Pharmacokinetic and Pharmacologic Characterization of the Dihydrotetrabenazine Isomers of Deutetrabenazine and Valbenazine.

Valbenazine and deutetrabenazine are vesicular monoamine transporter 2 (VMAT2) inhibitors approved for tardive dyskinesia. The clinical activity of valbenazine is primarily attributed to its only dihydrotetrabenazine (HTBZ) metabolite, [+]-α-HTBZ. Deutetrabenazine is a deuterated form of tetrabenazine and is metabolized to four deuterated HTBZ metabolites: [+]-α-deuHTBZ, [+]-ß-deuHTBZ, [-]-α-deuHTBZ, and [-]-ß-deuHTBZ. An open-label, crossover study characterized the pharmacokinetic profiles of the individual deuHBTZ metabolites, which have not been previously reported. VMAT2 inhibition and off-target interactions of the deuHTBZ metabolites were evaluated using radioligand binding. The only valbenazine HTBZ metabolite, [+]-α-HTBZ, was a potent VMAT2 inhibitor, with negligible affinity for off-target dopamine, serotonin, and adrenergic receptors. Following deutetrabenazine administration, [-]-α-deuHTBZ represented 66% of circulating deuHTBZ metabolites and was a relatively weak VMAT2 inhibitor with appreciable affinity for dopamine (D2S , D3 ) and serotonin (5-HT1A , 5-HT2B , 5-HT7 ) receptors. [+]-ß-deuHTBZ was the most abundant deuHTBZ metabolite that potently inhibited VMAT2, but it represented only 29% of total circulating deuHTBZ metabolites. The mean half-life of [+]-α-HTBZ (22.2 hours) was ∼3× longer than that of [+]-ß-deuHTBZ (7.7 hours). These findings are similar to studies with tetrabenazine, in that deutetrabenazine is metabolized to four deuHTBZ stereoisomers, the most abundant of which has negligible interaction with VMAT2 in vitro and appreciable affinity for several off-target receptors. In contrast, valbenazine's single HTBZ metabolite is a potent VMAT2 inhibitor in vitro with no discernible off-target activity. Determination of the effects of intrinsic/extrinsic variables on deutetrabenazine's safety/efficacy profile should incorporate assessment of the effects on all deuHTBZ metabolites.

Efficacy of Vesicular Monoamine Transporter 2 Inhibition and Synergy with Antipsychotics in Animal Models of Schizophrenia.

Antipsychotic medications function by blocking postsynaptic dopaminergic signaling in the central nervous system. Dopamine transmission can also be modulated presynaptically by inhibitors of vesicular monoamine transporter 2 (VMAT2), which inhibit loading of dopamine into presynaptic vesicles. Here we investigated the combination of these mechanisms in animal models of schizophrenia and weight gain (a primary side effect of antipsychotics). When dosed alone, the highly selective VMAT2 inhibitor RRR-dihydrotetrabenazine (RRR-DHTBZ, also known as [+]-α-HTBZ) elicited efficacy comparable to conventional antipsychotics in prepulse inhibition and conditioned avoidance models without eliciting weight gain. In combination experiments, synergy was observed: subthreshold doses of RRR-DHTBZ and risperidone or olanzapine produced robust efficacy, and in dose response experiments, RRR-DHTBZ increased the antipsychotic potency in the efficacy models but did not affect weight gain. The combinations did not affect plasma compound concentrations. The synergy is consistent with VMAT2 inhibition blocking the counterproductive presynaptic stimulation of dopamine by antipsychotics. These results suggest a therapeutic strategy of adding a VMAT2 inhibitor to lower the antipsychotic dose and reduce the side effect burden of the antipsychotic while maintaining and potentially enhancing its therapeutic effects. SIGNIFICANCE STATEMENT: Antipsychotics are often necessary and life-changing medications that reduce psychotic symptoms; however, these benefits come with a high side effect burden. This study shows that combining these postsynaptic dopaminergic modulators with a presynaptic dopamine modulator (vesicular monoamine transporter 2 [VMAT2] inhibitor) potentiates efficacy synergistically in animal models of schizophrenia without potentiating weight gain. Our data suggest that adding a VMAT2 inhibitor may be a viable therapeutic strategy for reducing antipsychotic side effects by lowering antipsychotic dose while maintaining therapeutic efficacy.

Effects of NBI-98782, a selective vesicular monoamine transporter 2 (VMAT2) inhibitor, on neurotransmitter efflux and phencyclidine-induced locomotor activity: Relevance to tardive dyskinesia and antipsychotic action.

Valbenazine, a vesicular monoamine transporter 2 (VMAT2, SLC18A2) inhibitor, is a newly approved treatment for tardive dyskinesia. VMAT2 is present in the membrane of secretory vesicles and transports dopamine (DA), norepinephrine (NE), serotonin (5-HT), histamine, glutamate (Glu), and GABA into vesicles for presynaptic release. We utilized microdialysis in awake, freely moving mice to determine the effect of NBI-98782, the active metabolite of valbenazine, alone, or in combination with several antipsychotic drugs (APDs), to influence neurotransmitter efflux in the medial prefrontal cortex (mPFC), dorsal striatum (dSTR), hippocampus and nucleus accumbens (NAC); we also compared it with tetrabenazine, the prototypical VMAT2 inhibitor. Acute NBI-98782 and tetrabenazine decreased mPFC, dSTR, hippocampus, and NAC DA, 5-HT, and NE efflux, while increasing that of DOPAC, HVA, and 5-HIAA. Sub-chronic NBI-98782 (7 days) decreased baseline DA and 5-HT efflux in both mPFC and dSTR. NBI-98782 elicited similar effects on neurotransmitter efflux in sub-chronic NBI-98782-treated mice but also enhanced ACh and GABA; the decrease in DA efflux in mPFC and dSTR was not significant in the sc-treated animals. NBI-98782 suppressed clozapine-, olanzapine- and risperidone-induced DA efflux in both mPFC and dSTR, and ACh efflux in mPFC. NBI-98782 suppressed the haloperidol-induced DA efflux in dSTR, with minimal effect on GABA efflux. NBI-98782 attenuated PCP-induced DA, 5-HT, NE and Glu efflux, and AMPH-induced DA and NE efflux, in both mPFC and dSTR, as well as PCP- and AMPH-induced hyperlocomotion, suggesting possible beneficial antipsychotic effects.

The importance of target binding kinetics for measuring target binding affinity in drug discovery: a case study from a CRF1 receptor antagonist program.

In drug discovery, it is essential to accurately measure drug-target binding affinity. Here, we revisit the fact that target binding kinetics impact the measurement of affinity, using a case study: development of corticotropin-releasing factor antagonists. Slow dissociation of the drug-target complex results in affinity assays being far from equilibrium, which results in erroneous estimates of affinity. This scenario can impair prediction of human dosing, assessment of target selectivity, identification of high-affinity ligands and determination of SAR. We describe strategies to detect lack of equilibration in affinity assays and methods to correctly measure affinity of slowly dissociating compounds. These considerations will facilitate drug discovery by ensuring reliable measurement of drug-target binding affinity.

VMAT2 Inhibitors and the Path to Ingrezza (Valbenazine).

The dopaminergic system plays a key role in the central nervous system, regulating executive function, arousal, reward, and motor control. Dysregulation of this critical monoaminergic system has been associated with diseases of the central nervous system including schizophrenia, Parkinson's disease, and disorders such as attention deficit hyperactivity disorders and addiction. Drugs that modify the dopaminergic system by modulating the activity of dopamine have been successful in demonstrating clinical efficacy by providing treatments for these diseases. Specifically, antipsychotics, both typical and atypical, while acting on a number of monoaminergic systems in the brain, primarily target the dopamine system via inhibition of postsynaptic dopamine receptors. The vesicular monoamine transporter 2 (VMAT2) is an integral presynaptic protein that regulates the packaging and subsequent release of dopamine and other monoamines from neuronal vesicles into the synapse. Despite acting on opposing sides of the synapse, both antipsychotics and VMAT2 inhibitors act to decrease the activity of central dopaminergic systems. Tardive dyskinesia is a disorder characterized by involuntary repetitive movements and thought to be a result of a hyperdopaminergic state precipitated by the use of antipsychotics. Valbenazine (NBI-98854), a novel compound that selectively inhibits VMAT2 through an active metabolite, has been developed for the treatment of tardive dyskinesia and is the first drug approved for the treatment of this disorder. This chapter describes the process leading to the discovery of valbenazine, its pharmacological characteristics, along with preclinical and clinical evidence of its efficacy.

Differences in Dihydrotetrabenazine Isomer Concentrations Following Administration of Tetrabenazine and Valbenazine.

BACKGROUND: Tetrabenazine (TBZ) activity is thought to result from four isomeric dihydrotetrabenazine (HTBZ) metabolites ([+]-α-HTBZ, [-]-α-HTBZ, [+]-ß-HTBZ, [-]-ß-HTBZ). Each isomer has a unique profile of vesicular monoamine transporter 2 (VMAT2) inhibition and off-target binding. Previously published data only report total isomer (α) and (ß) concentrations. We developed a method to quantify the individual HTBZ isomers in samples from patients with Huntington's disease receiving TBZ. For comparison, concentrations of [+]-α-HTBZ, the single active metabolite shared by valbenazine (VBZ) and TBZ, were determined in samples from patients with tardive dyskinesia receiving VBZ. METHODS: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitation of the four individual HTBZ isomers. Concentrations were determined in serum from patients with Huntington's disease administered TBZ and plasma from patients with tardive dyskinesia administered VBZ once daily. RESULTS: In patients administered TBZ, [-]-α-HTBZ and [+]-ß-HTBZ were the most abundant HTBZ isomers while [-]-ß-HTBZ and [+]-α-HTBZ were present as minor metabolites. Only [+]-α-HTBZ was observed in patients administered VBZ. CONCLUSIONS: Based on relative abundance and potency, [+]-ß-HTBZ appears to be the primary contributor to VMAT2 inhibition by TBZ, a finding in contrast with the generally held assertion that [+]-α-HTBZ is the major contributor. [-]-α-HTBZ, the other abundant TBZ metabolite, has much lower VMAT2 inhibitory potency than [+]-ß-HTBZ, but increased affinity for other CNS targets, which may contribute to off-target effects of TBZ. In contrast, pharmacological activity for VBZ is derived primarily from [+]-α-HTBZ. Individual HTBZ isomer concentrations provide a more clinically relevant endpoint for assessing on- and off-target effects of TBZ than total isomer concentrations.

Pharmacologic Characterization of Valbenazine (NBI-98854) and Its Metabolites.

The vesicular monoamine transporter 2 (VMAT2) is an integral presynaptic protein that regulates the packaging and subsequent release of dopamine and other monoamines from neuronal vesicles into the synapse. Valbenazine (NBI-98854), a novel compound that selectively inhibits VMAT2, is approved for the treatment of tardive dyskinesia. Valbenazine is converted to two significant circulating metabolites in vivo, namely, (+)-α-dihydrotetrabenazine (R,R,R-HTBZ) and a mono-oxy metabolite, NBI-136110. Radioligand-binding studies were conducted to assess and compare valbenazine, tetrabenazine, and their respective metabolites in their abilities to selectively and potently inhibit [3H]-HTBZ binding to VMAT2 in rat striatal, rat forebrain, and human platelet homogenates. A broad panel screen was conducted to evaluate possible off-target interactions of valbenazine, R,R,R-HTBZ, and NBI-136110 at >80 receptor, transporter, and ion channel sites. Radioligand binding showed R,R,R-HTBZ to be a potent VMAT2 inhibitor in homogenates of rat striatum (Ki = 1.0-2.8 nM), rat forebrain (Ki = 4.2 nM), and human platelets (Ki = 2.6-3.3 nM). Valbenazine (Ki = 110-190 nM) and NBI-136110 (Ki = 160-220 nM) also exhibited inhibitory effects on VMAT2, but with lower potency than R,R,R-HTBZ. Neither valbenazine, R,R,R-HTBZ, nor NBI-136110 had significant off-target interactions at serotonin (5-HT1A, 5-HT2A, 5-HT2B) or dopamine (D1 or D2) receptor sites. In vivo studies measuring ptosis and prolactin secretion in the rat confirmed the specific and dose-dependent interactions of tetrabenazine and R,R,R-HTBZ with VMAT2. Evaluations of potency and selectivity of tetrabenazine and its pharmacologically active metabolites were also performed. Overall, the pharmacologic characteristics of valbenazine appear consistent with the favorable efficacy and tolerability findings of recent clinical studies [KINECT 2 (NCT01733121), KINECT 3 (NCT02274558)].

Non-peptide CRF-Receptor Antagonists: Allosterism, Kinetics and Translation to Efficacy in Human Disease.

G-Protein coupled receptors (GPCRs) have been, and remain a key target of drug discovery programs for human disease. While many drugs have been developed that interact with these proteins in the simple classic manner - that is - physically blocking the cognate ligand from simply binding to its target receptor, drug discovery approaches have elucidated alternative more complex methods by which small molecules can interact with these receptors and block their function. This is most evident in the Class B GPCRs where the cognate ligands are relatively large peptides with multiple points of contact on the GPCR spanning both hydrophilic and hydrophobic domains on the same protein to elicit function. It has therefore been difficult to precisely determine not only the mechanism by which a small molecule can inhibit the function of a large peptide but also the nature of that mechanism that drives the differences in efficacy. This review will examine in detail the nature of small molecule inhibition of corticotropin-releasing factor receptors and illustrate the role that allosteric binding and kinetics play in the functional inhibition of this Class B GPCR.

The CRF1 Antagonist Verucerfont in Anxious Alcohol-Dependent Women: Translation of Neuroendocrine, But not of Anti-Craving Effects.

Blockade of corticotropin-releasing factor receptor 1 (CRF1) suppresses stress-induced alcohol seeking in rodents, but clinical translation remains. Here, we first showed that the CRF1 antagonist verucerfont potently blocks hypothalamic-pituitary adrenal (HPA) axis activation in adrenalectomized rats. We then evaluated verucerfont for its ability to block HPA axis activation and reduce stress-induced alcohol craving in alcohol-dependent patients. Anxious, alcohol-dependent women (age 21-65 years, n=39) were admitted to the NIH Clinical Center and completed withdrawal treatment before enrollment if needed. One-week single-blind placebo was followed by randomized double-blind verucerfont (350 mg per day) or placebo for 3 weeks. Verucerfont effects on the HPA axis were evaluated using the dexamethasone-CRF test. Craving was evaluated using two established protocols, one that combines a social stressor with physical alcohol cue exposure, and one that uses guided imagery to present personalized stress, alcohol, or neutral stimuli. An fMRI session examined brain responses to negative affective stimuli and alcohol cues. In contrast to our recent observations with another CRF1 antagonist, pexacerfont, verucerfont potently blocked the HPA axis response to the dexamethasone-CRF test, but left alcohol craving unaffected. Right amygdala responses to negative affective stimuli were significantly attenuated by verucerfont, but responses to alcohol-associated stimuli were increased in some brain regions, including left insula. Discontinuation rates were significantly higher in the verucerfont group. Our findings provide the first translational evidence that CRF1 antagonists with slow receptor dissociation kinetics may have increased efficacy to dampen HPA axis responses. The findings do not support a clinical efficacy of CRF1 blockade in stress-induced alcohol craving and relapse.

Single-Dose Study of a Corticotropin-Releasing Factor Receptor-1 Antagonist in Women With 21-Hydroxylase Deficiency.

CONTEXT: Treatment of 21-hydroxylase deficiency (21OHD) is difficult to optimize. Normalization of excessive ACTH and adrenal steroid production commonly requires supraphysiologic doses of glucocorticoids. OBJECTIVES: We evaluated the safety and tolerability of the selective corticotropin releasing factor type 1 (CRF1) receptor antagonist NBI-77860 in women with classic 21OHD and tested the hypothesis that CRF1 receptor blockade decreases early-morning ACTH and 17α-hydroxyprogesterone (17OHP) in these patients. PARTICIPANTS: The study enrolled eight classic 21OHD females, ages 18-58 years, seen at a single tertiary referral university setting. DESIGN: This was a phase Ib, single-blind, placebo-controlled, fixed-sequence, single-dose trial. During three treatment periods separated by 3-week washout intervals, patients sequentially received placebo, NBI-77860 300 mg, and NBI-77860 600 mg at 10 pm; glucocorticoid therapy was withheld for 20 hours. We evaluated ACTH, 17OHP, androstenedione, and testosterone as well as NBI-77860 pharmacokinetics over 24 hours. RESULTS: Dose-dependent reductions of ACTH and/or 17OHP were observed in six of eight subjects. Relative to placebo, NBI-77860 led to an ACTH and 17OHP reduction by a mean of 43% and 0.7% for the 300 mg dose, respectively, and by 41% and 27% for the 600 mg dose, respectively. Both NBI-77860 doses were well tolerated. CONCLUSION: The meaningful reductions in ACTH and 17OHP following NBI-77860 dosing in 21OHD patients demonstrate target engagement and proof of principle in this disorder. These promising data provide a rationale for additional investigations of CRF1 receptor antagonists added to physiologic doses of hydrocortisone and fludrocortisone acetate for the treatment of classic 21OHD.

Corticotropin-releasing factor receptor subtype 2 in human colonic mucosa: down-regulation in ulcerative colitis.

AIM: To assess corticotropin-releasing factor receptor 2 (CRF2) expression in the colon of healthy subjects and patients with ulcerative colitis (UC). METHODS: We examined CRF2 gene and protein expression in the distal/sigmoid colonic mucosal biopsies from healthy subjects and patients with UC (active or disease in remission), human immunodeficiency virus (HIV) and functional bowel disease (FBD) by reverse transcription-polymerase chain reaction and immunofluorescence. RESULTS: Gene expression of CRF2 was demonstrated in the normal human colonic biopsies, but not in the human colorectal adenocarcinoma cell line Caco2. Receptor protein localization showed immunoreactive CRF2 receptors in the lamina propria and in the epithelial cells of the distal/sigmoid biopsy samples. Interestingly, CRF2 immunoreactivity was no longer observed in epithelial cells of patients with mild-moderately active UC and disease in remission, while receptor protein expression did not change in the lamina propria. No differences in CRF2 expression profile were observed in distal/sigmoid intestinal biopsies from HIV infection and FBD patients, showing no signs of inflammation. CONCLUSION: The down-regulation of the CRF2 receptor in the distal/sigmoid biopsies of UC patients is indicative of change in CRF2 signalling associated with the process of inflammation.

Binding kinetics redefine the antagonist pharmacology of the corticotropin-releasing factor type 1 receptor.

Corticotropin-releasing factor (CRF) receptor antagonists are under preclinical and clinical investigation for stress-related disorders. In this study the impact of receptor-ligand binding kinetics on CRF1 receptor antagonist pharmacology was investigated by measuring the association rate constant (k1), dissociation rate constant (k₋1), and kinetically derived affinity at 37°C. Three aspects of antagonist pharmacology were reevaluated: comparative binding activity of advanced compounds, in vivo efficacy, and structure-activity relationships. Twelve lead compounds, with little previously noted difference of affinity, varied substantially in their kinetic binding activity with a 510-fold range of kinetically derived affinity (k₋1/k1), 170-fold range of k₋1, and 13-fold range of k1. The k₋1 values indicated previous affinity measurements were not close to equilibrium, resulting in compression of the measured affinity range. Dissociation was exceptionally slow for three ligands (k₋1 t(1/2) of 1.6-7.2 h at 37°C). Differences of binding behavior were consistent with in vivo pharmacodynamics (suppression of adrenocorticotropin in adrenalectomized rats). Ligand concentration-effect relationships correlated with their kinetically derived affinity. Two ligands that dissociated slowly (53 and 130 min) produced prolonged suppression, whereas only transient suppression was observed with a more rapidly dissociating ligand (16 min). Investigating the structure-activity relationship indicated exceptionally low values of k1, approaching 100,000-fold less than the diffusion-limited rate. Retrospective interpretation of medicinal chemistry indicates optimizing specific elements of chemical structure overcame kinetic barriers in the association pathway, for example, constraint of the pendant aromatic orthogonal to the ligand core. Collectively, these findings demonstrate receptor binding kinetics provide new dimensions for understanding and potentially advancing the pharmacology of CRF1 receptor antagonists.

Discovery of NBI-77860/GSK561679, a potent corticotropin-releasing factor (CRF1) receptor antagonist with improved pharmacokinetic properties.

Antagonists of the corticotropin-releasing factor (CRF) neuropeptide may prove effective in treating stress and anxiety related disorders. In an effort to identify antagonists with improved physico-chemical properties a new series of CRF(1) antagonists were designed to substitute the propyl groups at the C7 position of the pyrazolo[1,5-a]pyrimidine core of 1 with heterocycles. Compound (S)-8d was identified as a high affinity ligand with a pK(i) value of 8.2 and a functional CRF(1) antagonist with pIC(50) value of 7.0 in the in vitro CRF ACTH production assay.

Hippocampal dysfunction and cognitive impairments provoked by chronic early-life stress involve excessive activation of CRH receptors.

Chronic stress impairs learning and memory in humans and rodents and disrupts long-term potentiation (LTP) in animal models. These effects are associated with structural changes in hippocampal neurons, including reduced dendritic arborization. Unlike the generally reversible effects of chronic stress on adult rat hippocampus, we have previously found that the effects of early-life stress endure and worsen during adulthood, yet the mechanisms for these clinically important sequelae are poorly understood. Stress promotes secretion of the neuropeptide corticotropin-releasing hormone (CRH) from hippocampal interneurons, activating receptors (CRF(1)) located on pyramidal cell dendrites. Additionally, chronic CRF(1) occupancy negatively affects dendritic arborization in mouse organotypic slice cultures, similar to the pattern observed in middle-aged, early-stressed (CES) rats. Here we found that CRH expression is augmented in hippocampus of middle-aged CES rats, and then tested whether the morphological defects and poor memory performance in these animals involve excessive activation of CRF(1) receptors. Central or peripheral administration of a CRF(1) blocker following the stress period improved memory performance of CES rats in novel-object recognition tests and in the Morris water maze. Consonant with these effects, the antagonist also prevented dendritic atrophy and LTP attenuation in CA1 Schaffer collateral synapses. Together, these data suggest that persistently elevated hippocampal CRH-CRF(1) interaction contributes importantly to the structural and cognitive impairments associated with early-life stress. Reducing CRF(1) occupancy post hoc normalized hippocampal function during middle age, thus offering potential mechanism-based therapeutic interventions for children affected by chronic stress.

CRF system recruitment mediates dark side of compulsive eating.

Dieting to control body weight involves cycles of deprivation from palatable food that can promote compulsive eating. The present study shows that rats withdrawn from intermittent access to palatable food exhibit overeating of palatable food upon renewed access and an affective withdrawal-like state characterized by corticotropin-releasing factor-1 (CRF(1)) receptor antagonist-reversible behaviors, including hypophagia, motivational deficits to obtain less palatable food, and anxiogenic-like behavior. Withdrawal was accompanied by increased CRF expression and CRF(1) electrophysiological responsiveness in the central nucleus of the amygdala. We propose that recruitment of anti-reward extrahypothalamic CRF-CRF(1) systems during withdrawal from palatable food, analogous to abstinence from abused drugs, may promote compulsive selection of palatable food, undereating of healthier alternatives, and a negative emotional state when intake of palatable food is prevented.

Drugability of extracellular targets: discovery of small molecule drugs targeting allosteric, functional, and subunit-selective sites on GPCRs and ion channels.

Beginning with the discovery of the structure of deoxyribose nucleic acid in 1953, by James Watson and Francis Crick, the sequencing of the entire human genome some 50 years later, has begun to quantify the classes and types of proteins that may have relevance to human disease with the promise of rapidly identifying compounds that can modulate these proteins so as to have a beneficial and therapeutic outcome. This so called 'drugable space' involves a variety of membrane-bound proteins including the superfamily of G-protein-coupled receptors (GPCRs), ion channels, and transporters among others. The recent number of novel therapeutics targeting membrane-bound extracellular proteins that have reached the market in the past 20 years however pales in magnitude when compared, during the same timeframe, to the advancements made in the technologies available to aid in the discovery of these novel therapeutics. This review will consider select examples of extracellular drugable targets and focus on the GPCRs and ion channels highlighting the corticotropin releasing factor (CRF) type 1 and gamma-aminobutyric acid receptors, and the Ca(V)2.2 voltage-gated ion channel. These examples will elaborate current technological advancements in drug discovery and provide a prospective framework for future drug development.

Allosteric ligands for the corticotropin releasing factor type 1 receptor modulate conformational states involved in receptor activation.

Allosteric modulators of G-protein-coupled receptors can regulate conformational states involved in receptor activation ( Mol Pharmacol 58: 1412-1423, 2000 ). This hypothesis was investigated for the corticotropin-releasing factor type 1 (CRF(1)) receptor using a novel series of ligands with varying allosteric effect on CRF binding (inhibition to enhancement). For the G-protein-uncoupled receptor, allosteric modulation of CRF binding was correlated with nonpeptide ligand signaling activity; inverse agonists inhibited and agonists enhanced CRF binding. These data were quantitatively consistent with a two-state equilibrium underlying the modulation of CRF binding to the G-protein-uncoupled receptor. We next investigated the allosteric effect on CRF-stimulated G-protein coupling. Ligands inhibited CRF-stimulated cAMP accumulation regardless of their effect on the G-protein-uncoupled state. The modulators reduced CRF E(max) values, suggesting that they reduced the efficacy of a CRF-bound active state to couple to G-protein. Consistent with this hypothesis, the modulators inhibited binding to a guanine nucleotide-sensitive state. Together, the results are quantitatively consistent with a model in which 1) the receptor exists in three predominant states: an inactive state, a weakly active state, and a CRF-bound fully active state; 2) allosteric inverse agonists stabilize the inactive state, and allosteric agonists stabilize the weakly active state; and 3) antagonism of CRF signaling results from destabilization of the fully active state. These findings imply that nonpeptide ligands differentially modulate conformational states involved in CRF(1) receptor activation and suggest that different conformational states can be targeted in designing nonpeptide ligands to inhibit CRF signaling.

High-affinity CRF1 receptor antagonist NBI-34041: preclinical and clinical data suggest safety and efficacy in attenuating elevated stress response.

There is an extensive evidence that corticotropin releasing factor (CRF) is hypersecreted in depression and anxiety, and blockade of CRF could have therapeutic benefit. We report preclinical data and the results of a clinical Phase I study with the novel nonpeptide CRF(1) antagonist NBI-34041/SB723620. Preclinical data conducted with different cell lines expressing human CRF receptors and in Wistar and Sprague-Dawley rats indicate that NBI-34041 is effective in reducing endocrine responses to pharmacological and behavioral challenge mediated by CRF(1) receptors. These specific properties and its well-documented safety profile enabled a clinical Phase I study with 24 healthy male subjects receiving NBI-34041 (10, 50, or 100 mg) or placebo for 14 days. Regulation of the hypothalamic-pituitary-adrenocortical (HPA) axis was evaluated by intravenous stimulation with 100 microg of human CRF. Psychosocial stress response was investigated with the Trier Social Stress Test (TSST). Treatment with NBI-34041 did not impair diurnal adrenocorticotropic hormone (ACTH) and cortisol secretion or CRF evoked ACTH and cortisol responses but attenuated the neuroendocrine response to psychosocial stress. These results suggest that NBI-34041 is safe and does not impair basal regulation of the HPA system but improves resistance against psychosocial stress. NBI-34041 demonstrates that inhibition of the CRF system is a promising target for drug development against depression and anxiety disorders.

Cocaine withdrawal enhances long-term potentiation induced by corticotropin-releasing factor at central amygdala glutamatergic synapses via CRF, NMDA receptors and PKA.

Cocaine addiction is an enduring, relapsing, behavioural disorder in which stressors reinstate cocaine-seeking even after prolonged abstinence. Evidence suggests that the 'anxiety-like' behaviour and stress associated with protracted withdrawal may be mediated by increased corticotropin-releasing factor (CRF) in the central nucleus of the amygdala (CeA), a part of the limbic circuitry engaged in the coding and transmission of stimulus-reward associations. In the present study we describe a long-lasting potentiation of glutamatergic transmission induced at lateral amygdala (LA)-to-CeA synapses by rat/human CRF. After 2 weeks of withdrawal from repeated intermittent exposure to cocaine, CRF-induced long-term potentiation (LTP) was greatly enhanced compared to the respective saline control group while, after short-term withdrawal (24 h), there was no significant difference between the two treatment groups, indicating alterations in CRF systems during protracted withdrawal from chronic cocaine. After prolonged withdrawal, CRF-induced LTP was dependent on activation of CRF2, CaV2.3 (R-type) calcium channels and intracellular signalling through protein kinase C in both saline- and cocaine-treated groups. The enhanced CRF-induced LTP after 2 weeks of withdrawal was mediated through augmented CRF1 receptor function, associated with an increased signalling through protein kinase A, and required N-methyl-D-aspartate (NMDA) receptors. Accordingly, single-cell recordings revealed a significantly increased NMDA/AMPA ratio after prolonged withdrawal from the cocaine treatment. These results support a role for CRF1 receptor antagonists as plausible treatment options during withdrawal from chronic cocaine and suggest Ca(V)2.3 blockers as potential candidates for pharmaceutical modulation of CRF systems.

Corticotropin-releasing factor (CRF) receptor type 2 in the human stomach: protective biological role by inhibition of apoptosis.

Corticotropin-releasing factor agonists exert inhibitory effects in stomach functions possibly through peripheral routes. We have previously reported the expression of Urocortin (Ucn) I, an endogenous ligand of both CRF receptor types CRF(1) and CRF(2), in the human stomach. We examined CRF(1) and CRF(2) expression in the same tissue. Using RT-PCR, CRF(2) but not CRF(1) transcripts were detected in RNA extracts from normal human stomach. In addition, immunohistochemical analysis revealed receptor protein in epithelial gastric cells. In order to investigate the biological role of CRF(2) in these cells, an in vitro model was established, using the gastric cancer cell line AGS transiently transfected to express functional CRF(2). The effect of the CRF(2) endogenous ligands CRF, Ucns I and II on the growth parameters of the AGS/CRF(2) was examined. After 1 day of exposure, all three ligands reduced the degree of apoptosis (16%-19%, n = 9, P < 0.05) compared to non-treated controls and this effect was observed for 3 days of treatment. No such effect was detected in non-transfected cells, suggesting mediation through CRF(2) receptors. Administration of CRF, Ucns I and II had no effect on the proliferation rate of AGS/CRF(2) cells or on the release of PGE(2) by them. Our results demonstrate CRF(2) expression in the human gastric mucosa and indicate a physiological role of this receptor type in regulating apoptosis, an important parameter of gastric cell regeneration. Paracrine effects exerted by locally expressed endogenous ligands, such as Ucn I, support a significant role of the peripheral CRF system in gastric physiology. J. Cell. Physiol. 209: 905-911, 2006. (c) 2006 Wiley-Liss, Inc.