DMP 696

The pharmacology of DMP696 and DMP904, non-peptidergic CRF1 receptor antagonists

CRF(1) antagonists DMP696 and DMP904 were designed as drug development candidates for the treatment of anxiety and depression. Both compounds display nanomolar affinity for human CRF(1) receptors, and exhibit >1000-fold selectivity for CRF(1) over CRF(2) receptors and over a broad panel of other proteins. DMP696 and DMP904 block CRF-stimulated adenylyl cyclase activity in cortical homogenates and cell-lines expressing CRF(1) receptors. Both compounds inhibit CRF-stimulated ACTH release from rat pituitary corticotropes. Binding and functional studies indicate that DMP696 and DMP904 behave as noncompetitive full antagonists. DMP696 and DMP904 exhibit anxiolytic-like efficacy in several rat anxiety models. In the defensive withdrawal test, both compounds reduce exit latency with lowest effective doses of 3 and 1 mg/kg, respectively. The anxiolytic-like effect is maintained over 14 days of repeated dosing. In the context of a novel environment used in this test, DMP696 and DMP904 reverse mild stress-induced increases in plasma CORT secretion but at doses 3-4-fold greater than those required for anxiolyticlike efficacy. DMP696 and DMP904 are ineffective in three depression models including the learned helplessness paradigm at doses up to 30 mg/kg. At lowest anxiolytic-like doses, DMP696 and DMP904 occupy >50% CRF(1) receptors in the brain. The in vivo IC(50) values (plasma concentrations required for occupying 50% CRF(1) receptors) estimated based upon free, but not total, plasma concentrations are an excellent correlation with the in vitro IC(50) values. Neither compound produces sedation, ataxia, chlordiazepoxide-like subjective effects or adverse effects on cognition at doses 10-fold higher than anxiolytic-like doses. Neither compound produces physiologically significant changes in cardiovascular, respiratory, gastrointestinal or renal functions at anxiolytic-like doses. DMP696 and DMP904 have favorable pharmacokinetic profiles with good oral bioavailabilities. The overall pharmacological properties suggest that both compounds may be effective anxiolytics with low behavioral side effect liabilities.

Functional CRF receptors in BON cells stimulate serotonin release

BON cells are human, pancreatic carcinoid-derived, endocrine-like cells that share functional similarities with intestinal enterochromaffin (EC) cells. We investigated the presence of corticotropin-releasing factor (CRF) receptors, their signalling pathways and the functional effects of their stimulation in BON cells (clone #7). Expression analysis showed that BON cells contain mRNA for the CRF receptor types 1 and 2 (CRF1/2), although CRF2 mRNA levels were 23-fold higher than those of CRF1 mRNA. The CRF1/2 ligand, rat/human (r/h)CRF (EC50 = 233 nM), and the selective CRF2 ligand, human urocortin 3 (Ucn 3) (EC50 = 48 nM), induced a dose-dependent increase in cAMP formation. Effects of r/hCRF were blocked by 44% with the selective CRF1 antagonist DMP-696, while the selective CRF2 antagonist antisauvagine-30 had only marginal effects. Both ligands (100 nM) stimulated the release of serotonin with similar efficacy (3-fold increase over basal). Effects of r/hCRF, but not Ucn 3, were blocked by pre-incubation with antisauvagine-30. These observations demonstrate that the EC cell-related BON cells express functional CRF2 receptors linked to the release of serotonin. This suggests that EC cells may be a target for CRF and/or Ucn 3 in the intestine during stress-related responses. Actions of CRF/Ucn 3 and EC cell-derived mediators, such as serotonin, might underlie several motor, secretory and/or sensory disorders of the gastrointestinal (GI) tract which may play a role in the pathophysiology of functional GI disorders, such as irritable bowel syndrome.

Involvement of vasopressin 3 receptors in chronic psychological stress-induced visceral hyperalgesia in rats

Visceral hypersensitivity and stress have been implicated in the pathophysiology of functional gastrointestinal disorders. We used a selective vasopressin 3 (V(3)) receptor antagonist SSR149415 to investigate the involvement of the vasopressin (AVP)/V(3) signaling system in the development of stress-induced visceral hyperalgesia in rats. Rats were exposed to a daily 1-h session of water avoidance stress (WAS) or sham WAS for 10 consecutive days. The visceromotor response to phasic colorectal distension (CRD, 10-60 mmHg) was assessed before and after stress. Animals were treated daily with SSR149415 (0.3, 1, or 3 mg/kg ip 30 min before each WAS or sham WAS session), with a single dose of SSR149415 (1 mg/kg ip), or the selective corticotropin-releasing factor 1 (CRF(1)) antagonist DMP-696 (30 mg/kg po) before CRD at day 11. Effects of a single dose of SSR149415 (10 mg/kg iv) on acute mechanical sensitization during repetitive CRD (12 distensions at 80 mmHg) were also assessed. In vehicle-treated rats, repeated WAS increased the response to CRD, indicating visceral hypersensitivity. Repeated administration of SSR149415 at 1 or 3 mg/kg completely prevented stress-induced visceral hyperalgesia. Similarly, a single dose of DMP-696 or SSR149415 completely blocked hyperalgesic responses during CRD. In contrast, a single dose of SSR149415 did not affect the acute hyperalgesic responses induced by repeated, noxious distension. These data support a major role for V(3) receptors in repeated psychological stress-induced visceral hyperalgesia and suggest that pharmacological manipulation of the AVP/V(3) pathway might represent an attractive alternative to the CRF/CRF(1) pathway for the treatment of chronic stress-related gastrointestinal disorders.

Antidepressant-like activity of corticotropin-releasing factor type-1 receptor antagonists in mice

The development of selective corticotropin-releasing factor type-1 (CRF1) receptor antagonists represents a potential novel treatment for depression. These studies evaluated CRF1 receptor antagonists for antidepressant-like activity in mice. Subchronic dosing of both R 121919 (3-[6-(dimethylamino)-4-methyl-pyrid-3-yl]-2,5-dimethyl-N,N-dipropyl-pyrazolo[2,3-a]pyrimidin-7-amine) and DMP 696 (4-(1,3-dimethoxyprop-2-ylamino)-2,7-dimethyl-8-(2,4-dichlorophenyl)-pyrazolo[1,5-a]-1,3,5-triazine) significantly decreased immobility time in the tail suspension test (at 30 and at 3 and 10 mg/kg, i.p., respectively). These antidepressant-like effects were observed at doses that did not impair general locomotor activity. Neither antalarmin (N-butyl-N-ethyl-[2,5,6-trimethyl-7-(2,4,6)trimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]amine) nor DMP 904 (4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazolo-[1,5-a]-pyrimidine) had an effect indicative of antidepressant-like activity. These results suggest that the tail suspension assay may have utility to identify CRF1 receptor antagonists with antidepressant-like activity. Moreover, the results lend support to the theory that some nonpeptidic CRF1 receptor antagonists may possess antidepressant-like activity and therefore represent a promising novel pharmacotherapeutic strategy in the treatment of depression.

Mechanism of corticotropin-releasing factor type I receptor regulation by nonpeptide antagonists

Mechanisms of nonpeptide ligand action at family B G protein-coupled receptors are largely unexplored. Here, we evaluated corticotropin-releasing factor 1 (CRF(1)) receptor regulation by nonpeptide antagonists. The antagonist mechanism was investigated at the G protein-coupled (RG) and uncoupled (R) states of the receptor in membranes from Ltk(-) cells expressing the cloned human CRF(1) receptor. R was detected with the antagonist (125)I-astressin with 30 microM guanosine 5'-O-(3-thiotriphosphate present, and RG detected using (125)I-sauvagine. At the R state, nonpeptide antagonists antalarmin, NBI 27914, NBI 35965, and DMP-696 only partially inhibited (125)I-astressin binding (22-32% maximal inhibition). NBI 35965 accelerated (125)I-astressin dissociation and only partially increased the IC(50) value of unlabeled sauvagine, CRF, and urocortin for displacing (125)I-astressin binding (by 4.0-7.1-fold). Reciprocal effects at the R state were demonstrated using [(3)H]NBI 35965: agonist peptides only partially inhibited binding (by 13-40%) and accelerated [(3)H]NBI 35965 dissociation. These data are quantitatively consistent with nonpeptide antagonist and peptide ligand binding spatially distinct sites, with mutual, weak negative cooperativity (allosteric inhibition) between their binding. At the RG state the compounds near fully inhibited (125)I-sauvagine binding at low radioligand concentrations (79-94 pM). NBI 35965 did not completely inhibit (125)I-sauvagine binding at high radioligand concentrations (82 +/- 1%, 1.3-2.1 nM) and slowed dissociation of (125)I-sauvagine and (125)I-CRF. The antagonist effect at RG is consistent with either strong allosteric inhibition or competitive inhibition at one of the peptide agonist binding sites. These findings demonstrate a novel effect of R-G interaction on the inhibitory activity of nonpeptide antagonists: Although the compounds are weak inhibitors of peptide binding to the R state, they strongly inhibit peptide agonist binding to RG. Strong inhibition at RG explains the antagonist properties of the compounds.