Selective V1b Research Articles

V1b Receptor Antagonist SSR149415 and Naltrexone Synergistically Decrease Excessive Alcohol Drinking in Male and Female Mice.

A recent clinical trial found that pharmacological blockade of V1b receptors reduces alcohol relapse in alcohol-dependent patients. SSR149415 is a selective V1b receptor antagonist that has potential for development as an alcohol dependency treatment. In this study, we investigated whether SSR149415 alone or in combination with the mu-opioid receptor (MOP-r) antagonist naltrexone (NTN) would alter excessive alcohol drinking in mice. Both sexes of C57BL/6J (B6) mice were subjected to a chronic intermittent access (IA) drinking paradigm (2-bottle choice, 24-hour access every other day) for 3weeks. Sucrose and saccharin drinking were used as controls for alcohol-specific drug effects. Neuronal proopiomelanocortin (POMC) enhancer (nPE) knockout mice with hypothalamic-specific loss of POMC (including beta-endorphin, the main endogenous ligand of MOP-r) were used as a genetic control for the effects of NTN. Acute administration of SSR149415 (1 to 30mg/kg) reduced alcohol intake and preference in a dose-dependent manner in both male and female B6 mice after IA. To investigate potential synergistic effects between NTN and SSR149415, we tested 6 different combination doses of SSR149415 and NTN, and found that a combination of SSR149415 (3mg/kg) and NTN (1mg/kg) reduced alcohol intake profoundly at doses lower than the individual effective doses in both sexes of B6 mice. We confirmed the effect of SSR149415 on reducing alcohol intake in nPE-/- male mice, consistent with independent mechanisms by which SSR149415 and NTN decrease alcohol drinking. The combination of V1b antagonist SSR149415 with NTN at individual subthreshold doses shows potential in alcoholism treatment, possibly with less adverse effects.

Neuroanatomical distribution and function of the vasopressin V1B receptor in the rat brain deciphered using specific fluorescent ligands

It is now accepted that vasopressin, through V1A/V1B receptors, centrally regulates cognitive functions such as memory, affiliation, stress, fear and depression. However, the respective roles of these receptor isoforms and their contribution to stress-related pathologies remain uncertain. The development of new therapeutic treatments requires a precise knowledge of the distribution of these receptors within the brain, which has been so far hampered by the lack of selective V1B markers. In the present study, we have determined the pharmacological properties of three new potent rat V1B fluorescent ligands and demonstrated that they constitute valuable tools for simultaneous visualization and activation of native V1B receptors in living rat brain tissue. Thus, d[Leu4,Lys-Alexa 647)8]VP (analogue 3), the compound with the best affinity-selectivity/fluorescence ratio for the V1B receptor emerged as the most promising. The rat brain regions most concerned by stress such as hippocampus, olfactory bulbs, cortex and amygdala display the highest V1B fluorescent labelling with analogue 3. In the hippocampus CA2, V1B receptors are located on glutamatergic, not GABAergic neurones, and are absent from astrocytes. Using AVP-EGFP rats, we demonstrate the presence of V1B autoreceptors on AVP-secreting neurones not only in the hypothalamus, but also sparsely in the hippocampus. Finally, using both electrophysiology and visualization of ERK phosphorylation, we show analogue 3-induced activation of the V1B receptor in situ. This will help to analyse expression and functionality of V1B receptors in the brain and contribute to further explore the AVPergic circuitry in normal and pathological conditions.

Clinical safety and hypothalamic-pituitary-adrenal axis effects of the arginine vasopressin type 1B receptor antagonist ABT-436.

Arginine vasopressin type 1B receptor (V1B) receptor antagonism is considered a potential therapeutic for diseases with hypothalamic-pituitary-adrenal (HPA) axis dysregulation. The aim of the present study was to evaluate the safety and pharmacodynamics of ABT-436, a selective V1B antagonist, in healthy adults. Healthy adults received daily oral doses of ABT-436 in two clinical trials. In a dose escalation trial, nine subjects received each of 100, 500, or 800mg ABT-436, or placebo, in the morning for 7-14days. In a crossover trial on two 7-day regimens, 20 subjects received 200mg ABT-436 each morning or each evening. Pharmacokinetics, measures of basal HPA axis activity, and safety were assessed in both trials. Mild gastrointestinal intolerance was more common with ABT-436 treatment, compared to placebo, and showed dose dependence. Mean increases and decreases of systolic blood pressure (at different times), and mean pulse increases, were observed in subjects who received 800mg ABT-436. Mean decreases of plasma adrenocorticotrophic hormone (ACTH), serum cortisol, urine total glucocorticoids, and urine cortisol, compared to placebo, were observed following 7 daily doses of 500 and 800mg ABT-436. Statistically significant mean differences of plasma ACTH, serum cortisol, and urine total glucocorticoids were observed between morning and evening regimens of 200mg ABT-436. The largest observed differences were near the times of maximum post-dose ABT-436 plasma concentrations. ABT-436 regimens of 200-800mg once daily (QD) for 7days attenuated basal HPA axis activity. The results support further evaluation of ABT-436 for treatment of disorders in which HPA axis dysregulation may have an etiologic role.

Haemodynamic and renal sympathetic responses to V1b vasopressin receptor activation within the paraventricular nucleus.

What is the central question of this study? Does antagonism of V1b receptors prevent the haemodynamic and renal sympathetic nerve responses that occur with application of exogenous vasopressin into the paraventricular nucleus (PVN) of conscious, chronically instrumented rats? What is the main finding and its importance? Microinjection of vasopressin into the PVN increased mean arterial pressure, heart rate and renal sympathetic nerve activity, all of which were inhibited by pre-injection of the PVN with the V1b antagonist, nelivaptan. The administered vasopressin did not enter the peripheral circulation or increase plasma vasopressin. Ganglionic blockade prevented each of the responses, consistent with mediation by enhanced sympathetic output rather than an increase in circulating vasopressin. Vasopressin (VP) participates in regulation of haemodynamics and volume. Besides more classical actions as a circulating hormone, VP may act via release from axons and dendrites within the CNS. The paraventricular nucleus (PVN) possesses vasopressinergic neurons and a dense complement of VP receptors, including the V1b receptor, which has been implicated in several types of stress responses. We tested the hypothesis that antagonism of V1b receptors will prevent VP-induced increases in mean arterial pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA). Studies were performed in conscious male Sprague-Dawley rats chronically instrumented with vascular catheters, renal nerve electrodes and a cannula stereotaxically directed into the PVN. Unilateral microinjection of VP into the PVN significantly increased MAP, HR and RSNA, peaking at 10min. Pre-injection of the PVN with the selective V1b receptor antagonist, nelivaptan, did not alter baseline values but blocked the responses to VP. Ganglionic blockade with chlorisondamine decreased MAP and HR and abolished their increase in response to subsequent PVN application of VP. Injection of VP into the PVN did not alter plasma VP levels. Paraventricular nucleus injection with radiolabelled VP resulted in negligible radiolabelled VP in peripheral blood. These findings support the concept that, in basal conditions, PVN V1b receptor activation (rather than VP release into the periphery) may be implicated in the increases in MAP, HR and RSNA due to increased sympathetic outflow. While the role of V1a and oxytocin receptors cannot be excluded, these data suggest that further studies of the role of V1b receptor activation by endogenous VP during stress to effect neuroexcitation are warranted.

The effect of vasopressin 1b receptors (V1bRs) blockade on the HPA axis activity in rats exposed to acute heat stress

Thermal stressors such as low and high ambient temperature elicit an abundance of neuroendocrine responses including activation of the hypothalamo-pituitary-adrenal (HPA) axis and arginine vasopressin (AVP) release. The exposure to heat is a particularly interesting model for studying AVP action because this kind of stressor represents not only an unpleasant experience but also a threat to osmotic homeostasis. As AVP has long been recognized as a hormone involved in the modulation of HPA axis activity, the aim of this study was to elucidate the role of AVP in acutely heat-exposed rats using Nelivaptan, a selective vasopressin 1b receptor (V1bR) antagonist. Rats were exposed to high ambient temperature (38°C) for 60 min. The circulating hormones were determined by ELISA or chemiluminescence, and intrapituitary adrenocorticotropic hormone (ACTH) and V1bR level were determined by western blot. The results obtained show that V1bR blockade negatively affected the increase in blood ACTH caused by heat exposure. This treatment alone, or in combination with Nelivaptan, decreased intrapituitary V1bR levels while circulating AVP concentration was increased under the same conditions. Furthermore, a strong correlation was observed between blood ACTH and corticosterone concentration. In conclusion, our results directly confirm the positive role of AVP in the regulation of ACTH secretion from the pituitary in animals exposed to heat. Moreover, the results suggest that AVP from the general circulation influences pituitary V1bR.

Persistent Increase in Hypothalamic Arginine Vasopressin Gene Expression During Protracted Withdrawal from Chronic Escalating-Dose Cocaine in Rodents

Arginine vasopressin (AVP) from the paraventricular nucleus (PVN) of hypothalamus has important roles in regulation of the hypothalamic-pituitary-adrenal (HPA) axis and stress-related behaviors during chronic stress. It is unknown, however, whether AVP in the PVN is involved in the modulation of HPA activity after chronic cocaine exposure. Here, we examined the gene expression alterations of AVP in the hypothalamus, and V1b receptor and pro-opiomelanocortin (POMC) in the anterior pituitary, as well as HPA hormonal changes, in Fischer rats after chronic cocaine and withdrawal, using two different chronic (14-day) 'binge' pattern administration regimens: steady-dose cocaine (SDC, 45 mg/kg/day) and escalating-dose cocaine (EDC, 45 up to 90 mg/kg/day). There was a significant (7-fold) plasma adrenocorticotropic hormone (ACTH) elevation after chronic EDC (but not SDC), coupled with increased V1b and POMC mRNA levels in the anterior pituitary. From acute (1-day) to protracted (14-day) withdrawal from chronic EDC (but not from SDC), we found persistent elevations of both plasma ACTH and corticosterone levels and AVP mRNA levels in the PVN. Selective V1b antagonist SSR149415 (5 mg/kg) attenuated acute withdrawal-induced HPA activation after EDC. To study potential roles of endogenous opioids in modulating the AVP gene, we administered naloxone (1 mg/kg); we found that opioid receptor antagonism increased AVP mRNA levels in cocaine-naive rats, but not in cocaine-withdrawn rats, suggesting less tonic opioid inhibition of PVN AVP neurons after chronic EDC. To assess the effects of cocaine withdrawal on sub-populations of PVN AVP neurons, we utilized AVP-enhanced green fluorescent protein (EGFP) promoter transgenic mice and found that acute withdrawal following chronic EDC increased the number of AVP-EGFP neurons in the parvocellular PVN (pPVN). These results suggest that during protracted withdrawal, enhanced pPVN AVP gene expression is associated with persistent elevations of basal HPA activity; a hyposensitivity of PVN AVP gene expression to naloxone is indicative of reduced opioidergic tone. Our studies indicate that the AVP and its V1b receptor system may be a potential therapeutic target for treating anxiety and depressive symptoms associated with cocaine addiction.

Identification and optimisation of novel sulfonamide, selective vasopressin V1B receptor antagonists

The synthesis and preliminary structure-activity relationships (SAR) of a novel class of vasopressin V(1B) receptor antagonists are described. Hit compound 5, identified via high throughput screening of the corporate collection, showed good activity in a V(1B) binding assay (K(i) 63 nM) but did not possess the lead-like physicochemical properties typically required in a hit compound. A 'deletion approach' on the HTS hit 5 was performed, with the focus on improvement of physicochemical properties, yielding the selective V(1B) antagonist 9f (K(i) 190 nM), with improved druglike characteristics.

Design, Synthesis, and Pharmacological Characterization of Fluorescent Peptides for Imaging Human V1b Vasopressin or Oxytocin Receptors

Among the four known vasopressin and oxytocin receptors, the specific localization of the V1b isoform is poorly described because of the lack of selective pharmacological tools. In an attempt to address this need, we decided to design, synthesize, and characterize fluorescent selective V1b analogues. Starting with the selective V1b agonist [deamino-Cys(1),Leu(4),Lys(8)]vasopressin (d[Leu(4),Lys(8)]VP) synthesized earlier, we added blue, green, or red fluorophores to the lysine residue at position 8 either directly or by the use of linkers of different lengths. Among the nine analogues synthesized, two exhibited very promising properties. These are d[Leu(4),Lys(Alexa 647)(8)]VP (3) and d[Leu(4),Lys(11-aminoundecanoyl-Alexa 647)(8)]VP (9). They remained full V1b agonists with nanomolar affinity and specifically decorated the plasma membrane of CHO cells stably transfected with the human V1b receptor. These new selective fluorescent peptides will allow the cellular localization of V1b or OT receptor isoforms in native tissues.

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.

Proinflammatory role of vasopressin through V1b receptors in hapten-induced experimental colitis in rodents: implication in IBD

Vasopressin and its receptors modulate several gut functions, but their role in intestinal inflammation is unknown. Our aims were to determine 1) the localization of V1b receptors in human and rodent colon, 2) the role of vasopressin and V1b receptors in experimental colitis using two approaches: V1b⁻(/)⁻ mice and a selective V1b receptor antagonist, SSR149415, and 3) the mechanisms involved. V1b receptors were localized in normal and inflamed colon from humans and rats. Experimental colitis was induced in rats and mice and some groups were treated before or after colitis induction with oral SSR149415 (3-30 mg/kg). Other groups of mice were submitted to dehydration to increase vasopressin plasma levels, prior to colitis induction. Body weight, damage scores, MPO, and TNF-α tissue levels were determined. Finally, colonic segments of wild-type (WT) and V1b⁻(/)⁻ mice were mounted in Ussing chambers and paracellular permeability in response to vasopressin was studied. V1b receptors were expressed in enterocytes and ganglia cells of the enteric nervous system of human and rat intestine. Expression levels were independent from inflammatory status. Colitis was less severe in rodents treated by either preventive or curative SSR149415 and in V1b⁻(/)⁻ mice. 2,4,6-Trinitrobenzene sulfonic acid induced a strong mortality in dehydrated animals that was reversed by preventive SSR149415 or mast cell stabilizer. Vasopressin significantly increased paracellular permeability in WT, but not in V1b⁻(/)⁻ mice. Preincubation of colon tissues with SSR149415 abolished the vasopressin effect. Similarly, vasopressin had no effect in colonic preparations from WT mice pretreated with mast cell stabilizers. Vasopressin, through V1b receptor interaction, has proinflammatory properties linked to mast cell activation and downstream alterations of the colonic epithelial barrier. These findings underline the potential interest of V1b receptor blockers in gut inflammatory diseases.

Tetrahydroquinoline sulfonamides as vasopressin 1b receptor anatgonists

Vasopressin 1b (V1b) antagonists have been postulated as possible treatments for depression and anxiety. A novel series of potent and selective V1b antagonists has been identified starting from an in-house screen hit. The incorporation of a sulfonamide linker between a tetrahydroisoquinoline core and amino piperidine lead to the identification of a V1b antagonist with similar affinity for human and rat receptors. Further optimization of the right hand portion afforded potent V1b antagonists that possessed moderate to high selectivity over other receptors.

Effects of Vasopressin V1b Receptor Deficiency on Adrenocorticotropin Release from Anterior Pituitary Cells in Response to Oxytocin Stimulation

Oxytocin (OT) is one of the secretagogues for stress-induced ACTH release. OT-induced ACTH release is reported to be mediated by the vasopressin V1b receptor in the rat pituitary gland, which contains both OT and V1b receptors. We examined OT-induced ACTH release using primary cultures of anterior pituitary cells from wild-type (V1bR+/+) and V1b receptor knockout (V1bR-/-) mice. OT stimulated similar levels of ACTH release from pituitary cells of V1bR+/+ and V1bR-/- mice. OT-induced ACTH release was significantly inhibited by the selective V1b receptor antagonist SSR149415 and the OT receptor antagonist CL-14-26 in V1bR+/+ mice. In addition, cotreatment with SSR149415 at 10(-6) m and CL-14-26 at 10(-6) m inhibited OT-induced ACTH release to the control level inV1bR+/+ mice. In V1bR-/- mice, OT-induced ACTH release was significantly inhibited by CL-14-26 at 10(-8) m and completely inhibited at 10(-7)m. These results indicate that OT induces the ACTH response via OT and V1b receptors inV1bR+/+ mice but via only OT receptors in V1bR-/- mice. The gene expression level of the OT receptor was significantly higher in the anterior pituitary gland of V1bR-/- mice than in that of V1bR+/+ mice, suggesting that the OT receptor is up-regulated to compensate for ACTH release under conditions of V1b receptor deficiency.

Pharmacological and Physiological Characterization of d[Leu4, Lys8]Vasopressin, the First V1b-Selective Agonist for Rat Vasopressin/Oxytocin Receptors

Recently, we synthesized and characterized the first selective V(1b) vasopressin (VP)/oxytocin receptor agonist, d[Cha(4)]arginine vasopressin. However, this agonist was only selective for the human receptors. We thus decided to design a selective V(1b) agonist for the rodent species. We started from previous observations showing that modifying [deamino(1),Arg(8)]VP in positions 4 and 8 altered the rat VP/oxytocin receptor selectivity. We synthesized a series of 13 [deamino(1),Arg(8)]VP analogs modified in positions 4 and 8. Among them, one seemed very promising, d[Leu(4), Lys(8)]VP. In this paper, we describe its pharmacological and physiological properties. This analog exhibited a nanomolar affinity for the rat, human, and mouse V(1b) VP receptors and a strong V(1b) selectivity for the rat species. On AtT20 cells stably transfected with the rat V(1b) receptor, d[Leu(4), Lys(8)]VP behaved as a full agonist on both phospholipase C and MAPK assays. Additional experiments revealed its ability to induce the internalization of enhanced green fluorescent protein-tagged human and mouse V(1b) receptors as expected for a full agonist. Additional physiological experiments were performed to further confirm the selectivity of this peptide. Its antidiuretic, vasopressor, and in vitro oxytocic activities were weak compared with those of VP. In contrast, used at low doses, its efficiency to stimulate adrenocorticotropin or insulin release from mouse pituitary or perfused rat pancreas, respectively, was similar to that obtained with VP. In conclusion, d[Leu(4), Lys(8)]VP is the first selective agonist available for the rat V(1b) VP receptor. It will allow a better understanding of V(1b) receptor-mediated effects in rodents.

Mutual regulation of vasopressin- and oxytocin-induced glucagon secretion in V1b vasopressin receptor knockout mice

[Arg8]-vasopressin (AVP) and oxytocin (OT) are neurohypophysial hormones which exert various actions, including the control of blood glucose, in some peripheral tissues. To investigate the type of receptors involved in AVP- and OT-induced glucagon secretion, we investigated the effect of these peptides on glucagon secretion in islets of wild-type (V1bR+/+) and vasopressin V1b receptor knockout (V1bR-/-) mice. AVP-induced glucagon secretion was significantly inhibited by the selective V1b receptor antagonist, SSR149415 (30%), and OT-induced glucagon secretion by the specific OT receptor antagonist, d(CH2)5[Tyr(Me)2, Thr4, Tyr-NH(2)(9)]OVT (CL-14-26) (45%), in islets of V1bR+/+mice. AVP- and OT-induced glucagon secretions were not by the antagonist of each, but co-incubation with both 10(-6) M SSR149415 and 10(-6) M CL-14-26 further inhibited AVP- and OT-induced glucagon secretions in islets of V1bR+/+ mice (57 and 69% of the stimulation values respectively). In addition, both AVP and OT stimulated glucagon secretion with the same efficacy in V1bR-/- mice as in V1bR+/+ mice. AVP- and OT-induced glucagon secretion in V1bR-/- mice was significantly inhibited by CL-14-26. These results demonstrate that V1b receptors can mediate OT-induced glucagon secretion and OT receptors can mediate AVP-induced glucagon secretion in islets from V1bR+/+mice in the presence of a heterologous antagonist, while AVP and OT can stimulate glucagon secretion through the OT receptors in V1bR-/-mice, suggesting that the other receptor can compensate when one receptor is absent.

Design and Synthesis of the First Selective Agonists for the Rat Vasopressin V1bReceptor: Based on Modifications of Deamino-[Cys]arginine Vasopressin at Positions 4 and 8

The neurohypophyseal peptides arginine vasopressin (AVP) and oxytocin (OT) mediate a wide variety of peripheral and central physiological and behavioral effects by acting on four different G-protein coupled receptors, termed V1a (vascular), V1b (pituitary), V2 (renal), and OT (uterine). We recently reported that d[Cha4]AVP (A), d[Leu4]AVP (B), d[Orn4]AVP (C), and d[Arg4]AVP (D) have high affinity and are selective agonists for the human V1b receptor. However, peptides A-D were subsequently shown to be potent antidiuretic agonists in the rat and are, thus, not selective V1b agonists in the rat. Peptides A-D served as leads for the studies reported here. They were modified at position 8 by Lys, ornithine (Orn), diaminobutyric acid (Dab), and diaminopropionic acid (Dap) to give d[Cha4,Lys8]VP (1), d[Cha4,Orn8]VP (2), d[Cha4,Dab8]VP (3), d[Cha4,Dap8]VP (4), d[Leu4,Lys8]VP (5), d[Leu4,Orn8]VP (6), d[Leu4,Dab8]VP (7), d[Leu4,Dap8]VP (8), d[Orn4,Lys8]VP (9), d[Orn4,Orn8]VP (10), d[Arg4,Lys8]VP (11), d[Arg4,Orn8]VP (12), and d[Arg4,Dab8]VP (13). All peptides were synthesized by the Merrifield solid-phase method. Their binding and functional properties were evaluated in rat AVP V1a, V1b, and V2 receptors and on the rat OT receptor expressed either in native tissues or in stably transfected cells. They were also examined in rat vasopressor, antidiuretic, and in in vitro (no Mg++) oxytocic assays. Functional studies performed on chinese hamster ovary cells expressing the different AVP/OT receptors confirm that d[Cha4,Lys8]VP (1), d[Cha4,Dab8]VP (3), d[Leu4,Lys8]VP (5), and d[Leu4,Dap8]VP (8) are the first selective agonists for the rat V1b receptor. These selective V1b agonists are promising new tools for studies of the role of the V1b receptor in the rat.

Dual actions of vasopressin and oxytocin in regulation of water permeability in terminal collecting duct.

We conducted studies in isolated perfused terminal inner medullary collecting ducts (IMCD) from rats to investigate the roles of oxytocin and vasopressin in the regulation of osmotic water permeability. Vasopressin and oxytocin were found to have both stimulatory effects (at 0.1 nM) and inhibitory effects (at 10 nM) on osmotic water permeability. Measurements of adenosine 3',5'-cyclic monophosphate (cAMP) production demonstrated that both vasopressin and oxytocin increase cAMP production. Both the selective oxytocin-receptor agonist [Thr4,Gly7]oxytocin (10 nM) and the selective V1b agonist [deamino1,D-3-(pyridyl)Ala2,Arg8]vasopressin (10 nM) inhibited vasopressin-stimulated osmotic water permeability. In contrast, the selective V1a vasopressin-receptor agonist [Phe2,Ile3,Orn8]vasopressin (10 nM) had no effect on vasopressin-stimulated osmotic water permeability. These effects on water permeability correlated with the ability of the agents to transiently increase intracellular free calcium. The oxytocin/vasopressin-receptor antagonist [des-glycinamide9,d(CH2)5(1),O-Me-Tyr2,Thr4,Orn8]vasot ocin, which almost completely blocks vasopressin-induced calcium mobilization, also blocked the ability of 10 nM vasopressin to inhibit osmotic water permeability relative to that found with 0.1 nM vasopressin. We conclude the following. 1) Oxytocin, like vasopressin, has dual effects on osmotic water permeability, increasing it at subnanomolar concentrations and inhibiting it at suprananomolar concentrations. 2) Oxytocin, like vasopressin, can increase cAMP production, perhaps accounting for the increase in water permeability.(ABSTRACT TRUNCATED AT 250 WORDS)