Discovery and characterization of novel TRPML1 agonists.

Screening a library of >100,000 compounds identified the substituted tetrazole compound 1 as a selective TRPML1 agonist. Both enantiomers of compound 1 were separated and profiled in vitro and in vivo. Their selectivity, ready availability and CNS penetration should enable them to serve as the tool compounds of choice in future TRPML1 channel activation studies. SAR studies on conformationally locked macrocyclic analogs further improved the TRPML1 agonist potency while retaining the selectivity.

Discovery of oxazoline enhancers of cellular progranulin release.

Phenotypic screening of an annotated small molecule library and initial SAR studies identified compound 2 as a robust enhancer of progranulin secretion. Detailed SAR development on conformationally restricted carbamate isosteres led to the identification of compound 60 with a 3-fold improvement in BV-2 potency and a 9-fold decrease in hERG inhibition over compound 2, substantially improving this important margin of safety relative to compound 2.

Discovery of quinuclidine modulators of cellular progranulin.

Phenotypic screening of an annotated small molecule library identified the quinuclidine tetrahydroisoquinoline solifenacin (1) as a robust enhancer of progranulin secretion with single digit micromolar potency in a murine microglial (BV-2) cell line. Subsequent SAR development led to the identification of 29 with a 38-fold decrease in muscarinic receptor antagonist activity and a 10-fold improvement in BV-2 potency.

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 Alzheimer's 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.

Continuous infusion of the α7 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.

Discovery of the Oxadiazine FRM-024: A Potent CNS-Penetrant Gamma Secretase Modulator.

The recent approval of aducanumab for Alzheimer's disease has heightened the interest in therapies targeting the amyloid hypothesis. Our research has focused on identification of novel compounds to improve amyloid processing by modulating gamma secretase activity, thereby addressing a significant biological deficit known to plague the familial form of the disease. Herein, we describe the design, synthesis, and optimization of new gamma secretase modulators (GSMs) based on previously reported oxadiazine 1. Potency improvements with a focus on predicted and measured properties afforded high-quality compounds further differentiated via robust Aß42 reductions in both rodents and nonhuman primates. Extensive preclinical profiling, efficacy studies, and safety studies resulted in the nomination of FRM-024, (+)-cis-5-(4-chlorophenyl)-6-cyclopropyl-3-(6-methoxy-5-(4-methyl-1H-imidazole-1-yl)pyridin-2-yl)-5,6-dihydro-4H-1,2,4-oxadiazine, as a GSM preclinical candidate for familial Alzheimer's disease.

Effect of the Histone Deacetylase Inhibitor FRM-0334 on Progranulin Levels in Patients With Progranulin Gene Haploinsufficiency: A Randomized Clinical Trial.

Importance: Histone deacetylase inhibitors have been repeatedly shown to elevate progranulin levels in preclinical models. This report describes the first randomized clinical trial of a histone deacetylase inhibitor in frontotemporal dementia (FTD) resulting from progranulin (GRN) gene variations. Objective: To characterize the safety, tolerability, plasma pharmacokinetics, and pharmacodynamic effects of oral FRM-0334 on plasma progranulin and other exploratory biomarkers, including fluorodeoxyglucose (FDG)-positron emission tomography (PET), in individuals with GRN haploinsufficiency. Design, Setting, and Participants: In this randomized, double-blind, placebo-controlled, dose-escalating, phase 2a safety, tolerability, and pharmacodynamic clinical study, 2 doses of a histone deacetylase inhibitor (FRM-0334) were administered to participants with prodromal to moderate FTD with granulin variations. Participants were recruited from January 13, 2015, to April 13, 2016. The study included 27 participants with prodromal (n = 8) or mild-to-moderate symptoms of FTD (n = 19) and heterozygous pathogenic variations in GRN and was conducted at multiple centers in North America, the UK, and the European Union. Data were analyzed from June 9, 2019, to May 13, 2021. Interventions: Daily oral placebo (n = 5), 300 mg of FRM-0334 (n = 11), or 500 mg of FRM-0334 (n = 11) was administered for 28 days. Main Outcomes and Measures: Primary outcomes were safety and tolerability of FRM-0334 and its peripheral pharmacodynamic effect on plasma progranulin. Secondary outcomes were the plasma pharmacokinetic profile of FRM-0334 and its pharmacodynamic effect on cerebrospinal fluid progranulin. Exploratory outcomes were FDG-PET, FTD clinical severity, and cerebrospinal fluid biomarkers (neurofilament light chain [NfL], amyloid ß 1-42, phosphorylated tau 181, and total tau [t-tau]). Results: A total of 27 participants (mean [SD] age, 56.6 [10.5] years; 16 women [59.3%]; 26 White participants [96.3%]) with GRN variations were randomized and completed treatment. FRM-0334 was safe and well tolerated but did not affect plasma progranulin (4.3 pg/mL per day change after treatment; 95% CI, -10.1 to 18.8 pg/mL; P = .56), cerebrospinal fluid progranulin (0.42 pg/mL per day; 95% CI, -0.12 to 0.95 pg/mL; P = .13), or exploratory pharmacodynamic measures. Plasma FRM-0334 exposure did not increase proportionally with dose. Brain FDG-PET data were available in 26 of 27 randomized participants. In a cross-sectional analysis of 26 individuals, bifrontal cortical FDG hypometabolism was associated with worse Clinical Dementia Rating (CDR) plus National Alzheimer's Coordinating Center frontotemporal lobar degeneration sum of boxes score (b = -3.6 × 10-2 standardized uptake value ratio [SUVR] units/CDR units; 95% CI, -4.9 × 10-2 to -2.2 × 10-2; P < .001), high cerebrospinal fluid NfL (b = -9.2 × 10-5 SUVR units/pg NfL/mL; 95% CI, -1.3 × 10-4 to -5.6 × 10-5; P < .001), and high CSF t-tau (-7.2 × 10-4 SUVR units/pg t-tau/mL; 95% CI, -1.4 × 10-3 to -9.5 × 10-5; P = .03). Conclusions and Relevance: In this randomized clinical trial, the current formulation of FRM-0334 did not elevate PRGN levels, which could reflect a lack of efficacy at attained exposures, low bioavailability, or some combination of the 2 factors. Bifrontal FDG-PET is a sensitive measure of symptomatic GRN haploinsufficiency. International multicenter clinical trials of FTD-GRN are feasible. Trial Registration: ClinicalTrials.gov Identifier: NCT02149160.

BACE1 Inhibitor MK-8931 Alters Formation but Not Stability of Dendritic Spines.

Beta-site amyloid-precursor-protein cleaving enzyme 1 (BACE1) is the rate limiting protease in the production of the amyloid-beta peptide (Aß), which is considered to be the causative agent in the pathogenesis of Alzheimer's Disease (AD). Therefore, the therapeutic potential of pharmacological BACE1 inhibitors is currently tested in clinical trials for AD treatment. To ensure a positive clinical outcome it is crucial to identify and evaluate adverse effects associated with BACE1 inhibition. Preclinical studies show that chronic blockade of BACE1 activity alters synaptic functions and leads to loss of dendritic spines. To assess the mechanism of synapse loss, dendritic spine dynamics of pyramidal layer V cells were monitored by in vivo two-photon microscopy in the somatosensory cortex of mice, treated with the BACE1 inhibitor MK-8931. MK-8931 treatment significantly reduced levels of Aß40 and density of dendritic spines in the brain. However, the steady decline in dendritic spine density specifically resulted from a diminished formation of new spines and not from a loss of stable spines. Furthermore, the described effects on spine formation were transient and recovered after inhibitor withdrawal. Since MK-8931 inhibition did not completely abolish spine formation, our findings suggest that carefully dosed inhibitors might be therapeutically effective without affecting the structural integrity of excitatory synapses if given at an early disease stage.

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 Aß42 reductions and subsequent Aß37 increases in both rodent brain and CSF at 30 mg/kg dosed orally.

Effects of a novel phosphodiesterase 10A inhibitor in non-human primates: A therapeutic approach for schizophrenia with improved side effect profile.

Schizophrenia symptoms are associated with alterations in basal ganglia-cortical networks that include the cyclic nucleotides (cAMP/cGMP) signaling pathways. Phosphodiesterase 10A (PDE10A) inhibitors have been considered as therapeutic agents for schizophrenia because the regulation of cAMP and cGMP in the striatum by PDE10A plays an important role in the signaling mechanisms of the striatal-cortical network, and thereby in cognitive function. In the present study we assessed in non-human primates (NHPs) the effects of a novel PDE10A inhibitor (FRM-6308) that has demonstrated high potency and selectivity for human recombinant PDE10A in vitro. The behavioral effects of FRM-6308 in a dose range were determined in rhesus monkeys using a standardized motor disability scale for primates, motor tasks, and the "drug effects on the nervous system" (DENS) scale. The neuronal metabolic effects of FRM-6308 were determined with [(18)F]-fluorodeoxyglucose PET imaging. Results showed that FRM-6308 did not have any specific effects on the motor system at s.c. doses up to 0.32 mg/kg in NHPs, which induced a significant increase in the FDG-SUV in striatum (F 16.069, p < 0.05) and cortical (F 15.181, p < 0.05) regions. Higher doses induced sedation and occasional involuntary movements with clear development of tolerance after repeated exposures. These findings suggest that FRM-6308 has the adequate pharmacological profile to advance testing in clinical trials and demonstrate antipsychotic efficacy of PDE10A inhibition for the treatment of schizophrenia patients.

Hippocampal network dynamics in response to α7 nACh receptors activation in amyloid-ß overproducing transgenic mice.

Amyloid-ß (Aß) peptide overproduction is one of the pathomechanisms contributing to Alzheimer's disease (AD). Agonists of α7 nicotinic acetylcholine receptors (α7 nAChRs) are under development as symptomatic treatments for AD, and clinical findings suggest that α7 nAChR agonists may improve cognitive functions in AD patients. However, interactions between Aß and α7 nAChRs have been observed, implying that high levels of Aß may modify the effects of α7 nAChR agonists. Therefore, we tested the α7 nAChR agonist FRM-17874, an analogue of encenicline, in 8-month-old Aß overproducing 5xFAD mice in an in vivo neurophysiological assay with a high construct and predictive validity for testing procognitive drugs. By recording changes in brainstem-stimulation-elicited hippocampal oscillations, we identified previously undescribed neurophysiological impairments in 5xFAD mice, including significantly decreased power of theta and gamma oscillations and theta-phase-gamma-amplitude coupling. Compared with their saline controls, systemically administered FRM-17874 significantly increased stimulation-induced theta power by 30% in both 5xFAD and wild-type mice. However, FRM-17874 did not impact gamma oscillation or theta-phase-gamma-amplitude coupling in either wild type or 5xFAD mice, and it did not eliminate the significant differences in these parameters between the 2 groups.

Intracellular Retention of Three Quinuclidine Derivatives in Caco-2 Permeation Experiments: Mechanisms and Impact on Estimating Permeability and Active Efflux Ratio.

BACKGROUND: Three quinuclidine derivatives (FRM-1, FRM-2 and FRM-3) were subject to significant mass loss to cellular retention in Caco-2 permeation experiments. The apparent permeability coefficient (Papp) calculated with either 'sink' (Papp,sink) or 'non-sink' (Papp,nonsink) method was significantly biased. As a result, a simplified 3-compartmental distribution model was applied in this study to derive the 'intrinsic' Papp (Papp,int) and to understand the impact of cellular retention on estimating Papp and active efflux ratio (ER) values. METHODS: Time-courses of the amount of test compounds in the donor, receiver and cells were determined in the presence and absence of bafilomycin A1 (BFA, 100 nM) and / or cyclosporine A (CsA, 10 .M). A mathematical model was constructed to describe the mass transfer of test compounds among three compartments. The temporal profiles of directional Papp,sink, Papp,nonsink and the corresponding of ER values were compared with the counterpart parameters derived from data-fitting to the mathematical model. Simulations were performed for a better understanding of experimental observations. RESULTS: The mass recovery of test compounds deteriorated with incubation time and was direction dependent. Based on the directional Papp,sink values, the resulting ER is close to unity for FRM-1, and approximately 2 and 3.5 for FRM-2 and FRM-3. Treatment with BFA considerably enhanced mass recovery for FRM-1 and FRM-3 (by 5- and 2-fold) but elicited no impact on FRM-2, while ER values largely unchanged. Expectedly, Papp,nonsink was higher than Papp,sink, but the resulting ER was lower in most cases. In contrast, the model-derived Papp,int was much greater than the values of Papp,sink and Papp,nonsink. The model also quantitatively unveiled the respective contributions of lysosomal sequestration and nonspecific binding to the cellular retention of the compounds. CONCLUSION: Our work reveals the different mechanisms involved in cellular retention of these quinuclidine derivatives, and more importantly, demonstrates the value of kinetic analyses with mathematical modeling in minimizing the bias in Papp estimation when assumptions for conventional calculations are violated.

Characterization of FRM-36143 as a new γ-secretase modulator for the potential treatment of familial Alzheimer's disease.

BACKGROUND: Familial Alzheimer's disease (FAD) is caused by mutations in the amyloid precursor protein (APP) or presenilin (PS). Most PS mutations, which account for the majority of FAD cases, lead to an increased ratio of longer to shorter forms of the amyloid beta (Aß) peptide. The therapeutic rationale of γ-secretase modulators (GSMs) for Alzheimer's disease is based on this genetic evidence as well as on enzyme kinetics measurements showing changes in the processivity of the γ-secretase complex. This analysis suggests that GSMs could potentially offset some of the effects of PS mutations on APP processing, thereby addressing the root cause of early onset FAD. Unfortunately, the field has generated few, if any, molecules with good central nervous system (CNS) drug-like properties to enable proof-of-mechanism studies. METHOD: We characterized the novel GSM FRM-36143 using multiple cellular assays to determine its in vitro potency and off-target activity as well as its potential to reverse the effect of PS mutations. We also tested its efficacy in vivo in wild-type mice and rats. RESULTS: FRM-36143 has much improved CNS drug-like properties compared to published GSMs. It has an in vitro EC50 for Aß42 of 35 nM in H4 cells, can reduce Aß42 to 58 % of the baseline in rat cerebrospinal fluid, and also increases the non-amyloidogenic peptides Aß37 and Aß38. It does not inhibit Notch processing, nor does it inhibit 24-dehydrocholesterol reductase (DHCR24) activity. Most interestingly, it can reverse the effects of presenilin mutations on APP processing in vitro. CONCLUSIONS: FRM-36143 possesses all the characteristics of a GSM in terms of Aß modulation Because FRM-36143 was able to reverse the effect of PS mutations, we suggest that targeting patients with this genetic defect would be the best approach at testing the efficacy of a GSM in the clinic. While the amyloid hypothesis is still being tested with ß-site APP-cleaving enzyme inhibitors and monoclonal antibodies in sporadic AD, we believe it is not a hypothesis for FAD. Since GSMs can correct the molecular defect caused by PS mutations, they have the promise to provide benefits to the patients when treated early enough in the course of the disease.

Randomized, Double-Blind, Placebo-Controlled Study of Encenicline, an α7 Nicotinic Acetylcholine Receptor Agonist, as a Treatment for Cognitive Impairment in Schizophrenia.

Encenicline is a novel, selective α7 nicotinic acetylcholine receptor agonist in development for treating cognitive impairment in schizophrenia and Alzheimer's disease. A phase 2, double-blind, randomized, placebo-controlled, parallel-design, multinational study was conducted. Patients with schizophrenia on chronic stable atypical antipsychotics were randomized to encenicline 0.27 or 0.9 mg once daily or placebo for 12 weeks. The primary efficacy end point was the Overall Cognition Index (OCI) score from the CogState computerized battery. Secondary end points include MATRICS Consensus Cognitive Battery (MCCB) (in US patients), the Schizophrenia Cognition Rating Scale (SCoRS) total score, SCoRS global rating, and Positive and Negative Syndrome Scale (PANSS) total and subscale and cognition factor scores. Of 319 randomized patients, 317 were included in the safety population, and 307 were included in the intent-to-treat population. Notable trends in improvement were demonstrated across all cognition scales. For the OCI score, the LS mean difference for encenicline 0.27 mg vs placebo was significant (Cohen's d=0.257; P=0.034). Mean SCoRS total scores decreased showing improvement in function over time, and the difference was significant for encenicline 0.9 mg vs placebo (P=0.011). Furthermore, the difference between encenicline 0.9 mg and placebo was significant for the PANSS Cognition Impairment Domain (P=0.0098, Cohen's d=0.40) and for the PANSS Negative scale (P=0.028, Cohen's d=0.33). Treatment-emergent adverse events were reported at similar frequencies across all treatment groups (39.0% with placebo, 23.4% with encenicline 0.27 mg, and 33.3% with encenicline 0.9 mg). Overall, encenicline was generally well tolerated and demonstrated clinically meaningful improvements in cognition and function in patients with schizophrenia.

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 Alzheimer's 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.

Inhibition of phosphodiesterase 2 increases neuronal cGMP, synaptic plasticity and memory performance.

An essential element of the signalling cascade leading to synaptic plasticity is the intracellular second messenger molecule guanosine 3',5'-cyclic monophosphate (cGMP). Using the novel, potent, and selective inhibitor Bay 60-7550, we show that the enzyme 3',5'-cyclic nucleotide phosphodiesterase type 2 (PDE2) is responsible for the degradation of newly synthesized cGMP in cultured neurons and hippocampal slices. Inhibition of PDE2 enhanced long-term potentiation of synaptic transmission without altering basal synaptic transmission. Inhibition of PDE2 also improved the performance of rats in social and object recognition memory tasks, and reversed MK801-induced deficits in spontaneous alternation in mice in a T-maze. Our data provide strong evidence that inhibition of PDE2 can improve memory functions by enhancing neuronal plasticity.

The novel α7 nicotinic acetylcholine receptor agonist EVP-6124 enhances dopamine, acetylcholine, and glutamate efflux in rat cortex and nucleus accumbens.

BACKGROUND: Alpha7 and α4ß2 nicotinic acetylcholine receptor (nAChR) agonists have been shown to improve cognition in various animal models of cognitive impairment and are of interest as treatments for schizophrenia, Alzheimer's disease, and other cognitive disorders. Increased release of dopamine (DA), acetylcholine (ACh), glutamate (Glu), and γ-aminobutyric acid (GABA) in cerebral cortex, hippocampus, and nucleus accumbens (NAC) has been suggested to contribute to their beneficial effects on cognition. RESULTS: Using in vivo microdialysis, we found that EVP-6124 [(R)-7-chloro-N-quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide], a high-affinity α7 nAChR partial agonist, at 0.1 mg/kg, s.c., increased DA efflux in the medial prefrontal cortex (mPFC) and NAC. EVP-6124, 0.1 and 0.3 mg/kg, also increased efflux of ACh in the mPFC but not in the NAC. Similarly, EVP-6124, 0.1 mg/kg, but not 0.03 and 0.3 mg/kg, significantly increased mPFC Glu efflux. Thus, EVP-6124 produced an inverted U-shaped curve for DA and Glu release, as previously reported for other α7 nAChR agonists. The three doses of EVP-6124 did not produce a significant effect on GABA efflux in either region. Pretreatment with the selective α7 nAChR antagonist, methyllycaconitine (MLA, 1.0 mg/kg), significantly blocked cortical DA and Glu efflux induced by EVP-6124 (0.1 mg/kg), suggesting that the effects of EVP-6124 on these neurotransmitters were due to α7 nAChR agonism. MLA only partially blocked the effects of EVP-6124 on ACh efflux in the mPFC. CONCLUSION: These results suggest increased cortical DA, ACh, and Glu release, which may contribute to the ability of the α7 nAChR agonist, EVP-6124, to treat cognitive impairment and possibly other dimensions of psychopathology.

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.

Modulation of γ-secretase by EVP-0015962 reduces amyloid deposition and behavioral deficits in Tg2576 mice.

BACKGROUND: A hallmark of Alzheimer's disease is the presence of senile plaques in human brain primarily containing the amyloid peptides Aß42 and Aß40. Many drug discovery efforts have focused on decreasing the production of Aß42 through γ-secretase inhibition. However, identification of γ-secretase inhibitors has also uncovered mechanism-based side effects. One approach to circumvent these side effects has been modulation of γ-secretase to shift Aß production to favor shorter, less amyloidogenic peptides than Aß42, without affecting the overall cleavage efficiency of the enzyme. This approach, frequently called γ-secretase modulation, appears more promising and has lead to the development of new therapeutic candidates for disease modification in Alzheimer's disease. RESULTS: Here we describe EVP-0015962, a novel small molecule γ-secretase modulator. EVP-0015962 decreased Aß42 in H4 cells (IC50 = 67 nM) and increased the shorter Aß38 by 1.7 fold at the IC50 for lowering of Aß42. AßTotal, as well as other carboxyl-terminal fragments of amyloid precursor protein, were not changed. EVP-0015962 did not cause the accumulation of other γ-secretase substrates, such as the Notch and ephrin A4 receptors, whereas a γ-secretase inhibitor reduced processing of both. A single oral dose of EVP-0015962 (30 mg/kg) decreased Aß42 and did not alter AßTotal peptide levels in a dose-dependent manner in Tg2576 mouse brain at an age when overt Aß deposition was not present. In Tg2576 mice, chronic treatment with EVP-0015962 (20 or 60 mg/kg/day in a food formulation) reduced Aß aggregates, amyloid plaques, inflammatory markers, and cognitive deficits. CONCLUSIONS: EVP-0015962 is orally bioavailable, detected in brain, and a potent, selective γ-secretase modulator in vitro and in vivo. Chronic treatment with EVP-0015962 was well tolerated in mice and lowered the production of Aß42, attenuated memory deficits, and reduced Aß plaque formation and inflammation in Tg2576 transgenic animals. In summary, these data suggest that γ-secretase modulation with EVP-0015962 represents a viable therapeutic alternative for disease modification in Alzheimer's disease.

Neuronal store-operated calcium entry pathway as a novel therapeutic target for Huntington's disease treatment.

Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine expansion within Huntingtin (Htt) protein. In the phenotypic screen we identified a class of quinazoline-derived compounds that delayed a progression of a motor phenotype in transgenic Drosophila HD flies. We found that the store-operated calcium (Ca(2+)) entry (SOC) pathway activity is enhanced in neuronal cells expressing mutant Htt and that the identified compounds inhibit SOC pathway in HD neurons. The same compounds exerted neuroprotective effects in glutamate-toxicity assays with YAC128 medium spiny neurons primary cultures. We demonstrated a key role of TRPC1 channels in supporting SOC pathway in HD neurons. We concluded that the TRPC1-mediated neuronal SOC pathway constitutes a novel target for HD treatment and that the identified compounds represent a novel class of therapeutic agents for treatment of HD and possibly other neurodegenerative disorders.