Kayoko Mihara

Neuroprotective Efficacy of the Peroxisome Proliferator-Activated Receptor δ-Selective Agonists in Vitro and in Vivo

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily and function as ligand-modulated transcription factors that regulate gene expression in many important biological processes. The PPARδ subtype has the highest expression in the brain and is postulated to play a major role in neuronal cell function; however, the precise physiological roles of this receptor remain to be elucidated. Herein, we show that the high-affinity PPARδ agonists L-165041 [4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)-propoxyl]phenoxy]-acetic acid] and GW501516 [2-methyl4-((4-methyl-2-(4-trifluoromethylphenyl)-1,3-triazol-5-yl)-methylsulfanyl)phenoxy acetic acid] protect against cytotoxin-induced SH-SY5Y cell injury in vitro and both ischemic brain injury and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in vivo. In the SH-SY5Y studies, treatment with L-165041 or GW501516 significantly and concentration-dependently attenuated cell death following thapsigargin, 1-methyl-4-phenylpyridinium, or staurosporine exposure, with the extent of damage correlated with the level of caspase-3 inhibition. In the transient (90 min) middle cerebral artery occlusion model of ischemic brain injury in rats, i.c.v. infusion of L-165041 or GW501516 significantly attenuated the ischemic brain damage measured 24 h after reperfusion. Moreover, the PPARδ agonists also significantly attenuated MPTP-induced depletion of striatal dopamine and related metabolite contents in mouse brain. These results demonstrate that subtype-selective PPARδ agonists possess antiapoptotic properties in vitro, which may underlie their potential neuroprotective potential in in vivo experimental models of cerebral ischemia and Parkinsons disease (PD). These findings suggest that PPARδ agonists could be useful tools for understanding the role of PPARδ in other neurodegenerative disorders, as well as attractive therapeutic candidates for stroke and neurodegenerative diseases such as PD.

Pharmacological Characterization of a Novel, Potent Adenosine A1 and A2A Receptor Dual Antagonist, 5-[5-Amino-3-(4-fluorophenyl)pyrazin-2-yl]-1-isopropylpyridine-2(1H)-one (ASP5854), in Models of Parkinson's Disease and Cognition

Central adenosine A2A receptor is a promising target for drugs to treat Parkinsons disease (PD), and the central blockade of adenosine A1 receptor improves cognitive function. In the present study, we investigated the effect of a novel adenosine A1 and A2A dual antagonist, 5-[5-amino-3-(4-fluorophenyl) pyrazin-2-yl]-1-isopropylpyridine-2(1H)-one (ASP5854), in animal models of PD and cognition. The binding affinities of ASP5854 for human A1 and A2A receptors were 9.03 and 1.76 nM, respectively, with higher specificity and no species differences. ASP5854 also showed antagonistic action on A1 and A2A agonist-induced increases of intracellular Ca2+ concentration. ASP5854 ameliorated A2A agonist 2-[p-(2-carboxyethyl) phenethylamino]-5′-N-ethylcarboxamidoadenosine (CGS21680)- and haloperidol-induced catalepsy in mice, with the minimum effective doses of 0.32 and 0.1 mg/kg, respectively, and it also improved haloperidol-induced catalepsy in rats at doses higher than 0.1 mg/kg. In unilateral 6-hydroxydopamine-lesioned rats, ASP5854 significantly potentiated l-dihydroxyphenylalanine (l-DOPA)-induced rotational behavior at doses higher than 0.032 mg/kg. ASP5854 also significantly restored the striatal dopamine content reduced by 1-metyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment in mice at doses higher than 0.1 mg/kg. Furthermore, in the rat passive avoidance test, ASP5854 significantly reversed the scopolamine-induced memory deficits, whereas the specific adenosine A2A antagonist 8-((E)-2-(3,4-dimethoxyphenyl)ethenyl)-1,3-diethyl-7-methyl-3,7-dihydro-1H-purine-2,6-dione (KW-6002; istradefylline) did not. Scopolamine- or 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate) (MK-801)-induced impairment of spontaneous alternation in the mouse Y-maze test was ameliorated by ASP5854, whereas KW-6002 did not exert improvement at therapeutically relevant dosages. These results demonstrate that the novel, selective, and orally active dual adenosine A1 and A2A receptors antagonist ASP5854 improves motor impairments, is neuroprotective via A2A antagonism, and also enhances cognitive function through A1 antagonism.

Brain Adenosine A2A Receptor Occupancy by a Novel A1/A2A Receptor Antagonist, ASP5854, in Rhesus Monkeys: Relationship to Anticataleptic Effect

The purpose of the present study was to measure adenosine A2A receptor (A2AR) occupancy in the brain by a novel adenosine A1/A2A antagonist, 5-[5-amino-3-(4fluorophenyl)pyrazin-2-yl]-1-isopropylpyridine-2(1H)-one (ASP5854), and to determine the degree of receptor occupancy necessary to inhibit haloperidol-induced catalepsy in rhesus monkeys. Methods: A2AR occupancy by ASP5854 (0.001–0.1 mg/kg) was examined in the striatum using an A2AR-specific radiotracer, 11C-SCH442416, and PET in conscious rhesus monkeys. A2AR occupancy was monitored after a single intravenous administration of ASP5854 in 3 animals, and a dynamic PET scan was performed at 1, 4, and 8 h after an intravenous bolus injection of the tracer for approximately 740 MBq. Catalepsy was induced by haloperidol (0.03 mg/kg, intramuscularly) and examined for incidence and duration. Results: ASP5854 dose-dependently increased A2AR occupancy in the striatum and showed long-lasting occupancy even after the reduction of plasma concentration. Haloperidol induced severe catalepsy at 40 min after intramuscular injection. The incidence and duration of cataleptic posture were dose-dependently reduced by ASP5854 at 1 h after oral administration, and the minimum ED50 value was 0.1 mg/kg. Administration of a dose of 0.1 mg/kg yielded a plasma concentration of 97 ± 16.3 ng/mL, which corresponded to 85%–90% of A2AR occupancy. Conclusion: These results showed that ASP5854 antagonized A2AR in the striatum, and the dissociation from A2AR was relatively slow. In addition, more than 85% A2AR occupancy by ASP5854 resulted in an inhibition of haloperidol-induced catalepsy. Thus, such a pharmacodynamic study directly demonstrates both the kinetics of a drug in the brain and the relationship between dose-dependent receptor occupancy and plasma level.

FK419, a nonpeptide platelet glycoprotein IIb/IIIa antagonist, ameliorates brain infarction associated with thrombotic focal cerebral ischemia in monkeys: comparison with tissue plasminogen activator.

The binding of platelet glycoprotein (GP) IIb/IIIa to fibrinogen is the final common pathway in platelet aggregation, a process known to play a key role in the pathogenesis of ischemic brain damage. We compared the effects of FK419, a novel nonpeptide GPIIb/IIIa antagonist, with recombinant tissue plasminogen activator (rt-PA) on middle cerebral artery (MCA) patency and ischemic brain damage in a thrombotic stroke model in squirrel monkeys. FK419 not only inhibited in vitro platelet aggregation (IC50: 88 nmol/L), but also showed disaggregatory activity to aggregated platelet (EC50: 286 nmol/L). FK419 dose-dependently reduced the time to first reperfusion and total occlusion time of MCA blood flow when administered immediately after the termination of photoirradiation. FK419 reduced cerebral infarction and ameliorated neurologic deficits with similar dose-dependency. Although rt-PA reduced the time to first reperfusion, total occlusion time, and cerebral infarction, it did not significantly ameliorate neurologic deficits and induced petechial intracerebral hemorrhages. These results indicate: (1) FK419 restored cerebral blood flow after thrombotic occlusion of MCA, (2) FK419 reduced ischemic brain injury by its thrombolytic actions in a non-human primate stroke model, and (3) FK419 has superior antithrombotic efficacy and is safer than rt-PA.

An application of a new planar positron imaging system (PPIS) in a small animal: MPTP-induced parkinsonism in mouse

ObjectiveRecent animal PET research has led to the development of PET scanners for small animals. A planar positron imaging system (PPIS) was newly developed to study physiological function in small animals and plants in recent years. To examine the usefulness of PPIS for functional study in small animals, we examined dopaminergic images of mouse striata in MPTP-induced parkinsonism.MethodsMale C57BL/6NCrj mice were treated with MPTP 7 days before the PPIS study. Scans were performed to measure dopamine D1 receptor binding and dopamine transporter availability with [11C]SCH23390 (about 2 MBq) and [su11C]β -CFT (about 2 MBq), respectively. After the PPIS study, dopamine content in the striatum was measured by HPLC.ResultsThe MPTP treatment significantly reduced dopamine content in the striatum 7 days after treatment. In the MPTP-treated group, [11C]β -CFT binding in the striatum was significantly decreased compared with the control group, while striatal [11C]SCH23390 binding was not affected. Dopamine content in the striatum was significantly correlated with the striatal binding of [11C]β -CFT.ConclusionThe present results suggest that PPIS is able to determine brain function in a small animal. Using PPIS, high throughput imaging of small animal brain functions could be achieved.

FK419, a novel nonpeptide GPIIb/IIIa antagonist, restores microvascular patency and improves outcome in the guinea-pig middle cerebral artery thrombotic occlusion model: comparison with tirofiban

The antithrombotic efficacy of FK419, a novel nonpeptide platelet glycoprotein IIb/IIIa antagonist, was compared with tirofiban in guinea-pigs. FK419 and tirofiban similarly inhibited platelet aggregation in vitro (IC50 values: 0.43 ± 0.076 and 0.41 ± 0.053 μmol/L) and dispersed aggregated platelets (EC50 values: 2.3 ± 0.88 and 2.0 ± 0.81 μmol/L). FK419 inhibited retention of platelets and neutrophils in a collagen-coated bead column with greater potency than tirofiban (IC50 values of 0.90 ± 0.133 and 2.4 ± 0.21 μmol/L for platelet retention and 0.32 ± 0.078 and 0.57 ± 0.180 μmol/L for neutrophil retention). When FK419 or tirofiban were administered after photochemically induced middle cerebral artery (MCA) occlusion in guinea-pigs, they dose-dependently improved MCA patency. FK419 reduced neurological deficits and ischemic brain damage in a dose-dependent fashion, whereas tirofiban did not. Reduced regional cerebral blood flow in the striatum gradually returned to the preoccluded level with FK419 treatment; however, no restoration was observed with tirofiban even though the MCA was recanalized. These results indicate that FK419 ameliorates ischemic brain damage by not only lysing the obstructive thrombus in MCA but also preventing or restoring microcirculation deficits after occlusion/reperfusion, suggesting that FK419 would be an attractive intervention for the treatment of ischemic stroke patients.