BNC210, a negative allosteric modulator of the alpha 7 nicotinic acetylcholine receptor, demonstrates anxiolytic- and antidepressant-like effects in rodents.

This work describes the characterization of BNC210 (6-[(2,3-dihydro-1H-inden-2-yl)amino]-1-ethyl-3-(4-morpholinylcarbonyl)-1,8-naphthyridin-4(1H)-one), a selective, small molecule, negative allosteric modulator (NAM) of α7 nicotinic acetylcholine receptors (α7 nAChR). With the aim to discover a non-sedating, anxiolytic compound, BNC210 was identified during phenotypic screening of a focused medicinal chemistry library using the mouse Light Dark (LD) box to evaluate anxiolytic-like activity and the mouse Open Field (OF) (dark) test to detect sedative and/or motor effects. BNC210 exhibited anxiolytic-like activity with no measurable sedative or motor effects. Electrophysiology showed that BNC210 did not induce α7 nAChR currents by itself but inhibited EC80 agonist-evoked currents in recombinant GH4C1 cell lines stably expressing the rat or human α7 nAChR. BNC210 was not active when tested on cell lines expressing other members of the cys-loop ligand-gated ion channel family. Screening over 400 other targets did not reveal any activity for BNC210 confirming its selectivity for α7 nAChR. Oral administration of BNC210 to male mice and rats in several tests of behavior related to anxiety- and stress- related disorders, demonstrated significant reduction of these behaviors over a broad therapeutic range up to 500 times the minimum effective dose. Further testing for potential adverse effects in suitable rat and mouse tests showed that BNC210 did not produce sedation, memory and motor impairment or physical dependence, symptoms associated with current anxiolytic therapeutics. These data suggest that allosteric inhibition of α7 nAChR function may represent a differentiated approach to treating anxiety- and stress- related disorders with an improved safety profile compared to current treatments.

Novel therapeutic modalities to address nondrugable protein interaction targets.

Small molecule drugs are relatively effective in working on 'drugable' targets such as GPCRs, ion channels, kinases, proteases, etc but ineffective at blocking protein-protein interactions that represent an emerging class of 'nondrugable' central nervous system (CNS) targets. This article provides an overview of novel therapeutic modalities such as biologics (in particular antibodies) and emerging oligonucleotide therapeutics such as antisense, small-interfering RNA, and aptamers. Their key properties, overall strengths and limitations, and their utility as tools for target validation are presented. In addition, issues with regard to CNS targets as it relates to the blood-brain barrier penetration are discussed. Finally, examples of their application as therapeutics for the treatment of pain and some neurological disorders such as Alzheimer's disease, multiple sclerosis, Huntington's disease, and Parkinson's disease are provided.

Urocortin shares the memory modulating effects of corticotropin-releasing factor (CRF): mediation by CRF1 receptors.

Intracerebroventricular (i.c.v.) administration of corticotropin-releasing factor (CRF) biphasically affects performance in tests of learning and memory. In the present study, we used CRF, urocortin (Ucn), a recently cloned CRF homologue, and CRF receptor antagonists, to determine which CRF receptor subtype(s) mediate the memory modulating effects of CRF receptor agonists in male Wistar rats. Under difficult learning conditions (massed trials), i.c.v. pretreatment with CRF or Ucn facilitated the acquisition of spatial navigation in the Morris water maze in a non-dose-dependent fashion (optimal doses of 0.1 and 0.03 microg, respectively). Under less difficult learning conditions (spaced trials), both peptides impaired water maze performance. In addition, with i.c.v. posttraining treatment, the peptides were equipotent (1.0 microg) in facilitating the consolidation of passive avoidance learning. The performance-enhancing effects of Ucn in both water maze and passive avoidance paradigms were reversed by i.c.v. pretreatment with D-Phe CRF(12-41) (2.5, 5 microg), a broad CRF(1)/CRF(2) receptor antagonist, or antalarmin (10 microg), a potent, nonpeptide, CRF(1) selective receptor antagonist. Thus, Ucn shares CRF's memory-modulating effects, and these effects appear to be mediated via the CRF(1) receptor. These findings are consistent with the hypothesis that CRF receptor agonists affect performance in tests of learning and memory by increasing arousal.

Brain penetrance, receptor occupancy and antistress in vivo efficacy of a small molecule corticotropin releasing factor type I receptor selective antagonist.

The present studies were designed to evaluate the competitive binding properties and functional effects of a novel nonpeptide CRF1 receptor antagonist, R121919. R121919 administered in doses of 0.63 to 20 mg/kg p.o. 60 min pretest in Wistar rats dose dependently attenuated the swim stress-induced anxiogenic-like behavior in the elevated plus-maze model of anxiety. Moreover, receptor autoradiography revealed that R121919 dose-dependently occupied brain CRF1 receptors in subjects tested in the plus-maze experiment. Orally administered doses of up to 20 mg/kg R121919 also blunted basal and swim stress-induced pituitary-adrenocortical activation, produced additional anxiolytic-like behavioral actions in the defensive withdrawal and defensive burying paradigms, and functionally antagonized the locomotor stimulatory properties of exogenously administered CRF. Taken together, these results suggest that the anxiolytic-like efficacy of R121919 in attenuating the stress-, novelty-, shock-, and CRF-induced increases in behavioral arousal is correlated with competitive blockade of central CRF1 receptors.

Quantification of the soluble leptin receptor in human blood by ligand-mediated immunofunctional assay.

Panels of monoclonal antibodies (mAbs) were raised against recombinant human leptin and the recombinant human soluble leptin receptor. Using these mAbs, we established a ligand-mediated immunofunctional assay (LIFA) to quantify concentrations of the soluble leptin receptor, which has been shown to be a major binding protein for leptin in human serum. In performing the assay, a monoclonal antibody (mAb 2H6) against the soluble leptin receptor, which binds an epitope outside the leptin-binding site and equally recognizes both, free and leptin-occupied soluble leptin receptor, is used to capture the soluble leptin receptor on a microtiter plate. Recombinant human leptin is added to saturate all binding sites, and a biotinylated anti-leptin mAb (4D3) detects the amount of leptin (endogenous and exogenous) bound to the soluble leptin receptor. The same procedure, but without adding exogenous leptin, allows for measurement of the circulating endogenous leptin/soluble leptin receptor complexes. The LIFA assay has a linear working range of 0.5-200 microg/liter, intra- and interassay coefficients of variation ranged from 3.2-6.3% and from 5.2-7.9%, respectively. The assay has a linearity of 102.2 +/- 5.2% (mean +/- SD) and a recovery of 100.7 +/- 6.9%. Size-exclusion chromatography revealed that the assay measures a protein with a main peak eluted at 340 kDa. The soluble leptin receptor concentration (63.3 +/- 22.8 microg/liter (mean +/- SD), range 17.9-129.2 microg/liter, n = 43) in normal subjects (body mass index = 22.3 +/- 2.3 kg/m(2)) was not different from the concentration (54.4 +/- 19.8 microg/liter, range 23.7-104.8 microg/liter, n = 34, P > 0.05) found in obese subjects (body mass index = 40.9 +/- 15.7 kg/m(2)). However, the percentage of the total soluble leptin receptor complexed with endogenous leptin was significantly higher in obese subjects, compared with normal subjects (74.9% +/- 23.5% vs. 33.1% +/- 19.5%, P < 0.001). Higher serum leptin levels in obese subjects (38.4 +/- 23.7 microg/liter vs. 7.8 +/- 5.5 microg/liter in normal subjects, P < 0.001) together with comparable soluble leptin receptor levels result in a lower proportion of leptin bound to the soluble leptin receptor in obese subjects (19.3% +/- 19.4%, range 4.9-97.2%) than in normal subjects (39.0% +/- 22.5%, range 15.3-96.5%, P < 0.001). The development of this LIFA for the rapid and accurate quantification of total soluble leptin receptor and circulating leptin/soluble leptin receptor complexes provides a valuable tool for the further understanding of the role of leptin and its soluble receptor in health and disease.