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.     

Brain vasopressin signaling modulates aspects of maternal behavior in lactating rats

The brain vasopressin system mediates various social behaviors as has been studied mostly in males. Only recently, advances in social neuroscience revealed that central vasopressin signaling via its V1a and V1b receptors also facilitates female social behavior, including maternal behavior. In this review, we show how maternal care, maternal motivation and maternal aggression of lactating rat mothers are modulated in a V1 receptor subtype‐ and brain region‐specific manner. Measuring local release pattern of vasopressin via intracerebral microdialysis in the behaving rat mother as well as using pharmacological approaches to activate or block vasopressin receptors with subsequent behavioral observation provide detailed insight into the functional role of the vasopressin system in maternal behavior. In this context, the complementary rat animal model of high (HAB) and low anxiety‐related behavior (LAB) is particularly helpful due to the genetically determined high activity of the vasopressin gene in HAB rats, which also underlies their high levels of maternal behavior. Furthermore, first studies in humans indicate that the vasopressin system in general and the V1a receptor in more particular might mediate mothering.     

以田鼠作为动物模型来研究社会行为的进化:基因、脑与行为

田鼠属的一些近缘种间具有独特的社会行为多态性。例如Microtusochrogaster和M .pinetorum为一夫一妻制 ,而M .montanus和M .pennsylvanicus则为独居和一夫多妻制。无论是在野外还是人工饲养的条件下 ,单配制的田鼠其雌、雄成年个体一经交配即在两者之间形成长期的配偶关系并且双亲共同哺育后代。已证明神经多肽加压素 (Vasopressin)参与了田鼠单配制行为的神经调控。本篇综述了过去以及近期关于加压素调控田鼠配偶关系形成的研究结果和进展。首先 ,阐述了加压素V1a受体 (V1aR)在脑分布的种间差异 ,并以此来鉴别特定脑区在配偶关系形成中的功能 ;其次 ,探讨了运用V1aR拮抗物的药理学方法来决定究竟哪些脑区参与配偶关系的形成 ,还描述了田鼠种间V1aR基因结构和功能的不同 ,以及这些不同对V1aR在大脑的分布和行为调控潜在的作用机制 ;最后 ,讨论了最新的研究结果 ,即对一夫多妻制田鼠进行脑V1aR基因的改造 ,从而使之表现出一夫一妻制田鼠的行为。总之 ,了解复杂的社会性行为的遗传和神经机制可以加深我们对种间和种内行为分歧进化的理解     

Expression of vasopressin V2 receptor in Xenopus laevis oocytes by porcine kidney cell line (LLC-PK1) messenger RNA.

Vasopressin V2 receptor was expressed in Xenopus laevis oocytes which were injected with poly(A) +RNA from porcine kidney cell line LLC-PK1. Pharmacological antagonism of the expressed V2 receptor was observed between arginine vasopressin and two potent and selective vasopressin antagonists: [d(CH2)5, D2-Phe2 Ile4, Ala9-NH2]arginine vasopressin and [d(CH2)5,D-Ile2, Ile4]arginine vasopressin. Activation constant for arginine vasopressin concentration was 1.32 x 10(-10)M. The nucleotide length of the mRNA encoding for vasopressin V2 receptor was deduced to be approximately 2 kilobases.     

Iodinated photoreactive vasopressin antagonists: labelling of hepatic vasopressin receptor subunits

To identify and characterize V1 vasopressin receptors, photoreactive antagonists of the glycogenolytic and vasoconstrictor activity of vasopressin have been synthesized. The following analogues with 3-mercapto-3,3-cyclopentamethylene-propionic acid (Mca) and N-methylalanine (MeAla) in position 1 and 7 of vasopressin (VP) were effective V1 antagonists: [Mca1, D-Tyr2, MeAla7, Lys8]VP (1), [Mca1, MeAla7, Arg8, Lys9]VP (2), [Mca1, MeAla7, Arg8, D-Lys9]VP (3). Introduction of the photoreactive 4-azidophenylamidino group into the side-chain of Lys8 in analogue 1 or into Lys9 in analogues 2 and 3 increased the potency (for analogue 1 a tenfold increase in the antiglycogenolytic effect and a fivefold increase in the antivasopressor effect) and binding affinity for the rat hepatic V1 receptor. Mono-iodination at Tyr2 with 125I resulted in photoreactive antagonists of high specific radioactivity, which had roughly the same binding affinity as vasopressin for the rat hepatic V1 receptor (Kd = 0.9-1.8 nM). In photoaffinity labelling experiments with purified rat liver membranes, containing 2--3 pmol V1 receptor/mg protein, the analogues labelled specifically two proteins with the relative molecular masses (Mr) of 30,000 and 38,000. These results and the results of a recent study using 3H-labelled photoreactive vasopressin agonists [Boer, R. and Fahrenholz, F. (1985) J. Biol. Chem. 260, 15051-15054] provide evidence that both vasopressin agonists and antagonists can interact with the same two subunits of the heterodimeric hepatic V1 receptor. Furthermore the radioiodinated photoreactive V1 antagonists should be helpful to identify V1 receptor proteins in membranes of other cell types.     

Conformation of [8-arginine]vasopressin and V1 antagonists in dimethyl sulfoxide solution derived from two-dimensional NMR spectroscopy and molecular dynamics simulation

Structural and dynamic properties of [8-arginine]vasopressin and a class of highly potent vasopressin V1 antagonists which contain 3-mercapto-3,3-cyclopentamethylene propionic acid (Mca) in position 1 of the vasopressin sequence have been determined. On the basis of two-dimensional NMR experiments in dimethyl sulfoxide solution, interproton distances were derived according to which model conformations were built and refined using molecular dynamics simulations. The conformation of vasopressin and the V1 antagonists differ mainly in the region of the mutated residue. The antagonistic property was found to be related to an inversed chirality of the disulfide bridge. In all investigated molecules, characteristic beta-turn structure elements were found for the backbone conformation of the endocyclic residues Tyr2-Asn5. For this portion of the peptide sequence, various conformational equilibria were detected which matched different time scales. For [Arg8]vasopressin, averaged NMR parameters were obtained which could be explained by rapid interconversion between different beta-turn geometries, whereas multiple slowly exchanging conformations were observed for the V1 antagonists. V1 antagonists containing sarcosine in position 7 exhibited multiple spectral patterns for the exocyclic part attributed to cis/trans isomerization. The X-ray structure of deamino-oxytocin [Wood, S. P., Tickle, I. J., Treharne, A. M., Pitts, J. E., Mascarenhas, Y., Li, J. Y., Husain, J., Cooper, S., Blundell, T. L., Hruby, V. J., Buku, A., Fischman, A. J. & Wyssbrod, H. R. (1986) Science 232, 633-636] was found to represent one sample of the conformational space covered by the multiple conformations found for [Mca1, Arg8]vasopressin.     

Next-generation spirobenzazepines: identification of RWJ-676070 as a balanced vasopressin V1a/V2 receptor antagonist for human clinical studies

We have continued to explore spirobenzazepines as vasopressin receptor antagonists to follow up on RWJ-339489 (2), which had advanced into preclinical development. Further structural modifications were pursued to find a suitable backup compound for human clinical studies. Thus, we identified carboxylic acid derivative 3 (RWJ-676070; JNJ-17158063) as a potent, balanced vasopressin V(1a)/V(2) receptor antagonist with favorable properties for clinical development. Compound 3 is currently undergoing human clinical investigation.     

Characterization of intrathecal vasopressin-induced antinociception, scratching behavior, and motor suppression.

Intrathecal (IT) administration of vasopressin produces antinociception, scratching behavior, and motor suppression. The present experiments characterized these effects with regards to the following: 1) VP receptor specificity, 2) possible involvement of endogenous opiates, 3) possible involvement of seizure activity, and 4) whether the antinociception is due to direct actions of VP at the spinal cord. These studies showed that IT administration of a V1-specific vasopressin antagonist completely blocked the antinociception, scratching behavior, and motor suppression produced by 25 ng IT vasopressin. Furthermore, IT administration of the vasopressin metabolite, [pGlu4,Cyt6]AVP(4-9), produced none of the effects produced by vasopressin. Systemic administration of the opiate antagonists naloxone (1 mg/kg IP) and naltrexone (10 mg/kg IP) had no significant effect on the antinociception produced by IT vasopressin, whereas naltrexone potentiated the scratching behavior. Neither the IT vasopressin-induced antinociception nor scratching behavior was affected by pretreatment with the anticonvulsant sodium valproate. In addition, IT vasopressin inhibited the tail flick reflex in rats with transected spinal cords, demonstrating direct spinal effects of vasopressin. In conclusion, IT administration of vasopressin produces antinociception, scratching behavior, and motor suppression via activation of VP-specific receptors in the spinal cord.     

Potent nonpeptide vasopressin receptor antagonists based on oxazino- and thiazinobenzodiazepine templates

Vasopressin receptor antagonists can elicit ion-sparing diuretic effects (i.e., aquaresis) in vivo by blunting the action of the circulating hypophyseal hormone arginine vasopressin. We have identified two new series of basic tricyclic benzodiazepines, represented by general structure 1, which contain compounds that bind with high affinity to human V2 receptors. For example, (S)-(+)-8 and 5 are potent and selective V2 receptor antagonists with pronounced aquaretic activity in rats on oral administration.     

Vasopressin agonists and antagonists

In this article, the discrete modifications of the structure of the vasopressin molecule which led to the development of specific V1 vascular, V2 renal, and mixed V1/V2 analogs are reviewed. Particularly, the third generation of vasopressin antagonists produced by deletions and substitutions of the carboxy terminal, and the fourth generation of vasopressin antagonists obtained by deletions, substitutions, and the linearization of the molecule are presented. The potential advantages of these different compounds are illustrated by our work on V1 vascular vasopressin receptors of human platelets.     

The development of vasopressin antagonists

Antagonists of the physiological actions of vasopressin can be useful probes for detecting the influences of endogenous vasopressin on cardiovascular regulation. Antagonists that block vascular receptors of the V1 type have been widely used for this purpose. Recently effective antagonists of renal antidiuretic receptors of the V2 type have become available, and we have made progress toward improving their specificity for V2 receptors. Vasopressin antagonists of both types of responses could become useful in the diagnosis and treatment of pathophysiological states in which elevated levels of circulating vasopressin may disturb cardiovascular and renal functions.     

Association of vasopressin 1a receptor levels with a regulatory microsatellite and behavior

Vasopressin regulates complex behaviors such as anxiety, parenting, social engagement and attachment and aggression in a species-specific manner. The capacity of vasopressin to modulate these behaviors is thought to depend on the species-specific distribution patterns of vasopressin 1a receptors (V1aRs) in the brain. There is considerable individual variation in the pattern of V1aR binding in the brains of the prairie vole species, Microtus ochrogaster. We hypothesize that this individual variability in V1aR expression levels is associated with individual variation in a polymorphic microsatellite in the 5' regulatory region of the prairie vole v1ar gene. Additionally, we hypothesize that individual variation in V1aR expression contributes to individual variation in vasopressin-dependent behaviors. To test these hypotheses, we first screened 20 adult male prairie voles for behavioral variation using tests that measure anxiety-related and social behaviors. We then assessed the brains of those animals for V1aR variability with receptor autoradiography and used polymerase chain reaction to genotype the same animals for the length of their 5' microsatellite polymorphism in the v1ar gene. In this report, we describe the results of this discovery-based experimental approach to identify potential gene, brain and behavior interrelationships. The analysis reveals that V1aR levels, in some but not all brain regions, are associated with microsatellite length and that V1aR levels in those and other brain regions correlate with anxiety-related and social behaviors. These results generate novel hypotheses regarding neural control of anxiety-related and social behaviors and yield insight into potential mechanisms by which non-coding gene polymorphisms may influence behavioral traits.     

7-Chloro-5-hydroxy-1-[2-methyl-4-(2-methylbenzoyl-amino)benzoyl ]-2,3,4,5-tetrahydro-1H-1-benzazepine (OPC-41061): a potent, orally active nonpeptide arginine vasopressin V2 receptor antagonist.

We previously reported a series of benzazepine derivatives as orally active nonpeptide arginine vasopressin (AVP) V2 receptor antagonists. After the lead structure OPC-31260 was structurally evaluated and optimized, the introduction of the 7-Cl moiety on the benzazepine and 2-CH3 on the aminobenzoyl moiety enhanced its oral activity. The new AVP-V2 selective antagonist OPC-41061 was determined to be a potent and orally active agent.     

Repeated agonistic encounters in hamsters modulate AVP V1a receptor binding

Arginine vasopressin (AVP) regulates aggression in male Syrian hamsters. In this study, we used radioligand receptor autoradiography to examine whether changes in agonistic behavior following acute and repeated social defeat are accompanied by changes in AVP V1a receptor binding. Social defeat produced high levels of submissive behavior and a loss of territorial aggression when hamsters were subsequently tested with a novel intruder, and repeated agonistic encounters produced similar behavioral changes in subordinates. AVP V1a receptor binding was not reduced by acute social defeat but was affected by repeated agonistic encounters. Dominants had significantly more AVP V1a receptor binding in lateral portions of the ventromedial hypothalamus (VMHL) than did their subordinate opponents, but subordinates were no different from controls. In contrast, receptor binding did not differ in most other brain regions examined. The changes in receptor binding appear to be independent of testosterone levels, as testosterone levels did not differ among dominants, subordinates, and controls. Our results suggest that changes in AVP V1a receptors do not account for the changes in agonistic behavior produced by acute social defeat but AVP V1a binding in the VMHL correlates with, and may modulate, the behavioral changes that occur following repeated experiences of victory.     

An arginyl in the N-terminus of the V1a vasopressin receptor is part of the conformational switch controlling activation by agonist.

Defining how the agonist-receptor interaction differs from that of the antagonist-receptor and understanding the mechanisms of receptor activation are fundamental issues in cell signalling. The V1a vasopressin receptor (V1aR) is a member of a family of related G-protein coupled receptors that are activated by neurohypophysial peptide hormones, including vasopressin (AVP). It has recently been reported that an arginyl in the distal N-terminus of the V1aR is critical for binding agonists but not antagonists. To determine specific features required at this locus to support high affinity agonist binding and second messenger generation, Arg46 was substituted by all other 19 encoded amino acids. Our data establish that there is an absolute requirement for arginyl, as none of the [R46X]V1aR mutant constructs supported high affinity agonist binding and all 19 had defective signalling. In contrast, all of the mutant receptors possessed wildtype binding for both peptide and nonpeptide antagonists. The ratio of Ki to EC50, an indicator of efficacy, was increased for all substitutions. Consequently, although [R46X]V1aR constructs have a lower affinity for agonist, once AVP has bound all 19 are more likely than the wildtype V1aR to become activated. Therefore, in the wildtype V1aR, Arg46 constrains the inactive conformation of the receptor. On binding AVP this constraint is alleviated, promoting the transition to active V1aR. Our findings explain why arginyl is conserved at this locus throughout the evolutionary lineage of the neurohypophysial peptide hormone receptor family of G-protein coupled receptors.     

Novel,potent, selective and brain penetrant vasopressin 1b receptor antagonists

Herein we report the discovery of a novel oxindole-based series of vasopressin 1b (V1b) receptor antagonists. Introducing a substituted piperazine moiety and optimizing the southern and the northern aromatic rings resulted in potent, selective and brain penetrant V1b receptor antagonists. Compound 9c was found to be efficacious in a rat model of anti-depressant activity (3?mg/kg, ip). Interestingly, both moderate terminal half-life and moderate bioavailability could be achieved despite sub-optimal microsomal stability.     

Potent and selective oxindole-based vasopressin 1b receptor antagonists with improved pharmacokinetic properties

Herein we report the discovery of a novel series of vasopressin 1b (V1b) receptor antagonists, starting from potent but metabolically labile oxindole SSR149415. Masking the proline N,N-dimethyl amide moiety as an oxazole and attaching a benzylic amine moiety to the northern phenyl ring resulted in potent and selective V1b receptor antagonists with improved metabolic stability and improved pharmacokinetic properties in rat. Compound 18c was found to be efficacious in a rat model of anti-depressant activity.     

Social motivation is reduced in vasopressin 1b receptor null mice despite normal performance in an olfactory discrimination task

In this study, we characterized more thoroughly the social behavior of vasopressin 1b receptor null (V1bR-/-) mice. We confirmed that V1bR-/- males exhibit less social aggression than their wild-type (V1bR+/+) littermates. We tested social preference by giving male subjects a choice between pairs of soiled or clean bedding. In general, V1bR+/+ mice spent significantly more time engaged in chemoinvestigation of these social stimuli than V1bR-/- mice. Male V1bR+/+ mice preferred female-soiled bedding over male-soiled bedding, male-soiled bedding over clean bedding, and female-soiled bedding over clean bedding. In contrast, V1bR-/- males failed to exhibit a preference for any bedding. This difference in behavior is not explained by an anosmic condition as there were no differences between V1bR-/- and V1bR+/+ mice in their abilities to detect a cookie buried in clean bedding, or in their ability to perform in an operant conditioning task using a fully automated liquid dilution olfactometer. In the latter task, male V1bR-/- mice were fully capable of discriminating between male and female mouse urine. The latencies to learn this task did not differ between the two genotypes. Thus, a V1bR-/- male's ability to differentiate between male and female chemosensory cues appears no different than that of a V1bR+/+ male's. We propose that the V1bR plays an important role in social motivation, perhaps by coupling the processing, integration, and/or interpretation of chemosensory cues with the appropriate behavioral response.     

Polymorphism at the avpr1a locus in male prairie voles correlated with genetic but not social monogamy in field populations

Integrative studies of genetics, neurobiology and behaviour indicate that polymorphism in specific genes contributes to variation observed in some complex social behaviours. The neuropeptide arginine vasopressin plays an important role in the regulation of a variety of social behaviours, including social attachment of males to females, through its action on the vasopressin 1a receptor (V1aR). In socially monogamous prairie voles ( Microtus ochrogaster ), polymorphism in the length of microsatellite DNA within the regulatory region of the gene ( avpr1a ) encoding the V1aR predicts differences among males in neural expression of V1aRs and partner preference under laboratory conditions. However, understanding the extent to which V1aR mediates variation in prairie vole social and reproductive behaviour observed in nature requires investigating the consequences of avpr1a polymorphism and environmental influences under ecologically relevant conditions. We examined the relationship between avpr1a length polymorphism and monogamy among male prairie voles living in 0.1 ha enclosures during a time similar to their natural lifespan. We found no evidence that avpr1a genotype of males predicts variation in social monogamy measured in the field but some indices of social monogamy were affected by population density. Parentage data indicated that a male's avpr1a genotype significantly influenced the number of females with which he sired offspring and the total number of offspring sired. Total brain concentrations of V1aR mRNA were not associated with either male behaviour or avpr1a genotype. These data show that melding ecological field studies with neurogenetics can substantially augment our understanding of the effects of genes and environment on social behaviours.