Cliona P. MacSweeney

Effect of the glucocorticoid receptor antagonist Org 34850 on basal and stress-induced corticosterone secretion

The activity of the hypothalamic‐pituitary‐adrenal (HPA) axis is characterised both by an ultradian pulsatile pattern of glucocorticoid secretion and an endogenous diurnal rhythm. Glucocorticoid feedback plays a major role in regulating HPA axis activity and this mechanism occurs via two different receptors: mineralocorticoid (MR) and glucocorticoid receptors (GR). In the present study, the effects of both acute and subchronic treatment with the GR antagonist Org 34850 on basal and stress‐induced HPA axis activity in male rats were evaluated. To investigate the effect of Org 34850 on basal diurnal corticosterone rhythm over the 24‐h cycle, an automated blood sampling system collected samples every 10 min. Acute injection of Org 34850 (10 mg/kg, s.c.) did not affect basal or stress‐induced corticosterone secretion, but was able to antagonise the inhibitory effect of the glucocorticoid agonist methylprednisolone on stress‐induced corticosterone secretion. However, 5 days of treatment with Org 34850 (10 mg/kg, s.c., two times a day), compared to rats treated with vehicle (5% mulgofen in 0.9% saline, 1 ml/kg, s.c.), increased corticosterone secretion over the 24‐h cycle and resulted in changes in the pulsatile pattern of hormone release, but had no significant effect on adrenocorticotrophic hormone secretion or on stress‐induced corticosterone secretion. Subchronic treatment with Org 34850 did not alter GR mRNA expression in the hippocampus, paraventricular nucleus of the hypothalamus or anterior‐pituitary, or MR mRNA expression in the hippocampus. Our data suggest that a prolonged blockade of GRs is required to increase basal HPA axis activity. The changes observed here with ORG 34850 are consistent with inhibition of GR‐mediated negative feedback of the HPA axis. In light of the evidence showing an involvement of dysfunctional HPA axis in the pathophysiology of depression, Org 34850 could be a potential treatment for mood disorders.

Blockade of the V1b receptor reduces ACTH, but not corticosterone secretion induced by stress without affecting basal hypothalamic–pituitary–adrenal axis activity

Vasopressin (AVP), produced in parvocellular neurons of the hypothalamic paraventricular nucleus, regulates, together with CRH, pituitary ACTH secretion. The pituitary actions of AVP are mediated through the G protein receptor V(1b) (V(1b)|R). In man, hyperactivity of the hypothalamic-pituitary-adrenal axis has been associated with depression and other stress-related conditions. There are also clinical data suggesting a role for AVP in the dysfunctional HPA axis described in some depressed patients. In this study, we have investigated the effect of a recently synthesised selective antagonist of the V(1b)R both on exogenous AVP-induced ACTH and corticosterone secretion, and on basal and stress-induced pituitary-adrenal activity. Adult male Sprague-Dawley rats treated with the V(1b)R antagonist (Org, 30 mg/kg, s.c.) or vehicle (5% mulgofen in 0.9% saline, 2 ml/kg, s.c.). We found that blockade of the V(1b)R reduced the increase in both ACTH and corticosterone secretion induced by AVP (100 ng, i.v.). The same treatment had no effect either on basal ACTH and corticosterone levels or on the ultradian or diurnal rhythms of corticosterone secretion. Acute administration of the V(1b)R antagonist reduced ACTH secretion following both restraint and lipopolysaccharide, but did not antagonise the ACTH response to noise. The same treatment did not reduce corticosterone secretion in response to any of the three stressors used in this study. Our results confirm that this compound is an antagonist of the V(1b)R in the rat, and that its ability to reduce stress-induced ACTH responses is stressor dependent with differential modulation of pituitary and adrenal responses.

Effect of vasopressin 1b receptor blockade on the hypothalamic-pituitary-adrenal response of chronically stressed rats to a heterotypic stressor.

Exposure to chronic restraint (CR) modifies the hypothalamic-pituitary-adrenal (HPA) axis response to subsequent acute stressors with adaptation of the response to a homotypic and sensitization of the response to a heterotypic stressor. Since vasopressin (AVP) activity has been reported to change during chronic stress, we investigated whether this was an important factor in HPA facilitation. We therefore tested whether vasopressin 1b receptor (AVPR1B) blockade altered the ACTH and corticosterone response to heterotypic stressors following CR stress. Adult male rats were exposed to CR, single restraint, or were left undisturbed in the home cage. Twenty-four hours after the last restraint, rats were injected with either a AVPR1B antagonist (Org, 30 mg/kg, s.c.) or vehicle (5% mulgofen in saline, 0.2/kg, s.c.) and then exposed to either restraint, lipopolysaccharide (LPS) or white noise. CR resulted in the adaptation of the ACTH and corticosterone response to restraint and this effect was not prevented by pretreatment with Org. Although we found no effect of CR on LPS-induced ACTH and corticosterone secretion, both repeated and single episodes of restraint induced the sensitization of the ACTH, but not corticosterone response to acute noise. Pretreatment with Org reduced the exaggerated ACTH response to noise after both single and repeated exposure to restraint.

Characterization of a novel and selective V1B receptor antagonist

It has been argued that hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis is a major biological abnormality in patients suffering from psychiatric conditions such as major depression. Both arginine vasopressin (AVP) and corticotrophin releasing factor (CRF) are responsible for stimulating the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary. CRF is thought to be the predominant secretagogue under normal conditions but AVP may play a more important role in situations of aberrant/chronic stress. Studies in patients suffering from melancholic depression indicate a hyper-responsiveness to agonism at the vasopressin receptor type 1B (V(1B)); patients display a heightened ACTH release after challenge with the mixed V(1B)/V(2) (vasopressin receptor type 2) agonist desmopressin in comparison to control subjects. A V(1B) antagonist has been developed which has significant selectivity for the human V(1B) receptor over the other members of the vasopressin receptor sub-family. The compound acts as an effective antagonist at both the human recombinant receptor (stably expressed in Chinese hamster ovary (CHO) cells) and the native rat V(1B) receptor (using isolated anterior pituitary cells), blocking the induction of luciferase and the release of ACTH, respectively. In vivo the compound can block the release of ACTH after challenge with a variety of V(1B) agonists. It can also attenuate the ACTH response to acute stressors in rats. Interestingly, this compound does not modulate the activity of the HPA axis under normal basal conditions.

Differential effect of glucocorticoid receptor antagonists on glucocorticoid receptor nuclear translocation and DNA binding

The effects of RU486 and S-P, a more selective glucocorticoid receptor antagonist from Schering-Plough, were investigated on glucocorticoid receptor nuclear translocation and DNA binding. In the in vitro study, AtT20 cells were treated with vehicle or with RU486, S-P or corticosterone (3–300 nM) or co-treated with vehicle or glucocorticoid receptor antagonists (3–300 nM) and 30 nM corticosterone. Both glucocorticoid receptor antagonists induced glucocorticoid receptor nuclear translocation but only RU486 induced DNA binding. RU486 potentiated the effect of corticosterone on glucocorticoid receptor nuclear translocation and DNA binding, S-P inhibited corticosterone-induced glucocorticoid receptor nuclear translocation, but not glucocorticoid receptor-DNA binding. In the in vivo study, adrenalectomized rats were treated with vehicle, RU486 (20 mg/kg) and S-P (50 mg/kg) alone or in combination with corticosterone (3 mg/kg). RU486 induced glucocorticoid receptor nuclear translocation in the pituitary, hippocampus and prefrontal cortex and glucocorticoid receptor-DNA binding in the hippocampus, whereas no effect of S-P on glucocorticoid receptor nuclear translocation or DNA binding was observed in any of the areas analysed. These findings reveal differential effects of RU486 and S-P on areas involved in regulation of hypothalamic–pituitary–adrenal axis activity in vivo and they are important in light of the potential use of this class of compounds in the treatment of disorders associated with hyperactivity of the hypothalamic–pituitary–adrenal axis.

Effect of the glucocorticoid receptor antagonist Org 34850 on fast and delayed feedback of corticosterone release

We investigated the effect of the glucocorticoid receptor (GR) antagonist Org 34850 on fast and delayed inhibition of corticosterone secretion in response to the synthetic glucocorticoid methylprednisolone (MPL). Male rats were implanted with a catheter in the right jugular vein, for blood sampling and MPL administration, and with an s.c. cannula for Org 34850 administration. All experiments were conducted at the diurnal hormonal peak in the late afternoon. Rats were connected to an automated sampling system and blood samples were collected every 5 or 10 min. Org 34850 (10 mg/kg, s.c.) or vehicle (5% mulgofen in saline) was injected at 1630 h; 30 min later, rats received an injection of MPL (500 microg/rat, i.v.) or saline (0.1 ml/rat). We found that an acute administration of MPL rapidly decreased the basal corticosterone secretion and this effect was not prevented by acute pretreatment with Org 34850. However, blockade of GR with Org 34850 prevented delayed inhibition of MPL on corticosterone secretion measured between 4 and 12 h after MPL administration. Our data suggest an involvement of GR in modulating delayed, but not fast, inhibition induced by MPL on basal corticosterone secretion.

Synthesis and SAR studies of novel 2-(6-aminomethylaryl-2-aryl-4-oxo-quinazolin-3(4H)-yl)acetamide vasopressin V1b receptor antagonists.

Synthesis and structure-activity relationships (SAR) of a novel series of vasopressin V(1b) antagonists are described. 2-(6-Aminomethylaryl-2-aryl-4-oxo-quinazolin-3(4H)-yl)acetamide have been identified with low nanomolar affinity for the V(1b) receptor and good selectivity with respect to related receptors V(1a), V(2) and OT. Optimised compound 16 shows a good pharmacokinetic profile and activity in a mechanistic model of HPA dysfunction.

Synthesis and SAR studies of novel 2-(4-oxo-2-aryl-quinazolin-3(4H)-yl)acetamide vasopressin V1b receptor antagonists

Synthesis and structure-activity relationships (SAR) of a novel series of vasopressin V(1b) (V(3)) antagonists are described. 2-(4-Oxo-2-aryl-quinazolin-3(4H)-yl)acetamides have been identified with low nanomolar affinity for the V(1b) receptor and good selectivity with respect to related receptors V(1a), V(2) and oxytocin (OT). Optimised compound 12j demonstrates a good pharmacokinetic profile and activity in a mechanistic model of HPA dysfunction.

The discovery of novel 8-azabicyclo[3.2.1]octan-3-yl)-3-(4-chlorophenyl) propanamides as vasopressin V1A receptor antagonists

The discovery of a novel series of 8-azabicyclo[3.2.1]octan-3-yl)-3-(4-chlorophenyl) propanamide antagonists of the vasopressin V(1A) receptor is disclosed. Compounds 47 and 48 were found to be high affinity, selective vasopressin V(1A) antagonists.