.

In aripiprazole-treated PC12 cells, we previously showed that the mitochondrial membrane potential (Δψm) was rather increased in spite of lowered cytochrome c oxidase activity. To address these inconsistent results, we focused the NADPH generation by glucose-6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme of the pentose phosphate pathway (PPP), to titrate reactive oxygen species (ROS) that results in the Δψm maintenance. G6PD may be also involved in another inconsistent result of lowered intracellular lactate level in aripiprazole-treated PC12 cells, because PPP competes glucose-6-phosphate with the glycolytic pathway, resulting in the downregulation of glycolysis. Therefore, we assayed intracellular amounts of NADPH, ROS, and the activities of the enzymes generating or consuming NADPH (G6PD, NADP(+)-dependent isocitrate dehydrogenase, NADP(+)-dependent malic enzyme, glutathione reductase, and NADPH oxidase [NOX]) and estimated glycolysis in 50 μM aripiprazole-, clozapine-, and haloperidol-treated PC12 cells. NADPH levels were enhanced only in aripiprazole-treated ones. Only haloperidol increased ROS. However, the enzyme activities did not show significant changes toward enhancing NADPH level except for the aripiprazole-induced decrease in NOX activity. Thus, the lowered NOX activity could have contributed to the aripiprazole-induced increase in the NADPH level by lowering ROS generation, resulting in maintained Δψm. Although the aforementioned assumption was invalid, the ratio of fructose-1,6-bisphosphate to fructose-6-phosphate was decreased by all antipsychotics examined. Pyruvate kinase activity was enhanced only by aripiprazole. In summary, these observations indicate that aripiprazole possibly possesses the pharmacological superiority to clozapine and haloperidol in the ROS generation and the adjustment of glycolytic pathway.

Tetrahydrobiopterin (BH4) is essential for the biosynthesis of dopamine, noradrenaline, and serotonin, which serve as cofactors for tyrosine hydroxylase (TH) and tryptophan hydroxylase. GTP cyclohydrolase (GCH) is the first and rate-limiting enzyme for BH4 biosynthesis. Genetic defects in an allele of the GCH gene can result in dopa-responsive dystonia due to partial BH4 deficiency. To explore the transcriptional control of the GCH gene, we analyzed the signaling pathway. Bacterial lipopolysaccharide (LPS) greatly enhanced the expression of GCH in RAW264 cells, and the induction of GCH by LPS was suppressed by treatment with either a MEK1/2 inhibitor or an inhibitor for the NF-κB pathway. Next, we analyzed two types of biopterin-deficient transgenic mice. We found that both mice exhibited motor disorders with slight differences. Dopamine and TH protein levels were markedly and concurrently increased from birth (P0) to P21 in wild-type mice, and these increases were abolished in both types of biopterin-deficient mice. Our results suggest that the developmental manifestation of psychomotor symptoms in BH4 deficiency might be attributable at least partially to the high dependence of dopaminergic development on the availability of BH4.

Intimal thickening in the femoral artery of spontaneously hypertensive rats (SHR) was initiated by endothelial damage induced by the photochemical reaction between green light and systemic rose bengal. This model represents a non-mechanical method of producing vessel wall denudation. Neointima formation was assessed by calculating the cross-sectional area of intima, media and lumen, using computer analysis. Tranilast (30, 100 and 300 mg/kg, p.o.), administered 2 days prior to endothelial injury, reduced intimal area by 29, 62 and 87%, respectively, compared to that of vehicle-treated controls. In cultured SHR-derived vascular smooth muscle cells, tranilast produced concentration-dependent inhibition of mitogenesis, whether stimulated by platelet-derived growth factor, basic fibroblast growth factor, insulin-like growth factor or fetal bovine serum. These results suggest that tranilast may be effective in preventing coronary restenosis.

SummaryThe inhibitory effect of a novel orally active platelet membrane glycoprotein receptor complex IIb/IIIa (GP IIb/IIIa) inhibitor, SC-54684A is studied on intimal thickening in the guinea pig femoral artery. A segment of the femoral artery was occluded by a platelet-rich thrombus induced by photochemical reaction between systemically administered Rose Bengal and transluminal green light which causes endothelial injury followed by platelet adhesion and aggregation at the site of photochemical reaction. Three weeks after successful thrombolysis by tissue-type plasminogen activator, intimal thickening occurred at the irradiated site. SC-54684A (30 mg/kg), administered 2 h before photochemical reaction, significantly (P