Akinori Iwashita

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.

Inhibitor-induced structural change of the active site of human poly(ADP-ribose) polymerase

The crystal structure of human recombinant poly(ADP‐ribose) polymerase (PARP) complexed with a potent inhibitor, FR257517, was solved at 3.0 Å resolution. The fluorophenyl part of the inhibitor induces an amazing conformational change in the active site of PARP by motion of the side chain of the amino acid, Arg878, which forms the bottom of the active site. Consequently, a corn‐shaped hydrophobic subsite, which consists of the side chains of Leu769, Ile879, Pro881, and the methylene chain of Arg878, newly emerges from the well‐known active site.

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.

Discovery of quinazolinone and quinoxaline derivatives as potent and selective poly(ADP-ribose) polymerase-1/2 inhibitors.

Two classes of quinazolinone derivatives and quinoxaline derivatives were identified as potent and selective poly(ADP‐ribose) polymerase‐1 and 2 (PARP‐1) and (PARP‐2) inhibitors, respectively. In PARP enzyme assays using recombinant PARP‐1 and PARP‐2, quinazolinone derivatives displayed relatively high selectivity for PARP‐1 and quinoxaline derivatives showed superior selectivity for PARP‐2. SBDD analysis via a combination of X‐ray structural study and homology modeling suggested distinct interactions of inhibitors with PARP‐1 and PARP‐2. These findings provide a new structural framework for the design of selective inhibitors for PARP‐1 and PARP‐2.

Antinociceptive Effects of AS1892802, a Novel Rho Kinase Inhibitor, in Rat Models of Inflammatory and Noninflammatory Arthritis

Rho kinase (ROCK) is involved in various physiological functions, including cell motility, vasoconstriction, and neurite extension. Although a functional role of ROCK in nociception in the central nervous tissue has been reported in neuropathy, the peripheral function of this protein in hyperalgesia is not known. In this study, antinociceptive effects of AS1892802 [1-[(1S)-2-hydroxy-1-phenylethyl]-3-[4-(pyridin-4-yl)phenyl]urea], a novel and highly selective ROCK inhibitor, were investigated in two rat models of arthritis. Orally administered AS1892802 exhibited potent antinociceptive effect in both an adjuvant-induced arthritis (AIA) model (inflammatory arthritis model) and a monoiodoacetate-induced arthritis (MIA) model (noninflammatory arthritis model), with an ED50 of 0.15 mg/kg (MIA model). Fasudil, a ROCK inhibitor, and tramadol were also effective in both models; however, diclofenac was effective only in the AIA model. The onset of antinociceptive effect of AS1892802 was as fast as those of tramadol and diclofenac. AS1892802 did not induce gastric irritation or abnormal behavior. Because AS1892802 rarely penetrates the central nervous tissue and is also effective by intra-articular administration, it seemed to function peripherally. These results suggest that AS1892802 has an attractive analgesic profile for the treatment of severe osteoarthritis pain.

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.

A novel adenosine A1 and A2A receptor antagonist ASP5854 ameliorates motor impairment in MPTP-treated marmosets: Comparison with existing anti-Parkinson's disease drugs

Recent evidence indicates that adenosine A(2A) receptor antagonists hold therapeutic potential for the treatment of Parkinsons disease (PD). A study on the novel adenosine A(1) and A(2A) receptor dual antagonist 5-[5-amino-3-(4-fluorophenyl)pyrazin-2-yl]-1-isopropylpyridine-2(1H)-one (ASP5854) showed it to be effective in various rodents models of PD and cognition. In the present study, we further investigated the potential of ASP5854 as an anti-PD drug using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated common marmosets, which is a highly predictive model of clinical efficacy in PD, and compared its effect with those of existing anti-PD drugs. ASP5854 significantly and dose-dependently improved the total motor disability score for 7h at doses higher than 1mg/kg, and significantly increased total locomotor activity at doses higher than 0.1mg/kg without adverse effects. l-3,4-Dihydroxyphenylalanine+benserazide and bromocriptine also significantly improved the motor disability score and the hypolocomotion caused by MPTP treatment in a dose-dependent fashion. This amelioration was significant at 32+8 and 10-32 mg/kg, respectively, although bromocriptine induced severe emesis. Trihexiphenidyl also significantly improved the total motor disability score at doses of 10-32 mg/kg; however, while a significant increase in the total locomotor activity was observed at 10mg/kg, the drug induced ataxia-like behavior at 32 mg/kg. On the other hand, neither selegiline nor amantadine improved the total motor disability and hypolocomotion. These data substantiate the evidence that the novel adenosine antagonist ASP5854 exerts comparable anti-PD activity with existing anti-PD drugs, which indicates that ASP5854 might have potential to ameliorate motor deficits in PD.

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.