Neuroprotective effects of a novel poly(ADP-ribose) polymerase-1 inhibitor, 2-[3-[4-(4-chlorophenyl)-1-piperazinyl] propyl]-4(3H)-quinazolinone (FR255595), in an in vitro model of cell death and in mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease

The massive activation of poly(ADP-ribose) polymerase-1 (PARP-1) by DNA-damaging stimuli, such as exposure to reactive oxygen species (ROS), can lead to cell injury via severe, irreversible depletion of the NAD and ATP pool, and PARP-1 inhibitors have been expected to rescue neurons from degeneration in a number of disease models. We have recently identified 2-[3-[4-(4-chlorophenyl)-1-piperazinyl] propyl]-4(3H)-quinazolinone (FR255595) as a novel and potent PARP-1 inhibitor through structure-based drug design and high-throughput screening. This compound potently inhibited PARP activity with an IC(50) value of 11 nM and was orally active and highly brain penetrable. Here, we show that prevention of PARP activation by FR255595 protects against both ROS-induced cells injury in vitro and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigrostriatal dopaminergic damage in an in vivo Parkinson's disease (PD) model. In cell death models in vitro, exposure of hydrogen peroxide induced cell death with PARP overactivation in PC12 cells and SH-SY5Y cells, and pre- and post-treatment with FR255595 (10(-9)-10(-5) M) significantly reduced PARP activation and cell death. In mouse MPTP model, MPTP (20 mg/kg i.p.) intoxication lead to PARP activation and cell damage in the nigrostriatal dopaminergic pathway, which was significantly ameliorated by oral administration of FR255595 (10-32 mg/kg), both in the substantia nigra and in the striatum via marked reduction of PARP activation, even with delayed treatment. These findings clearly indicate that the novel PARP-1 inhibitor FR255595 exerts neuroprotective effect through its potent PARP-1 inhibitory actions in PD model, suggesting that the drug could be an attractive candidate for several neurodegenerative disorders, including PD.     

A novel and potent poly(ADP-ribose) polymerase-1 inhibitor, FR247304 (5-chloro-2-[3-(4-phenyl-3,6-dihydro-1(2H)-pyridinyl)propyl]-4(3H)-quinazolinone), attenuates neuronal damage in in vitro and in vivo models of cerebral ischemia

The activation of poly(ADP-ribose) polymerase-1 (PARP-1) after exposure to nitric oxide or oxygen-free radicals can lead to cell injury via severe, irreversible depletion of NAD. Genetic deletion or pharmacological inhibition of PARP-1 attenuates brain injury after focal ischemia and neurotoxicity in several neurodegenerative models in animals. FR247304 (5-chloro-2-[3-(4-phenyl-3,6-dihydro-1(2H)-pyridinyl)propyl]-4(3H)-quinazolinone) is a novel PARP-1 inhibitor that has recently been identified through structure-based drug design. In an enzyme kinetic analysis, FR247304 exhibits potent and competitive inhibition of PARP-1 activity, with a K(i) value of 35 nM. Here, we show that prevention of PARP activation by FR247304 treatment protects against both reactive oxygen species-induced PC12 cell injury in vitro and ischemic brain injury in vivo. In cell death model, treatment with FR247304 (10(-8)-10(-5) M) significantly reduced NAD depletion by PARP-1 inhibition and attenuated cell death after hydrogen peroxide (100 microM) exposure. After 90 min of middle cerebral artery occlusion in rats, poly(ADP-ribosy)lation and NAD depletion were markedly increased in the cortex and striatum from 1 h after reperfusion. The increased poly(ADP-ribose) immunoreactivity and NAD depletion were attenuated by FR247304 (32 mg/kg i.p.) treatment, and FR247304 significantly decreased ischemic brain damage measured at 24 h after reperfusion. Whereas other PARP inhibitors such as 3-aminobenzamide and PJ34 [N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylactamide] showed similar neuroprotective actions, they were less potent in in vitro assays and less efficacious in an in vivo model compared with FR247304. These results indicate that the novel PARP-1 inhibitor FR247304 exerts its neuroprotective efficacy in in vitro and in vivo experimental models of cerebral ischemia via potent PARP-1 inhibition and also suggest that FR247304 or its derivatives could be attractive therapeutic candidates for stroke and neurodegenerative disease.     

A new poly(ADP-ribose) polymerase inhibitor, FR261529 [2-(4-chlorophenyl)-5-quinoxalinecarboxamide], ameliorates methamphetamine-induced dopaminergic neurotoxicity in mice

Methamphetamine (METH) administration in mice, results in a chronic dopamine (DA) depletion associated with nerve terminal damage, with DA oxidation and generation of reactive oxygen species (ROS) primarily mediating this neurotoxicity. The oxidative stress induced by METH putatively activates nuclear enzyme poly(ADP-ribose) polymerase (PARP), with excessive PARP activation eventually leading to cell death. In this study, we show that prevention of PARP activation by treatment with FR261529 [2-(4-chlorophenyl)-5-quinoxalinecarboxamide], the compound that was recently identified as a novel PARP inhibitor (IC50 for PARP-1 = 33 nM, IC50 for PARP-2 = 7 nM), protects against both ROS-induced cells injury in vitro and METH-induced dopaminergic neuronal damage in an in vivo Parkinson's disease (PD) model. In PC12 cells, exposure of hydrogen peroxide or METH markedly induced PARP activation, and treatment with FR261529 (1 microM) significantly reduced PARP activation and attenuated cell death. In the mouse METH model, METH (15 mg/kg x 2 i.p., 2 h apart) intoxication accelerated DA metabolism and oxidation in the striatum, with subsequent cell damage in nigrostriatal dopaminergic neurons after 4 days. Oral administration of FR261529 (10 or 32 mg/kg) attenuated the damage of dopaminergic neurons via marked reduction of PARP activity and not via changes in dopamine metabolism or body temperature. These findings indicate that the neuroprotective effects of a novel PARP inhibitor, FR261529, were accompanied by inhibition of METH-induced PARP activation, suggesting that METH induces nigrostriatal dopaminergic neurodegeneration involving PARP activation and also orally active and brain-penetrable PARP inhibitor FR261529 could be a novel attractive therapeutic candidate for neurodegenerative disorders such as PD.     

A newly synthesized poly(ADP-ribose) polymerase inhibitor, DR2313 [2-methyl-3,5,7,8-tetrahydrothiopyrano[4,3-d]-pyrimidine-4-one]: pharmacological profiles, neuroprotective effects, and therapeutic time window in cerebral ischemia in rats

We investigated the pharmacological profiles of DR2313 [2-methyl-3,5,7,8-tetrahydrothiopyrano[4,3-d]pyrimidine-4-one], a newly synthesized poly(ADP-ribose) polymerase (PARP) inhibitor, and its neuroprotective effects on ischemic injuries in vitro and in vivo. DR2313 competitively inhibited poly(ADP-ribosyl)ation in nuclear extracts of rat brain in vitro (K(i) = 0.23 microM). Among several NAD(+)-utilizing enzymes, DR2313 was specific for PARP but not selective between PARP-1 and PARP-2. DR2313 also showed excellent profiles in water solubility and rat brain penetrability. In in vitro models of cerebral ischemia, exposure to hydrogen peroxide or glutamate induced cell death with overactivation of PARP, and treatment with DR2313 reduced excessive formation of poly(ADP-ribose) and cell death. In both permanent and transient focal ischemia models in rats, pretreatment with DR2313 (10 mg/kg i.v. bolus and 10 mg/kg/h i.v. infusion for 6 h) significantly reduced the cortical infarct volume. To determine the therapeutic time window of neuroprotection by DR2313, the effect of post-treatment was examined in transient focal ischemia model and compared with that of a free radical scavenger, MCI-186 (3-methyl-1-phenyl-2-pyrazolone-5-one). Pretreatment with MCI-186 (3 mg/kg i.v. bolus and 3 mg/kg/h i.v. infusion for 6 h) significantly reduced the infarct volume, whereas the post-treatment failed to show any effects. In contrast, post-treatment with DR2313 (same regimen) delaying for 2 h after ischemia still prevented the progression of infarction. These results indicate that DR2313 exerts neuroprotective effects via its potent PARP inhibition, even when the treatment is initiated after ischemia. Thus, a PARP inhibitor like DR2313 may be more useful in treating acute stroke than a free radical scavenger.     

Novel isoquinolinone-derived inhibitors of poly(ADP-ribose) polymerase-1: pharmacological characterization and neuroprotective effects in an in vitro model of cerebral ischemia

Excessive activation of poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme catalyzing the transfer of ADP-ribose units from NAD to acceptor proteins, induces cellular energy failure by NAD and ATP depletion and has been proposed to play a causative role in a number of pathological conditions, including ischemia/reperfusion injury. In this study, we used an in vitro enzyme activity assay to characterize a series of newly synthesized isoquinolinone derivatives as potential PARP-1 inhibitors. Several compounds displayed powerful inhibitory activity: thieno[2,3-c]isoquinolin-5-one (TIQ-A) displayed a submicromolar IC50 of 0.45 +/- 0.1 microM, whereas the 5-hydroxy and 5-methoxy TIQ-A derivatives had IC50 values of 0.39 +/- 0.19 and 0.21 +/- 0.12 microM, respectively. We then examined the neuroprotective effects of the newly characterized compounds in cultured mouse cortical cells exposed to 60 min of oxygen and glucose deprivation (OGD). When PARP-1 inhibitors were present in the incubation medium during OGD and the subsequent 24-h recovery period, they significantly attenuated neuronal injury. TIQ-A provided neuroprotection even when added to the culture 30 min after OGD and was able to reduce the early activation of PARP induced by OGD as detected by flow cytometry. When the IC50 values observed in the PARP-1 activity assay for selected compounds were compared with their IC50 values for the neuroprotective activity, a significant correlation (r = 0.93, P < 0.01) was observed. Our results suggest that TIQ-A and its derivatives are a new class of neuroprotectants that may be helpful in studies aimed at understanding the involvement of PARP-1 in physiology and pathology.     

Activation of poly(ADP-ribose) polymerase contributes to development of doxorubicin-induced heart failure

Activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) by oxidant-mediated DNA damage is an important pathway of cell dysfunction and tissue injury in conditions associated with oxidative stress. Increased oxidative stress is a major factor implicated in the cardiotoxicity of doxorubicin (DOX), a widely used antitumor anthracycline antibiotic. Thus, we hypothesized that the activation of PARP may contribute to the DOX-induced cardiotoxicity. Using a dual approach of PARP-1 suppression, by genetic deletion or pharmacological inhibition with the phenanthridinone PARP inhibitor PJ34, we now demonstrate the role of PARP in the development of cardiac dysfunction induced by DOX. PARP-1+/+ and PARP-1-/- mice received a single injection of DOX (25 mg/kg i.p). Five days after DOX administration, left ventricular performance was significantly depressed in PARP-1+/+ mice, but only to a smaller extent in PARP-1-/- ones. Similar experiments were conducted in BALB/c mice treated with PJ34 or vehicle. Treatment with a PJ34 significantly improved cardiac dysfunction and increased the survival of the animals. In addition PJ34 significantly reduced the DOX-induced increase in the serum lactate dehydrogenase and creatine kinase activities but not metalloproteinase activation in the heart. Thus, PARP activation contributes to the cardiotoxicity of DOX. PARP inhibitors may exert protective effects against the development of severe cardiac complications associated with the DOX treatment.     

Pharmacological properties of 3-amino-5,6,7,8-tetrahydro-2-[4-[4-(quinolin-2-yl)piperazin-1-yl]butyl]quinazolin-4(3H)-one (TZB-30878), a novel therapeutic agent for diarrhea-predominant irritable bowel syndrome (IBS) and its effects on an experimental IBS model

3-Amino-5,6,7,8-tetrahydro-2-[4-[4-(quinolin-2-yl)piperazin-1-yl]butyl]quinazolin-4(3H)-one (TZB-30878) is a novel compound with both 5-hydroxytryptamine (5-HT)(1A) agonism and 5-HT(3) antagonism effects. We hypothesized that TZB-30878 might have benefits from these dual effects as a medication for diarrhea-predominant irritable bowel syndrome (d-IBS), and these studies were designed to confirm the pharmacological properties of TZB-30878 and its efficacy in an IBS-like animal model. The binding assays demonstrated that [(3)H]TZB-30878 selectively binds to human 5-HT(1A) and 5-HT(3) receptors, with K(d) values of 0.68 +/- 0.03 and 8.90 +/- 1.73 nM, respectively. Systemic administration of TZB-30878 inhibited 5-HT-induced bradycardia in a dose-dependent manner in rats. In behavioral assays TZB-30878 produced signs of 5-HT syndrome in rats. These results suggest that TZB-30878 has dual effects as a 5-HT(1A) receptor agonist and a 5-HT(3) receptor antagonist. Finally, we evaluated the effects of TZB-30878 on wrap restraint stress-induced defecation in an IBS-like model in rats. TZB-30878 (1-10 mg/kg p.o.) normalized stress-induced defecation in a dose-dependent manner, whereas the 5-HT(1A) agonist tandospirone (30 and 100 mg/kg p.o.) and the 5-HT(3) antagonist alosetron (1-10 mg/kg p.o.) did not show such effects. Furthermore, this efficacy of TZB-30878 was partly antagonized by a 5-HT(1A) antagonist, [O-methyl-3H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride (WAY-100635). These results suggest that 5-HT(1A) receptor agonism and 5-HT(3) receptor antagonism contribute to the efficacy of TZB-30878 in the IBS-like model. The efficacy of TZB-30878 supports the concept that the presence of both actions, namely 5-HT(1A) receptor agonism and 5-HT(3) receptor antagonism, could be an important mechanism in the treatment of d-IBS.     

ARD-353 [4-((2R,5S)-4-(R)-(4-diethylcarbamoylphenyl)(3-hydroxyphenyl)methyl)-2,5-dimethylpiperazin-1-ylmethyl)benzoic acid], a novel nonpeptide delta receptor agonist, reduces myocardial infarct size without central effects

A novel delta-receptor selective compound, ARD-353 [4-((2R,5S)-4-(R)-(4-diethylcarbamoylphenyl)(3-hydroxyphenyl)methyl)-2, 5-dimethylpiperazin-1-ylmethyl)benzoic acid], was evaluated for activity on infarct size in a rat model of acute myocardial infarction. ARD-353 was characterized as having delta receptor selectivity using radioligand binding and had no apparent selectivity between delta receptor subtypes as determined by [(3)H] cyclic [D-Pen(2),D-Pen(5)]enkephalin (delta(1)) and [(3)H]Deltorphin II (delta(2)) competition binding. ARD-353 also showed selective delta receptor agonist activity in mouse-isolated vas deferens. There was no evidence of any seizure-like convulsions when ARD-353 was administered to mice either i.v. or p.o., implying minimal penetration of the blood-brain barrier. ARD-353 decreased infarct size in a left anterior descending coronary artery (LAD) occlusion model of myocardial infarction. In animals pretreated with ARD-353 (i.v.) and then subjected to 30 min of LAD occlusion followed by 90 min of reperfusion, infarct size was reduced in a dose-dependent manner compared with vehicle-treated controls. The effects of ARD-353 on infarct size were blocked by the delta(1)-opioid selective antagonist 7-benzylidenenaltrexone, indicating a significant role for the delta(1)-opioid receptor in the cardioprotective mechanism of ARD-353. ARD-353 (0.3 mg/kg i.v.) produced significant protection when administered 5 min and 12 and 48 h before ischemic insult or when given immediately after the ischemic insult (at the start of reperfusion). Given the lack of central nervous system effects and beneficial efficacy in the rat model of myocardial ischemia, it is felt that ARD-353 is the first nonpeptide delta-receptor agonist with true potential for clinical use before surgically induced ischemia or in an emergency setting.     

ADX47273 [S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]-oxadiazol-5-yl]-piperidin-1-yl}-methanone]: a novel metabotropic glutamate receptor 5-selective positive allosteric modulator with preclinical antipsychotic-like and procognitive activities

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGlu5) enhance N-methyl-d-aspartate receptor function and may represent a novel approach for the treatment of schizophrenia. ADX47273 [S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidin-1-yl}-methanone], a recently identified potent and selective mGlu5 PAM, increased (9-fold) the response to threshold concentration of glutamate (50 nM) in fluorometric Ca(2+) assays (EC(50) = 170 nM) in human embryonic kidney 293 cells expressing rat mGlu5. In the same system, ADX47273 dose-dependently shifted mGlu5 receptor glutamate response curve to the left (9-fold at 1 microM) and competed for binding of [(3)H]2-methyl-6-(phenylethynyl)pyridine (K(i) = 4.3 microM), but not [(3)H]quisqualate. In vivo, ADX47273 increased extracellular signal-regulated kinase and cAMP-responsive element-binding protein phosphorylation in hippocampus and prefrontal cortex, both of which are critical for glutamate-mediated signal transduction mechanisms. In models sensitive to antipsychotic drug treatment, ADX47273 reduced rat-conditioned avoidance responding [minimal effective dose (MED) = 30 mg/kg i.p.] and decreased mouse apomorphine-induced climbing (MED = 100 mg/kg i.p.), with little effect on stereotypy or catalepsy. Furthermore, ADX47273 blocked phencyclidine, apomorphine, and amphetamine-induced locomotor activities (MED = 100 mg/kg i.p.) in mice and decreased extracellular levels of dopamine in the nucleus accumbens, but not in the striatum, in rats. In cognition models, ADX47273 increased novel object recognition (MED = 1 mg/kg i.p.) and reduced impulsivity in the five-choice serial reaction time test (MED = 10 mg/kg i.p.) in rats. Taken together, these effects are consistent with the hypothesis that allosteric potentiation of mGlu5 may provide a novel approach for development of antipsychotic and procognitive agents.     

Anxiolytic-like effects of the corticotropin-releasing factor1 (CRF1) antagonist DMP904 [4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazolo-[1,5-a]-pyrimidine] administered acutely or chronically at doses occupying central CRF1 receptors in rats

Corticotropin-releasing factor(1) (CRF(1)) antagonists may be effective in the treatment of anxiety disorders with fewer side effects compared with classic benzodiazepines. The behavioral effects of DMP904 [4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazolo-[1,5-a]-pyrimidine] and its effects on the hypothalamic-pituitary-adrenal (HPA) axis were related to its levels in plasma and estimated occupancy of central CRF(1) receptors. DMP904 (10-30 mg/kg, p.o.) and alprazolam (10 mg/kg, p.o.) increased time spent in open arms of an elevated-plus maze. In addition, acutely or chronically (14 days) administered DMP904 (1.0-30 mg/kg, p.o.) and acute alprazolam (1.0-3.0 mg/kg, p.o.) significantly reduced exit latency in the defensive withdrawal model of anxiety in rats, suggesting that tolerance may not develop to the anxiolytic-like effects of DMP904 in this model of anxiety. Acutely, DMP904 reversed the stress-induced increase in plasma corticosterone levels in defensive withdrawal at doses of 3.0 mg/kg and higher. These doses also resulted in levels of DMP904 in plasma similar to (for anxiolytic-like effects) or 4-fold higher (for effects on the HPA axis) than the in vitro IC(50) value for binding affinity at CRF(1) receptors and greater than 50% occupancy of CRF(1) receptors. Unlike alprazolam, DMP904 did not produce sedation, ataxia, or chlordiazepoxide-like subjective effects (as measured by locomotor activity, rotorod performance, and chlordiazepoxide discrimination assays, respectively) at doses at least 3-fold higher than anxiolytic-like doses. In conclusion, anxiolytic-like effects and effects on the stress-activated HPA axis of DMP904 in the defensive withdrawal model of anxiety required 50% or greater occupancy of central CRF(1) receptors. This level of CRF(1) receptor occupancy resulted in fewer motoric side effects compared with classic benzodiazepines.     

Increased poly(ADP-ribosyl)ation in peripheral leukocytes and the reperfused myocardium tissue of rats with ischemia/reperfusion injury: prevention by 3-aminobenzamide treatment

The overactivation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) is considered a final common effector in ischemia/reperfusion (I/R) injury. The aim of the current study was to examine the precise time course of the activation of PARP in peripheral leukocytes and the reperfused myocardium tissue on myocardial I/R injury from the same rat and to identify the relationship between myocardial infarct size and the degree of PARP activation in circulating leukocytes. Another aim of the study was to test the effect of 3-aminobenzamide (a well-known and widely used PARP inhibitor) on the activation of PARP in the reperfused myocardium and peripheral leukocytes. Poly(ADP-ribose) polymerase activation was measured by Western blotting for its product, poly(ADP-ribose) (PAR). The localization of PARP activation was determined by PAR immunohistochemistry. The results showed that poly(ADP-ribosyl)ation was detected 15 min, peaked 2 to 6 h, and remained markedly detectable 24 h in the reperfused heart after I/R model. Similarly, PAR content of the leukocytes increased in cells isolated just after reperfusion from the same rat. Immunohistochemical studies localized the staining of PAR primarily to the cardiac myocytes and vascular endothelial cells. At 6 h, there was a significant linear correlation between infarct size and PARP activity, whereas at 2 and 24 h, no relationship was found. The PARP inhibitor 3-aminobenzamide (3-AB, 20 mg kg?1 i.v. injection 15 min before reperfusion, and every 2 h thereafter for 6 h) markedly reduced infarct size through depressing the activation of the enzyme in myocytes and peripheral leukocytes even when the treatment is initiated at 2 h after reperfusion.     

Activation of poly(ADP-ribose) polymerase in circulating leukocytes during myocardial infarction

Myocardial ischemia-reperfusion can lead to increased oxidative stress both locally and in circulating leukocytes. Oxidant-mediated DNA single strand breaks are known to activate the nuclear enzyme poly(ADP-ribose) polymerase (PARP) in various forms of shock, inflammation, and ischemia-reperfusion injury. The aim of the current study was to investigate whether a local insult such as myocardial ischemia-reperfusion is sufficient to lead to activation of PARP in circulating leukocytes. In anesthetized rats myocardial ischemia-reperfusion was induced by transient ligation of the left anterior descending coronary artery. There was a marked increase in poly(ADP-ribosyl)ation of proteins in homogenates of leukocytes isolated from rats at the end of the reperfusion period. Poly(ADP-ribosyl)ation was inhibited by administration of the pharmacologic PARP inhibitor INO-1001 (30 mg/kg) to the rats. We conclude that local insults, such as myocardial reperfusion injury, are sufficient to activate PARP in circulating leukocytes. PARP activation in circulating cells may mediate certain systemic effects of local ischemia-reperfusion injury such as inflammatory mediator production and remote organ injury.     

SSR240600 [(R)-2-(1-[2-[4-[2-[3,5-bis(trifluoromethyl)phenyl]acetyl]-2-(3,4-dichlorophenyl)-2-morpholinyl]ethyl]-4-piperidinyl)-2-methylpropanamide], a centrally active nonpeptide antagonist of the tachykinin neurokinin 1 receptor: II. Neurochemical and behavioral characterization

SSR240600 [(R)-2-(1-[2-[4-[2-[3,5-bis(trifluoromethyl)phenyl]acetyl]-2-(3,4-dichlorophenyl)-2-morpholinyl]ethyl]-4-piperidinyl)-2-methylpropanamide], a new nonpeptide tachykinin neurokinin 1 (NK1) receptor antagonist, was evaluated against the neurochemical, electrophysiological, and behavioral effects provoked by direct activation of brain tachykinin NK1 receptors or by stress in guinea pigs. SSR240600 (0.1-10 mg/kg i.p. or p.o.) antagonized the excitatory effect of i.c.v. infusion of [Sar(9),Met(O2)(11)]substance P (SP) on the release of acetylcholine in the striatum of anesthetized and awake guinea pigs. This antagonistic action was still observed after repeated administration of SSR240600 (5 days, 10 mg/kg p.o., once a day). SSR240600 (10 mg/kg i.p.) inhibited the phosphorylation of the cAMP response element-binding protein in various brain regions induced by i.c.v. administration of [Sar9,Met(O2)(11)]SP. In slice preparations, neuronal firing of the locus coeruleus (LC) neurons elicited by the application of [Sar9,Met(O2)(11)]SP was suppressed by SSR240600 at 100 nM. Norepinephrine release in the prefrontal cortex, elicited either by an intra-LC application of [Sar9,Met(O2)(11)]SP or by an i.c.v administration of corticotropin-releasing factor, was reduced by SSR240600 (0.3-1 mg/kg and 1-10 mg/kg i.p., respectively). SSR240600 (1-10 mg/kg i.p.) inhibited vocalizations induced in adult guinea pigs by an i.c.v. administration of the NK1 receptor agonist, GR73632 [D-Ala-[L-Pro9,Me-Leu8]substance P(7-11)]. Furthermore, SSR240600 (1-10 mg/kg i.p.) inhibited distress vocalizations produced in guinea pig pups by maternal separation. SSR240600 also reduced maternal separation-induced increase in the number of neurons displaying NK1 receptor internalization in the amygdala. Finally, SSR240600 counteracted the increase in body temperature induced by isolation stress. In conclusion, SSR240600 is able to antagonize various NK1 receptor-mediated as well as stress-mediated effects in the guinea pig.     

In vivo efficacy in airway disease models of N-(3,5-dichloropyrid-4-yl)-[1-(4-fluorobenzyl)-5-hydroxy-indole-3-yl]-glyoxylic acid amide (AWD 12-281), a selective phosphodiesterase 4 inhibitor for inhaled administration

N-(3,5-Dichloro-pyrid-4-yl)-[1-(4-fluorobenzyl)-5-hydroxy-indole-3-yl]-glyoxylic acid amide (AWD 12-281) is a highly potent and selective phosphodiesterase 4 (PDE4) inhibitor that was designed to have a metabolic profile that was optimized for topical administration. The aim of the current study was to explore the pharmacological profile of intratracheally administered AWD 12-281 in different models of asthma and chronic obstructive pulmonary disease (COPD) in comparison with steroids. To assess the anti-inflammatory potential of AWD 12-281, the antigen-induced cell infiltration in bronchoalveolar lavage fluid (BALF) of Brown Norway rats was determined. AWD 12-281 (ID50 of 7 microg/kg i.t.) as well as beclomethasone (0.1microg/kg i.t.) suppresses late-phase eosinophilia when administered intrapulmonary. Furthermore, AWD 12-281 has also strong anti-inflammatory properties when tested in lipopolysaccharide-induced acute lung neutrophilia in Lewis rats (ID50 of 0.02 microg/kg i.t.), ferrets (ID50 of 10 microg/kg i.t.), and domestic pigs (2-4 mg/pig i.t. or 1 mg/kg i.v.). In pigs, AWD 12-281 was as effective as beclomethasone (0.4 mg/pig i.t.) and dexamethasone (0.28 mg/kg i.v.), although at 3 to 10 times the dosage. The bronchodilatory activity of AWD 12-281 was assessed in sensitized guinea pigs. AWD 12-281 (1.5 mg/kg i.t., 1-h pretreatment) inhibited allergen-induced bronchoconstriction by 68% (parameter airway resistance). In sensitized BP-2 mice AWD 12-281 abolished the allergen-induced bronchial hyperresponsiveness and eosinophilia in BALF, showing dose dependence. When given orally, i.v. or i.t., AWD 12-281 has a considerably lower emetic potential than cilomilast in ferrets and roflumilast in pigs. When given topically by inhalation, no emesis could be induced in dogs up to the highest feasible dose (15 mg/kg in 50% lactose blend). These results indicate that AWD 12-281 is a unique potential new drug for the topical treatment of asthma and COPD.     

A novel selective peroxisome proliferator-activated receptor alpha agonist, 2-methyl-c-5-[4-[5-methyl-2-(4-methylphenyl)-4-oxazolyl]butyl]-1,3-dioxane-r-2-carboxylic acid (NS-220), potently decreases plasma triglyceride and glucose levels and modifies lipoprotein profiles in KK-Ay mice

2-Methyl-c-5-[4-[5-methyl-2-(4-methylphenyl)-4-oxazolyl]butyl]-1,3-dioxane-r-2-carboxylic acid (NS-220) was newly synthesized and demonstrated to be a novel potent peroxisome proliferator-activated receptor alpha (PPARalpha) agonist with high subtype selectivity. In cell-based reporter gene assays, the EC(50) values of NS-220 for human PPARalpha, PPARgamma, and PPARdelta were 1.9 x 10(-8), 9.6 x 10(-6), and >10(-4) M, respectively, and for mouse PPARalpha, PPARgamma, and PPARdelta were 5.5 x 10(-8), 3.3 x 10(-5), and >10(-4) M, respectively. In addition, [(3)H]NS-220 bound to the ligand-binding domain of human PPARalpha with a K(D) value of 1.85 x 10(-7) M. Fenofibric acid and bezafibrate showed weak agonist activity for PPARalpha (EC(50), 2-8 x 10(-5) M), with poor subtype selectivity. NS-220 (0.1-3 mg/kg p.o.) decreased plasma triglyceride levels in ddY mice in a dose-dependent manner, but its hypolipidemic activity was abolished in PPARalpha-deficient mice. In KK-A(y) mice, an animal model of type-2 diabetes, NS-220 (0.3-1 mg/kg p.o.; 4 days) and fenofibrate (100-300 mg/kg p.o.; 4 days) decreased plasma triglyceride and glucose levels in a dose-dependent manner. In a 2-week repeated administration test, NS-220 (0.3-1 mg/kg p.o.) decreased plasma glucose levels markedly without increasing in plasma insulin levels. Furthermore, NS-220 increased high-density lipoprotein levels and decreased triglyceride-rich lipoprotein levels. In conclusion, a newly synthesized dioxanecarboxylic acid derivative, NS-220, is a potent and highly selective PPARalpha agonist that ameliorates metabolic disorders in diabetic mice. These results strongly suggest that it will be a promising drug for the treatment of hyperlipidemia or metabolic disorders in type-2 diabetes.     

Pharmacological profile of (2R-trans)-4-[1-[3,5-bis(trifluoromethyl)benzoyl]-2-(phenylmethyl)-4-piperidinyl]-N-(2,6-dimethylphenyl)-1-acetamide (S)-Hydroxybutanedioate (R116301), an orally and centrally active neurokinin-1 receptor antagonist

In comparison with a series of reference compounds, (2R-trans)-4-[1-[3,5-bis(trifluoromethyl)benzoyl]-2-(phenylmethyl)-4-piperidinyl]-N-(2,6-dimethylphenyl)-1-acetamide (S)-Hydroxybutanedioate (R116301) was characterized as a specific, orally, and centrally active neurokinin-1 (NK(1)) receptor antagonist with subnanomolar affinity for the human NK(1) receptor (K(i): 0.45 nM) and over 200-fold selectivity toward NK(2) and NK(3) receptors. R116301 inhibited substance P (SP)-induced peripheral effects (skin reactions and plasma extravasation in guinea pigs) and a central effect (thumping in gerbils) at low doses (0.08-0.16 mg/kg, s.c. or i.p.), reflecting its high potency as an NK(1) receptor antagonist and excellent brain disposition. Higher doses blocked various emetic stimuli in ferrets, cats, and dogs (ED(50) values: 3.2 mg/kg, s.c.; 0.72-2.5 mg/kg, p.o.). Even higher doses (11-25 mg/kg, s.c.) were required in mice (capsaicin-induced ear edema) and rats (SP-induced extravasation and salivation), consistent with lower affinity for the rodent NK(1) receptor and known species differences in NK(1) receptor interactions. R116301 inhibited the ocular discharge (0.034 mg/kg) but not the dyspnoea, lethality, or cough (>40 mg/kg, s.c.) induced by [betaALA(8)]-neurokinin A (NKA) (4-10) in guinea pigs, attesting to NK(1) over NK(2) selectivity. R116301 did not affect senktide-induced miosis (>5 mg/kg, s.c.) in rabbits, confirming the absence of an interaction with the NK(3) receptor. R116301 was inactive in guinea pigs against skin reactions induced by histamine, platelet-aggregating factor, bradykinin, or Ascaris allergens (>10 mg/kg, s.c.). In all species, R116301 showed excellent oral over parenteral activity (ratio, 0.22-2.7) and a relatively long duration (6.5-16 h, p.o.). The data attest to the specificity and sensitivity of the animal models and support a role of NK(1) receptors in various diseases.     

Peripheral phosphodiesterase 4 inhibition produced by 4-[2-(3,4-Bis-difluoromethoxyphenyl)-2-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-phenyl]-ethyl]-3-methylpyridine-1-oxide (L-826,141) prevents experimental autoimmune encephalomyelitis

Administration of phosphodiesterase 4 (PDE4) inhibitors suppresses the pathogenesis associated with experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). In the present study, we compared the effects of rolipram and 4-[2-(3,4-bis-difluoromethoxyphenyl)-2-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-phenyl]-ethyl]-3-methylpyridine-1-oxide (L-826,141), a novel nonbrain penetrant PDE4 inhibitor, on the onset and severity of clinical signs in a chronic, nonrelapsing/remitting model of EAE. Both rolipram (10 mg/kg p.o.) and L-826,141 (3 mg/kg p.o.) reduced the severity of EAE relative to controls, whereas L-826,141 (3 mg/kg p.o.) also delayed disease onset. To assess whether L-826,141 prevented EAE progression after the first signs of clinical onset, rolipram (10 mg/kg p.o.) or L-826,141 (3 or 30 mg/kg p.o.) were administered 24 h after the first signs of EAE were observed. Only L-826,141 at a dose of 30 mg/kg p.o. significantly decreased the clinical severity of EAE compared with vehicle controls. Immunohistochemical detection of the neuronal activity marker Fos confirmed that L-826,141 did not reach concentrations in the central nervous system sufficient to activate central neurons. Lipopolysaccharide-induced tumor necrosis factor-alpha in whole blood and plasma concentrations of L-826,141 revealed that only the 30-mg/kg dose resulted in levels sufficient to produce a near complete inhibition of PDE4 activity in immune cells. Taken together, these results demonstrate that peripheral PDE4 inhibition, produced by L-826,141, prevents the progression of EAE after the first onset of clinical signs, and suggest that similar compounds may have clinical efficacy in the treatment of MS.     

2-[4-(7-chloro-2-quinoxalinyloxy)phenoxy]-propionic acid (XK469) inhibition of topoisomerase IIbeta is not sufficient for therapeutic response in human Waldenstrom's macroglobulinemia xenograft model

The role of DNA topoisomerase (Topo) IIbeta in cancer chemotherapy remains unclear, although this particular isoform has been implicated in drug resistance. In this study, we investigated Topo IIbeta as a target for 2-[4-(7-chloro-2-quinoxalinyloxy)phenoxy]-propionic acid (XK469), a novel synthetic quinoxaline phenoxypropionic acid derivative, in a Waldenstrom's macroglobulinemia (WM) model. In vitro, the WSU-WM cell line was exposed to 1.0, 2.0, 5.0, 8.0, and 10 microM XK469. Our results demonstrate a concentration-dependent cell growth inhibition with a concentration-independent inhibition of Topo IIbeta, as determined by band depletion assay. The cell growth inhibition of cells correlated well with increase in Bax:Bcl-2 ratio and poly(ADP-ribose) polymerase (PARP) cleavage. We used our established WSU-WM severe combined immunodeficient mouse xenograft model to test the efficacy and effect of XK469 on Topo IIbeta in vivo. Topo IIbeta was inhibited equally using two different dose schedules (20 and 40 mg/kg, i.v., for a total of 120 and 240 mg/kg, respectively); however, there was no significant decrease in tumor weight. Western blot analysis of cells isolated from s.c. tumors showed no induction of the Bax protein and a very low Bax:Bcl-2 ratio of approximately 0.3 in correlation with minimum PARP cleavage. Our study shows that XK469 inhibits Topo IIbeta in WSU-WM cells both in vitro and in vivo at or below the maximum tolerated dose in severe combined immunodeficient mice. However, there was no change of apoptosis-related molecules such as PARP, Bax, and Bcl-2 or reduction in tumor weight in association with Topo IIbeta inhibition. We conclude that Topo IIbeta inhibition by XK469 as a target is not sufficient for therapeutic effects in WSU-WM.     

Radiosensitization by the poly(ADP-ribose) polymerase inhibitor 4-amino-1,8-naphthalimide is specific of the S phase of the cell cycle and involves arrest of DNA synthesis

Radiosensitization caused by the poly(ADP-ribose) polymerase (PARP) inhibitor 4-amino-1,8-naphthalimide (ANI) was investigated in 10 asynchronously growing rodent (V79, CHO-Xrs6, CHO-K1, PARP-1+/+ 3T3, and PARP-1-/- 3T3) or human (HeLa, MRC5VI, IMR90, M059J, and M059K) cell lines, either repair proficient or defective in DNA-PK (CHO-Xrs6 and M059J) or PARP-1 (PARP-1-/- 3T3). Pulse exposure to ANI (1-hour contact) potentiated radiation response in rodent cells except in PARP-1(-/-) 3T3 fibroblasts. In contrast, ANI did not significantly enhance radiation susceptibility in asynchronously dividing human cells; yet, single-strand break rejoining was lengthened by ca. 7-fold in all but mouse PARP-1-/- 3T3s. Circumstantial evidence suggested that radiosensitization by ANI occurs in rapidly dividing cells only. Experiments using synchronized HeLa cells consistently showed that ANI-induced radiosensitization is specific of the S phase of the cell cycle and involves stalled replication forks. Under these conditions, prolonged contact with ANI ended in the formation of de novo DNA double-strand breaks hours after irradiation, evoking collision with uncontrolled replication forks of DNA lesions whose repair was impaired by inhibition of the PARP catalytic activity. The data suggest that increased response to radiotherapy by PARP inhibitors may be achieved only in rapidly growing tumors with a high S-phase content.