Discovery and Characterization of RGH-122, a Potent, Selective, and Orally Bioavailable V1a Receptor Antagonist.

The V1a receptor is a major contributor in mediating the social and emotional effects of arginine-vasopressin (AVP); therefore it represents a promising target in the treatment of several neuropsychiatric conditions. The aim of this research was to design and synthesize novel and selective V1a antagonists with improved in vitro and in vivo profiles. Through optimization and detailed SAR studies, we developed low nanomolar antagonists, and further characterizations led to the discovery of the clinical candidate compound 43 (RGH-122). The CNS activity of the compound was determined in a 3-chamber social preference test of autism in which RGH-122 successfully enhanced social preference with the lowest effective dose of 1.5 mg/kg.

Discovery of New Heterocyclic Ring Systems as Novel and Potent V1A Receptor Antagonists.

Autism spectrum disorder is a neurodevelopmental disease with increasing occurrence. Recent studies focus on the development of novel V1A receptor antagonists which can influence the core symptoms of autism through the AVP pathway. In this study, we describe the synthesis of new heterocyclic ring systems. These are a novel class of brain-penetrating V1A antagonists with improved metabolic stability and in vivo potency. The efficacy of the compounds was strongly influenced by the position of the chlorine atom, suggesting halogen bond formation between the ligands and the V1A receptor.

New V1a receptor antagonist. Part 1. Synthesis and SAR development of urea derivatives.

Solid preclinical evidence links vasopressin to social behavior in animals, so, extensive work has been initiated to find new vasopressin V1a receptor antagonists which can improve deteriorated social behavior in humans and can treat the core symptoms of autistic behavior, as well. Our aim was to identify new chemical entities with antagonizing effects on vasopressin V1a receptors. Starting from a moderately potent HTS hit (7), we identified a molecule (49) having nanomolar binding strength and functional activity, which is in the same range as the potency of clinically tested V1a antagonists.

New V1a receptor antagonist. Part 2. Identification and optimization of triazolobenzazepines.

Solid preclinical evidence links vasopressin to social behavior in animals, so, extensive work has been initiated to find new vasopressin V1a receptor antagonists which can improve deteriorated social behavior in humans and can treat the core symptoms of autistic behavior, as well. Our aim was to identify new chemical entities with antagonizing effects on vasopressin V1a receptors. Continuing our previous work, we found an in vitro and in vivo orally active V1a selective antagonist molecule (40) among [1,2,4]triazolo[4,3-a][1]benzazepines.

Measuring sociability of mice using a novel three-chamber apparatus and algorithm of the LABORAS™ system.

BACKGROUND: The social approach and social novelty tests utilizing the three-chamber apparatus are widely accepted to measure social behavior of rodents. The LABORAS™ system offers a possibility to assess sociability of mice in a reliable and objective manner. NEW METHOD: We assessed the capability of the LABORAS™ sociability cage and algorithm (2.6.6) to detect social behaviors in mice. Furthermore, we investigated whether the system is able to detect various levels of sociability due to genetic background or after pharmacological treatments. RESULTS: By comparing manual scoring with various detection zone settings of the automated registration, the most fitting algorithm with a detection zone radius of 90 mm was identified. When different strains were investigated, C57Bl/6 J and NMRI mice proved to be social, while CD1 mice were found asocial. The system was able to detect the sociability increasing effect of R-baclofen (0.5 mg/kg i.p.) and oxytocin (12 ng i.c.v.) in asocial CD1 mice. The negative control PCP impaired social behavior of C57Bl/6 J mice (1 mg/kg i.p.) and increased social avoidance in CD1 mice (0.3 mg/kg i.p.). COMPARISON WITH EXISTING METHOD(S): This setup, in contrast to video frame analysis softwares, determines signal changes caused by movements of rodents allowing accurate detection and analysis of trajectories. Parallel automated measurements also allow replacing time and labor intensive, highly subjective human observational work. CONCLUSIONS: The set-up provides a fast and reliable method to examine social behavior of mice in the three-chamber apparatus. The system is capable of detecting pro or antisocial activity of pharmacological agents.

Assessment of the effects of NS11394 and L-838417, α2/3 subunit-selective GABA(A) [corrected] receptor-positive allosteric modulators, in tests for pain, anxiety, memory and motor function.

The aim of the present paper was to study the effects of GABAA receptor-positive modulators (L-838417 and NS11394) showing a preference for α2/3 subunits of the GABAA receptor, in models of pain, anxiety, learning, memory and motor function. These compounds have been suggested to have a favourable therapeutic profile over nonselective compounds such as diazepam. In this study, we tested both compounds for their effects in rat models of formalin-induced pain, spinal nerve-ligation-induced mechanical allodynia, plus maze, open field, rotarod, balance beam walking, contextual fear conditioning and Morris water maze. Both compounds exerted analgesic, but no anxiolytic effects. However, they induced motor side-effects, and learning and memory impairment at similar doses. Therefore, the anxiolytic effect and the lack of side-effects of these compounds, as described in the literature, could not be confirmed in the present study.

Synergistic interaction of endogenous platelet-activating factor and vasopressin in generating angina in rats.

We examined the involvement of endogenous vasopressin and platelet-activating factor (PAF) in the pathogenesis of two types of experimental angina in urethane-anaesthetised male Wistar rats. In the first model, epinephrine (10 microg kg(-1)) was injected into the tail vein, followed at the development of the maximum blood pressure response, i.e., 30 s later, by phentolamine (15 mg kg(-1)). In the second model, the vasopressin V1 receptor agonist ornithine-vasopressin (ornipressin; 0.5 IU kg(-1), i.v.) was administered. The heart rate, mean arterial blood pressure and surface electrocardiogram (ECG, standard lead II) were registered simultaneously. As a measure of myocardial ischaemia, at 1 min after phentolamine or ornipressin administration, we found significant ST-segment depression, lasting for more than 10 or 5 min, respectively. Pretreatment (15 min, s.c.) with the vasopressin V1 receptor antagonist Mca1,Tyr(Me)2AVP (the Manning peptide; 0.02-0.2 microg kg(-1)) or the platelet-activating factor receptor antagonist ginkgolide B (BN 52021; 0.25-2.5 mg kg(-1)) alone caused a dose-dependent reduction of the ST-segment depression. Concurrent administration of the two antagonists in their threshold doses (0.02 microg kg(-1) and 0.25 mg kg(-1)) also attenuated the ST-segment depression in both models. Neither antagonist affected the blood pressure or heart rate changes throughout the studies. Our results suggest that endogenous vasopressin and platelet-activating factor interact synergistically in provoking myocardial ischaemia in vivo in experimental angina in the rat.

Raloxifene lowers ischaemia susceptibility by increasing nitric oxide generation in the heart of ovariectomized rats in vivo.

We studied the effects of a 2-week period of oral raloxifene therapy on the cardiac level of nitric oxide (NO) and on the susceptibility to angina in ovariectomized rats. Ovariectomy decreased the activity of Ca2+-dependent nitric oxide synthase (NOS) in the left ventricle, an effect restored by raloxifene (0.2-5 mg kg(-1) day(-1)) or 17beta-oestradiol (0.3 mg kg(-1) day(-1)). Ovariectomy led to a significant ST segment depression after the injection of (1) ornithine-vasopressin (0.5 IU kg(-1), i.v.) or (2) epinephrine (10 microg kg(-1), i.v.), followed 30 s later by phentolamine (15 mg kg(-1), i.v.); both effects were reversed by raloxifene or 17beta-oestradiol treatment. Inhibition of nitric oxide synthase (with NG-nitro-L-arginine methyl ester [L-NAME]; 5 mg kg(-1), s.c.) augmented the ST segment depression in the ovariectomized rat and abolished the anti-ischaemic effect of 17beta-oestradiol or raloxifene. Thus, an oestrogen deficiency down-regulates the cardiac constitutive nitric oxide synthase, which increases the susceptibility of the heart to ishaemia because both actions can be blocked by exogenous administration of the natural oestrogen 17beta-oestradiol or the selective oestrogen-receptor modulator (SERM) raloxifene. In the present in vivo system, raloxifene exerts oestrogen-agonist properties.

ATP and ATPase secretion by exocrine pancreas in rat, guinea pig, and human.

ATP is an extracellular regulator in numerous physiological and pathologic processes. Recently, 7 different subtypes of purinoceptors were identified on either the basolateral or the luminal membrane of pancreatic duct cells. However, the in vivo regulatory role of ATP in pancreatic function has not been established. We investigated the possible regulatory role of endogenous ATP in pancreatic function by measuring ATP concentrations and ATPase activity in pancreatic juice obtained from anesthetized rats and guinea pigs and from human patients undergoing endoscopy. Juice was collected from the main pancreatic duct in rats and guinea pigs under basal conditions or during stimulation with CCK, bombesin, or secretin. In guinea pigs, CCK, bombesin, and secretin did not affect ATP output, although they did stimulate fluid secretion. ATPase activity in the juice was evaluated by measuring the rate of hydrolysis of added ATP. Consistent with the low ATP concentrations in rat pancreatic juice, we found high levels of ATPase activity in this species. This was confirmed by HPLC, which also showed the metabolites of ATP hydrolysis. Ecto-ATPase activity was demonstrated by enzyme histochemistry in both the pancreatic acini and ducts in rats, but it was not detectable in guinea pigs and humans. These differences in ATP levels and ATPase expression may indicate significant species differences in the purinergic regulation of pancreatic secretion.

Localization of nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) and NTPDase2 in pancreas and salivary gland.

Ectonucleoside triphosphate diphosphohydrolases (NTPDases) are membrane-bound ectoenzymes that hydrolyze extracellular nucleotides. We investigated the distribution of NTPDase1 and NTPDase2 in murine salivary gland and pancreas. Histochemistry and immunostaining (by both light and electron microscopy), combined with functional assays, were used to describe the localization patterns and enzyme activities in the organs of wild-type and NTPDase1/cd39-null mice. Pancreatic acinar cells and salivary gland acinar/myoepithelial cells were positive for NTPDase1 and NTPDase2. Ecto-ATPase activity was slightly higher in salivary glands. Ductal epithelial cells expressed ecto-ATPase activity but NTPDase1 and NTPDase2 expression were weak at best. ATPase activity was found in blood vessels of both tissues and its localization pattern overlapped with NTPDase1 staining. In these structures, NTPDase2 antibodies stained the basolateral aspect of endothelial cells and the supporting cells. Biochemical assays and histochemical staining showed relatively high levels of ATPase activity in both glands of cd39(-/-) mice. Our data therefore support a physiological role for NTPDase2 and other ectonucleotidases in the pancreas and salivary glands. Because NTPDase1 is expressed in non-vascular cell types, this finding suggests that NTPDase1 may have functions in the gastrointestinal tract that differ from those demonstrated in the vascular system.