Gerhard Koenig

The Novel 7 Nicotinic Acetylcholine Receptor Agonist N-((3R)-1-Azabicyclo(2.2.2)oct-3-yl)-7-(2-(methoxy)phenyl)-1- benzofuran-2-carboxamide Improves Working and Recognition Memory in Rodents

The relative contribution of α4β2, α7 and other nicotinic acetylcholine receptor (nAChR) subtypes to the memory enhancing versus the addictive effects of nicotine is the subject of ongoing debate. In the present study, we characterized the pharmacological and behavioral properties of the α7 nAChR agonist N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-7-[2-(methoxy)phenyl]-1-benzofuran-2-carboxamide (ABBF). ABBF bound to α7 nAChR in rat brain membranes (Ki = 62 nM) and to recombinant human 5-hydroxytryptamine (5-HT)3 receptors (Ki = 60 nM). ABBF was a potent agonist at the recombinant rat and human α7 nAChR expressed in Xenopus oocytes, but it did not show agonist activity at other nAChR subtypes. ABBF acted as an antagonist of the 5-HT3 receptor and α3β4, α4β2, and muscle nAChRs (at higher concentrations). ABBF improved social recognition memory in rats (0.3–1 mg/kg p.o.). This improvement was blocked by intracerebroventricular administration of the α7 nAChR antagonist methyllycaconitine at 10 μg, indicating that it is mediated by α7 nAChR agonism. In addition, ABBF improved working memory of aged rats in a water maze repeated acquisition paradigm (1 mg/kg p.o.) and object recognition memory in mice (0.3–1 mg/kg p.o.). Rats trained to discriminate nicotine (0.4 mg/kg s.c.) from vehicle did not generalize to ABBF (0.3–30 mg/kg p.o.), suggesting that the nicotine cue is not mediated by the α7 nAChR and that selective α7 nAChR agonists may not share the abuse liability of nicotine. Our results support the hypothesis that α7 nAChR agonists may provide a novel therapeutic strategy for the treatment of cognitive deficits with low abuse potential.

Evp-6124: Safety, Tolerability And Cognitive Effects Of A Novel A7 Nicotinic Receptor Agonist In Alzheimer'S Disease Patients On Stable Donepezil Or Rivastigmine Therapy

raphy (PET) tracers have been developed for in vivo imaging of AD pathology. Of these ligands, [C]PIB and [F]FDDNP have been used most widely. [C]PIB was designed to measure the amount of fibrillar Ab deposits while [F]FDDNP has been reported to label not only amyloid but also other proteins including neurofibrillary tangles. As these two PET ligands are currently considered potential surrogate markers of disease, investigating associations with the hallmark symptom of AD, cognitive impairment, is essential. Objective: To investigate the relationship between [C]PIB and [F]FDDNP binding using positron emission tomography (PET) and measures of cognitive performance in patients with Alzheimer’s disease (AD), mild cognitive impairment (MCI) and controls. Methods: Twelve AD patients, 13 MCI patients and 15 controls were included in this study. Paired [C]PIB and [F]FDDNP scans were performed in all subjects. Global cortical binding potential (BPND) was calculated using parametric images of BPND. Cognitive functions were assessed using a battery of neuropsychological tests. Associations between [C]PIB and [F]FDDNP binding with cognitive measures were assessed using linear regression analyses. Results: Adjusted for age, sex and diagnosis, higher global [C]PIB binding was associated with lower scores on delayed recall of the Rey Auditory Verbal Learning Task (RAVLT) (standardized b1⁄4-0.46, p1⁄40.003). Higher global [F]FDDNP binding was associated with worse performance on immediate and delayed recall of RAVLT (standardized b1⁄4-0.34, p1⁄40.02 and b1⁄4-0.27, p1⁄40.02). No relationship was found for either ligand with impairment on cognitive tests other than memory. Within diagnostic groups, higher [C]PIB binding was correlated with lower scores on delayed recall of RAVLT in MCI patients only. Higher global [F]FDDNP binding was correlated with lower scores on immediate recall of RAVLT in AD patients. Conclusion: Binding of both ligands was related to memory impairment, but not to impairment in other cognitive domains. [C]PIB is especially associated with memory at the stage of MCI while [F]FDDNP has strongest value in patients with AD.

Concentration-response relationship of the α7 nicotinic acetylcholine receptor agonist FRM-17874 across multiple in vitro and in vivo assays

Pharmacological activation of α7 nicotinic acetylcholine receptors (α7 nAChRs) may improve cognition in schizophrenia and Alzheimers disease. The present studies describe an integrated pharmacological analysis of the effects of FRM-17874, an analogue of encenicline, on α7 nAChRs in vitro and in behavioral and neurophysiological assays relevant to cognitive function. FRM-17874 demonstrated high affinity binding to human α7 nAChRs, displacing [(3)H]-methyllacaconitine (Ki=4.3nM). In Xenopus laevis oocytes expressing human α7 nAChRs, FRM-17874 acted as an agonist, evoking inward currents with an EC50 of 0.42μM. Lower concentrations of FRM-17874 (0.01-3nM) elicited no detectable current, but primed receptors to respond to sub-maximal concentrations of acetylcholine. FRM-17874 improved novel object recognition in rats, and enhanced memory acquisition and reversal learning in the mouse water T-maze. Neurophysiological correlates of cognitive effects of drug treatment, such as synaptic transmission, long-term potentiation, and hippocampal theta oscillation were also evaluated. Modulation of synaptic transmission and plasticity was observed in rat hippocampal slices at concentrations of 3.2 and 5nM. FRM-17874 showed a dose-dependent facilitation of stimulation-induced hippocampal theta oscillation in mice and rats. The FRM-17874 unbound brain concentration-response relationship for increased theta oscillation power was similar in both species, exhibited a biphasic pattern peaking around 3nM, and overlapped with active doses and exposures observed in cognition assays. In summary, behavioral and neurophysiological assays indicate a bell-shaped effective concentration range and this report represents the first attempt to explain the concentration-response function of α7 nAChR-mediated pro-cognitive effects in terms of receptor pharmacology.

Continuous infusion of the alpha 7 nicotinic acetylcholine receptor agonist EVP-6124 produces no signs of tolerance at memory-enhancing doses in rats: a pharmacokinetic and behavioral study

We investigated whether the effects of acutely administered EVP-6124, an α7 nicotinic acetylcholine receptor (α7 nAChR) agonist, on cognition were maintained after 6-day continuous minipump administration. Performance in a delay-dependent forgetting test was measured in the object recognition task after single-oral doses of 0.3 or 1 mg/kg, or at plasma steady-state concentrations (Css) of 0.6 or 2 ng/ml, which were similar to the efficacious plasma concentrations after single-oral dosing. The 0.3 mg/kg acute dose enhanced memory at a total plasma concentration of ∼0.3 ng/ml at 1-4 h after dosing. Continuous treatment produced total plasma Css values of 0.48 and 1.93 ng/ml on day 6 and enhanced memory. At EVP-6124 plasma concentrations that optimally enhance memory in the object recognition task, tolerance did not develop after 6 days of continuous treatment.

Neuropharmacokinetics of two investigational compounds in rats: Divergent temporal profiles in the brain and cerebrospinal fluid

Two investigational compounds (FRM-1, (R)-7-fluoro-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide and FRM-2, (R)-7-cyano-N-(quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide) resided in rat brain longer than in systemic circulation. In Caco-2 directional transport studies, they both showed good intrinsic passive permeability but differed significantly in efflux susceptibility (efflux ratio of <2 and ∼7, respectively), largely attributed to P-glycoprotein (P-gp). Capitalizing on these interesting properties, we investigated how cerebrospinal fluid (CSF) concentration (CCSF) would be shaped by unbound plasma concentration (Cu,p) and unbound brain concentration (Cu,b) in disequilibrium conditions and at steady state. Following subcutaneous administration, FRM-1CCSF largely followed Cu,p initially and leveled between Cu,p and Cu,b. However, it gradually approached Cu,b and became lower than, but parallel to Cu,b at the terminal phase. In contrast, FRM-2CCSF temporal profile mostly paralleled the Cu,p but was at a much lower level. Upon intravenous infusion to steady state, FRM-1CCSF and Cu,b were similar, accounting for 61% and 69% of the Cu,p, indicating a case of largely passive diffusion-governed brain penetration where CCSF served as a good surrogate for Cu,b. On the contrary, FRM-2CCSF and Cu,b were remarkably lower than Cu,p (17% and 8% of Cu,p, respectively), suggesting that FRM-2 brain penetration was severely impaired by P-gp-mediated efflux and CCSF underestimated this impact. A semi-physiologically based pharmacokinetic (PBPK) model was constructed that adequately described the temporal profiles of the compounds in the plasma, brain and CSF. Our work provided some insight into the relative importance of blood-brain barrier (BBB) and blood-CSF barrier (BCSFB) in modulating CCSF.

EVP-6124, a selective alpha-7 partial agonist, has positive effects on cognition and clinical function in mild-to-moderate Alzheimer's disease patients: Results of a six-month, double-blind, placebo controlled, dose ranging study

62% were female. 57 participants were evaluated at the 6 week final visit, with 50 participants completing 6 weeks of treatment with study drug. Adherence was very good with 95% of pill bottles returned and 88% of pills being taken according to pill counts. After 6 weeks mean (SD) change in AES scores were -1.9 (1.5) for Met vs. 0.6 (1.4) for Plb (p1⁄40.23). The odds ratio (95% CI) for improvement in CGI-C was 3.7 (1.3, 10.8) (p1⁄40.02), with 21% of Met vs. 3% of Plb rated as moderately or markedly improved. The improvement in NPI Apathy scores was 1.8 points (95% CI 0.3, 3.4) greater in Met vs. Plb (p1⁄40.02). Met treatment was well tolerated with a trend toward greater anxiety and weight loss > 2% in Met vs. Plb. Conclusions: In this proof-of-concept trial Met treatment of apathy in AD was associated with significant improvement in two of three preplanned outcomes, with a third outcome favoring. These results support the efficacy of Met treatment for apathy in AD and warrant a definitive, hypothesis-testing randomized controlled trial.

α7-nAChR agonist enhances neural plasticity in the hippocampus via a GABAergic circuit.

Agonists of the α7-nicotinic acetylcholine receptor (α7-nAChR) have entered clinical trials as procognitive agents for treating schizophrenia and Alzheimers disease. The most advanced compounds are orthosteric agonists, which occupy the ligand binding site. At the molecular level, agonist activation of α7-nAChR is reasonably well understood. However, the consequences of activating α7-nAChRs on neural circuits underlying cognition remain elusive. Here we report that an α7-nAChR agonist (FRM-17848) enhances long-term potentiation (LTP) in rat septo-hippocampal slices far below the cellular EC50 but at a concentration that coincides with multiple functional outcome measures as we reported in Stoiljkovic M, Leventhal L, Chen A, Chen T, Driscoll R, Flood D, Hodgdon H, Hurst R, Nagy D, Piser T, Tang C, Townsend M, Tu Z, Bertrand D, Koenig G, Hajós M. Biochem Pharmacol 97: 576-589, 2015. In this same concentration range, we observed a significant increase in spontaneous γ-aminobutyric acid (GABA) inhibitory postsynaptic currents and a moderate suppression of excitability in whole cell recordings from rat CA1 pyramidal neurons. This modulation of GABAergic activity is necessary for the LTP-enhancing effects of FRM-17848, since inhibiting GABAA α5-subunit-containing receptors fully reversed the effects of the α7-nAChR agonist. These data suggest that α7-nAChR agonists may increase synaptic plasticity in hippocampal slices, at least in part, through a circuit-level enhancement of a specific subtype of GABAergic receptor.

GSM treatment after the onset of Aβ deposition in mice reduces total Aβ load

Background:Alzheimer’s disease (AD) is a common, debilitating neurodegenerative disorder afflicting the aging population. Synaptic accumulation of Aß-protein oligomers and tau might be responsible for the neurological damage in AD. Thus, special interest has emerged in developing treatments that reduce the formation or facilitate the clearance of these oligomers. Utilizing molecular modeling and structure based design techniques we generated a series of novel organic heterocyclic compounds that are capable of recognizing the aggregated Aß-protein molecule and promoting clearance. Using cell-free and cell-based assays, a compound denominated NPT-400 with activity in the high nanomolar range was identified for pharmacokinetic (PK) and preliminary efficacy studies in APP transgenic (tg) mice. Methods: The mThy1-hAPP751 transgenic mouse was utilized for efficacy studies. This mouse combines the Swedish (K670M/N671L) and London (V717I) APPmutations, and these animals develop AD-like neuropathology and behavioral deficits starting at 3-4 months of age. Female non-tg and tg mice (w5 mo of age) received a single injection of vehicle or NPT-400-3 (IP, 10 mg/kg) three times per week for 6.5 weeks. Behavioral assessments were conducted during week 4 of treatment including locomotor activity and water maze. At the completion of treatment, brains were hemisected and tissue sections were processed for immunolabeling and confocal analysis, and homogenates were analyzed for levels of Abeta-protein by ELISA and immunoblot. For PK studies, wildtype C57Bl6 mice received IVor PO NPT-400-3 compound at 10 mg/kg and blood and plasma levels were analyzed at 0-24 hrs. Results: Treatment with NPT-400-3 resulted in a statistically significant normalization of locomotor activity accompanied by amelioration of the synaptic and dendritic pathology in the neocortex and hippocampus. By immunoblot and ELISA the levels of monomeric and oligomeric Aß-protein were statistically significantly reduced in the brains of the APP tg mice. PK studies showed that NPT400-3 is stable in plasma, is orally bioavailable and penetrates the brain with a B/P ratio of 0.8. Conclusions:NPT-400-3 is an orally bioavailable, brain penetrating Aß-protein stabilizing organic compound that reduces the accumulation of Aß-protein oligomers and ameliorates behavioral deficits and neuropathology in APP tg mice without overt adverse effects.

Pharmacokinetic and pharmacodynamic analysis of the gamma-secretase modulator (GSM) EVP-0015962

Background: CAD106 is an active immunotherapy developed for Alzheimer’s disease. It comprises amino acids 1-6 of Ab coupled to a virus-like particle derived from E. coli bacteriophage Qb. In APP overexpressing mice, administration of CAD106 induced Ab antibodies as well as a reduction of amyloid accumulation in the brain. Infusion of antibodies or active immunization against Ab have been shown to increase the level of total Ab in plasma indicating target engagement by the antibodies. In the present study we have investigated the active Ab immunotherapy CAD106 for its effect on plasma Ab. Methods: Cynomolgus monkeys aged 3 to 6 years were administered 600 mg CAD106 per subcutaneous or intramuscular injection at days 1, 14, 28 and every 4 weeks thereafter for a total of 24 weeks. Each dose contained either Alum or MF58 as adjuvant. Controls received adjuvant only. Total plasma Ab was determined at baseline and 12 to 14 days after the injections at week 8 and 24 using two different ELISAs and Western blotting. To minimize an interference of the CAD106-induced antibodies samples were SDSdenatured prior to quantification with an ELISA using a N-terminal capturing antibody. Alternatively, an assay combining C-terminal capture and mid-region detection antibodies was used. In addition, results were verified by direct SDS-PAGE separation of plasma followed by Western blotting. Results: Following active immunotherapy with CAD106, total plasma Ab levels increased compared to baseline and further between the intermediate and final time points of the study. Individual animals showed increases of up to about 50-fold over baseline. Median group increases reached up to 25-fold. Very similar results were obtained with the different assays. Overall the Ab levels in plasma correlated well with the Ab antibody titers. Conclusions: The increase in plasma Ab indicates an interaction of CAD106 induced antibodies with Ab in vivo. Its extent is in line with observations made with Nterminal monoclonal antibodies.

Design, Synthesis, and Evaluation of a Novel Series of Oxadiazine Gamma Secretase Modulators for Familial Alzheimer’s Disease

Herein we describe the design, synthesis, and evaluation of a novel series of oxadiazine-based gamma secretase modulators obtained via isosteric amide replacement and critical consideration of conformational restriction. Oxadiazine lead 47 possesses good in vitro potency with excellent predicted CNS drug-like properties and desirable ADME/PK profile. This lead compound demonstrated robust Aβ42 reductions and subsequent Aβ37 increases in both rodent brain and CSF at 30 mg/kg dosed orally.