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.

Development of stable liquid glucagon formulations for use in artificial pancreas.

A promising approach to treat diabetes is the development of fully automated artificial/bionic pancreas systems that use both insulin and glucagon to maintain euglycemia. A physically and chemically stable liquid formulation of glucagon does not currently exist. Our goal is to develop a glucagon formulation that is stable as a clear and gel-free solution, free of fibrils and that has the requisite long-term shelf life for storage in the supply chain, short-term stability for at least 7 days at 37°C, and pump compatibility for use in a bihormonal pump. We report the development of two distinct families of stable liquid glucagon formulations which utilize surfactant or surfactant-like excipients (LMPC and DDM) to "immobilize" the glucagon in solution potentially through the formation of micelles and prevention of interaction between glucagon molecules. Data are presented that demonstrate long-term physical and chemical stability (~2 years) at 5°C, short-term stability (up to 1 month) under accelerated 37°C testing conditions, pump compatibility for up to 9 days, and adequate glucose responses in dogs and diabetic swine. These stable glucagon formulations show utility and promise for further development in artificial pancreas systems.

Ultra-rapid absorption of recombinant human insulin induced by zinc chelation and surface charge masking.

BACKGROUND: In order to enhance the absorption of insulin following subcutaneous injection, excipients were selected to hasten the dissociation rate of insulin hexamers and reduce their tendency to reassociate postinjection. A novel formulation of recombinant human insulin containing citrate and disodium ethylenediaminetetraacetic acid (EDTA) has been tested in clinic and has a very rapid onset of action in patients with diabetes. In order to understand the basis for the rapid insulin absorption, in vitro experiments using analytical ultracentrifugation, protein charge assessment, and light scattering have been performed with this novel human insulin formulation and compared with a commercially available insulin formulation [regular human insulin (RHI)]. METHOD: Analytical ultracentrifugation and dynamic light scattering were used to infer the relative distributions of insulin monomers, dimers, and hexamers in the formulations. Electrical resistance of the insulin solutions characterized the overall net surface charge on the insulin complexes in solution. RESULTS: The results of these experiments demonstrate that the zinc chelating (disodium EDTA) and charge-masking (citrate) excipients used in the formulation changed the properties of RHI in solution, making it dissociate more rapidly into smaller, charge-masked monomer/dimer units, which are twice as rapidly absorbed following subcutaneous injection than RHI (Tmax 60 ± 43 versus 120 ± 70 min). CONCLUSIONS: The combination of rapid dissociation of insulin hexamers upon dilution due to the zinc chelating effects of disodium EDTA followed by the inhibition of insulin monomer/dimer reassociation due to the charge-masking effects of citrate provides the basis for the ultra-rapid absorption of this novel insulin formulation.

A review of a family of ultra-rapid-acting insulins: formulation development.

This review summarizes the clinical development of a family of ultra-rapid-acting recombinant human insulin formulations. These formulations use ethylenediaminetetraacetic acid (EDTA) to chelate zinc and thereby destabilize insulin hexamers. In addition, insulin monomer surface charges are chemically masked with citrate to prevent reaggregation. The first phase 1 trials were performed using BIOD-090, an acidic 25 unit U/ml insulin formulation, which contained disodium-EDTA (NaEDTA). When compared with regular human insulin (RHI) and/or insulin lispro in multiple phase 1 studies, BIOD-090 consistently showed more rapid absorption and/or onset of action. A standard meal challenge study also demonstrated improved postprandial glucose profiles associated with BIOD-090. However, increased patient exposure in larger phase 3 trials showed that this formulation was associated with an increased incidence of local injection site reactions, most commonly pain. A next generation formulation, BIOD-100, contained the same excipients as a standard insulin concentration of 100 U/ml. BIOD-100 maintained an ultra-rapid action profile and was associated with modest but significantly improved toleration when compared with BIOD-090. In order to further improve toleration, the hypothesis that NaEDTA contributed to discomfort by chelating endogenous calcium was tested by either substituting calcium-EDTA for NaEDTA or by adding calcium chloride to the NaEDTA formulation. These calcium formulations essentially eliminated the excess discomfort associated with BIOD-090 but were associated with less optimal pharmacokinetic profiles in humans. Recent efforts have succeeded in developing ultra-rapid-acting human insulin formulations with acceptable injection site toleration by optimizing concentrations of calcium (BIOD-125) and with the use of magnesium sulfate to mitigate discomfort (BIOD-123). Similar formulation technology has also been shown to accelerate absorption of insulin analogs in animal models.

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.