Preclinical and clinical biomarker studies of CT1812: A novel approach to Alzheimer's disease modification

Abstract

Introduction: Amyloid beta (Aβ) oligomers are one of the most toxic structural forms of the Aβ protein and are hypothesized to cause synaptotoxicity and memory failure as they build up in Alzheimer's disease (AD) patients' brain tissue. We previously demonstrated that antagonists of the sigma-2 receptor complex effectively block Aβ oligomer toxicity. CT1812 is an orally bioavailable, brain penetrant small molecule antagonist of the sigma-2 receptor complex that appears safe and well tolerated in healthy elderly volunteers. We tested CT1812's effect on Aβ oligomer pathobiology in preclinical AD models and evaluated CT1812's impact on cerebrospinal fluid (CSF) protein biomarkers in mild to moderate AD patients in a clinical trial (ClinicalTrials.gov NCT02907567).

Methods: Experiments were performed to measure the impact of CT1812 versus vehicle on Aβ oligomer binding to synapses in vitro, to human AD patient post mortem brain tissue ex vivo, and in living APPSwe /PS1dE9 transgenic mice in vivo. Additional experiments were performed to measure the impact of CT1812 versus vehicle on Aβ oligomer-induced deficits in membrane trafficking rate, synapse number, and protein expression in mature hippocampal/cortical neurons in vitro. The impact of CT1812 on cognitive function was measured in transgenic Thy1 huAPPSwe/Lnd+ and wild-type littermates. A multicenter, double-blind, placebo-controlled parallel group trial was performed to evaluate the safety, tolerability, and impact on protein biomarker expression of CT1812 or placebo given once daily for 28 days to AD patients (Mini-Mental State Examination 18-26). CSF protein expression was measured by liquid chromatography with tandem mass spectrometry or enzyme-linked immunosorbent assay in samples drawn prior to dosing (Day 0) and at end of dosing (Day 28) and compared within each patient and between pooled treated versus placebo-treated dosing groups.

Results: CT1812 significantly and dose-dependently displaced Aβ oligomers bound to synaptic receptors in three independent preclinical models of AD, facilitated oligomer clearance into the CSF, increased synaptic number and protein expression in neurons, and improved cognitive performance in transgenic mice. CT1812 significantly increased CSF concentrations of Aβ oligomers in AD patient CSF, reduced concentrations of synaptic proteins and phosphorylated tau fragments, and reversed expression of many AD-related proteins dysregulated in CSF.

Discussion: These preclinical studies demonstrate the novel disease-modifying mechanism of action of CT1812 against AD and Aβ oligomers. The clinical results are consistent with preclinical data and provide evidence of target engagement and impact on fundamental disease-related signaling pathways in AD patients, supporting further development of CT1812.

Keywords: Abeta oligomers; Alzheimer's disease; clinical trial; sigma-2 receptor; synapse.

Conflict of interest statement

N.I., K.M.L., C.L., C.R., R.Y., L.W., G.L., G.R., H.S., M.H., C.W., K.S., and S.C. are employees of Cognition Therapeutics, Inc. C.Y., J.C., H.L., M.S., M.G., and T.S.‐J. are paid consultants of Cognition Therapeutics, Inc. L.S. reports grants and personal fees from Eli Lilly; personal fees from Avraham, Ltd; personal fees from Boehringer Ingelheim; grants and personal fees from Merck; personal fees from Neurim, Ltd; personal fees from Neuronix, Ltd; personal fees from Cognition Therapeutics, Inc.; personal fees from Eisai; personal fees from Takeda; personal fees from vTv; grants and personal fees from Roche/Genentech; grants from Biogen; grants from Novartis; personal fees from Abbott; grants from Biohaven, outside the submitted work. S.D. is a member of the Neuroscience Advisory Board for Amgen; Chair of the Medical Scientific Advisory Board for Acumen; Chair of the Drug Monitoring Committee for Biogen; Chair of the Medical Advisory Board for Cognition Therapeutics, Inc.; and Editor for Dementia for UpToDate. H.Z. has served at scientific advisory boards for Cognition Therapeutics, Roche Diagnostics, Wave, and Samumed; has given lectures in symposia sponsored by Biogen and Alzecure; and is a co‐founder of Brain Biomarker Solutions in Gothenburg AB, a GU Ventures‐based platform company at the University of Gothenburg. T.S. is a scientific advisory board member of Cognition Therapeutics, Inc. and receives collaborative grant funding from two pharmaceutical companies. K.B. has served as a consultant or at advisory boards for Alector, Biogen, Cognition Therapeutics, Lilly, MagQu, Novartis, and Roche Diagnostics, and is a co‐founder of Brain Biomarker Solutions in Gothenburg AB, a GU Venture‐based platform company at the University of Gothenburg. International Patent WO 15/116923 pertains to the results presented in this paper.

© 2021 The Authors. Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.

Figures

FIGURE 1

CT1812 is a selective sigma‐2 receptor ligand that prevents and treats amyloid beta (Aβ) oligomer‐induced membrane trafficking deficits. A, CT1812 binds sigma‐2 receptors with a 8.5 nM Kd and sigma‐1 receptors with a 63 nM Kd as measured in radioligand displacement studies (n = 2; means ± standard deviation [SD]). B, Effects of synthetic Aβ oligomers on vesicle trafficking in primary cultures of neurons and glia. Quantification is shown of the amount of formazan within intracellular vesicles in Aβ oligomer‐treated (red dot) or vehicle‐treated (blue square) conditions, and the dose‐dependent restoration of trafficking rate to normal vehicle‐treated values after CT1812 addition 1 hour before addition of Aβ oligomers (gray squares, EC50 = 6.7 μM, n = 11 experiments) or 1 hour after addition of Aβ oligomers (black squares, EC50 = 0.36 μM, n = 12 experiments). Vesicle trafficking was assayed 24 hours after addition of Aβ oligomers (1.75 μM, total peptide; means ± standard error of the mean). N represents the number of replicate experiments from separate cell culture preparations. C, Human Alzheimer's disease brain derived Aβ oligomers (1‐hour treatment) caused a 27 ± 11% (SD; P < 0.001, n = 3) decrease in trafficking rate in cultured neurons and glia. Pretreatment of cultures for 1 hour with 15 μM CT1812 before addition of human derived Aβ oligomers blocked the trafficking deficits (P < 0.001 for Aβ oligomers vs. Aβ oligomers + CT1812, analysis of variance with Tukey's post hoc test, n = 3 experiments). Each point represents results from quadruple replicates from Aβ oligomer preparations isolated from three separate donor brains with the mean of all the points represented by the horizontal lines

FIGURE 2

CT1812 prevents amyloid beta (Aβ) oligomer binding to and displaces bound oligomers from neuronal receptors, leading to restoration of synaptic density and protein expression in cultured mouse neurons. A, Aβ oligomers (750 nM, > 80% of Kd) bind specifically and saturably to a single receptor site on some, but not all, neurons in mature (> 21 DIV) primary hippocampal and cortical neuronal cultures. B, CT1812 [10 uM] prevents Aβ binding by 59 ± 8% (standard error of the mean [SEM]) when added 1 hour prior to addition of Aβ oligomers. C, CT1812 [10 uM] displaces Aβ oligomer binding by 42 ± 9% (SEM) when added 1 hour after addition of Aβ oligomers. Scale bar = 20 microns. D, CT1812 prevents binding and more than doubles the Kd of oligomer binding, from 430 nM (at 0 μM CT1812) to 1120 nM (at 10 μM CT1812; n = 6; means ± SEM). E, CT1812 displaces binding and nearly doubles the Kd of oligomer binding, from 610 nM (at 0 μM CT1812) to 1160 nM (for 10 μM CT1812; n = 6; means ± SEM). F, top, Aβ oligomers bind to receptors at neuronal synapses (red, 6E10 immunodetection), resulting in a significant loss of synapses (green, drebrin immunodetection, 12.8 ± 2.6% [SEM] decrease in synapse density, P < 0.01 vs. vehicle‐treated, Student t test [G]; the blue square represents synapse density in vehicle‐treated neurons). F, bottom,CT1812 displaces bound Aβ oligomers and restores synaptic numbers to normal in a dose‐dependent manner. Scale bar = 5 microns. G, CT1812 restores synapse number to normal whether added before (prevention, EC50 = 68 nM) or after (treatment, EC50 = 127 nM) Aβ oligomers (*P < 0.5 vs. Aβ oligomers alone for both conditions, Student's t test, means ± SEM). Treatment with CT1812 restores synaptic protein expression in cultured rat neurons. H, Neurogranin is expressed at high levels in postsynaptic dendrites and synapses in a subset of neurons in primary hippocampal/cortical cultures. I, Addition of Aβ oligomers (0.75 μM, 24 hours) causes a 28% loss of neurons expressing high levels of neurogranin (P = 0.014). J, Treatment with 4.8 μM CT1812 1 hour after addition of Aβ oligomers restores the expression of neurogranin to normal levels (Aβ oligomers vs. Aβ oligomers + CT1812 P = 0.031, vehicle vs. Aβ oligomers + CT1812, n.s., analysis of variance [ANOVA] with Tukey's post hoc test, n = 20). K, Synaptotagmin‐1 is expressed in presynaptic terminals. L, Addition of Aβ oligomers (3.5 μM, 48 hours) cause a 37% loss of synaptotagmin‐1 presynaptic terminals in primary hippocampal/cortical cultures (P = 0.0068, N = 24). M, Treatment with 4.8 μM CT1812 1 hour after addition of Aβ oligomers blocks these losses and restores expression of synaptotagmin‐1 levels (vehicle vs. Aβ oligomers + CT1812, n.s). H–M, ANOVA with Tukey's post hoc test, n = 20. For all cell culture experiments, n represents the number of replicate experiments from separate cell culture preparations, and data points represent means ± SD or SEM as noted

FIGURE 3

CT1812 treatment displaces amyloid beta (Aβ) oligomers from mouse hippocampal receptors and post mortem Alzheimer's disease human patient tissue and facilitates their clearance into cerebrospinal fluid (CSF). A, B, Microimmunoelectrodes (MIE) coated with oligomer‐specific antibody A11 (A) or mAβ40 antibody HJ2 (B) in 12‐month‐old transgenic hAPP/PS1 mice detect soluble Aβ in the hippocampal interstitial fluid (ISF). A, After intravenous injection of 0.3 or 3 mg/kg CT1812 (vertical dashed line), soluble Aβ oligomers are significantly elevated in hippocampal ISF in CT1812‐treated mice (n = 7) compared to vehicle‐treated mice (n = 4, means ± standard error of the mean [SEM]; *** P < 0.001, **** P < 0.0001, two‐way analysis of variance [ANOVA] with Sidak's post hoc test for effect of drug vs. vehicle). B, CT1812 treatment does not cause a change in total Aβ levels in the hippocampus (n = 5) compared to vehicle treatment (n = 4, means ± SEM). C–E, Aβ oligomers displaced from post mortem brain tissue sections from 8 AD patients were quantified by enzyme‐linked immunosorbent assay (ELISA; D) and native western blots (C [representative individual patient], E), and show the expected dose‐dependent increase in concentration in CT1812‐treated compared to the vehicle‐treated condition. For western blots, the sum of the intensity of discrete Aβ protein bands at 22, 33, 43, 48, and 85 Kd was quantified for each donor tissue section (C). In (D) and (E), values for Aβ measured by ELISA for each brain tissue section and for intensity of western blot bands for each brain tissue section normalized to vehicle‐treated controls (dashed line) from the same donor are shown, with the mean of all the points represented by the horizontal lines (*P < 0.05, ANOVA with Tukey's post hoc test for each treatment vs. vehicle treated controls). F (representative individual patient), G, H, Aβ oligomers (red) located in a 2 μM halo surrounding compact thioflavin‐S positive plaques (green) are displaced from frozen post mortem human AD brain tissue sections (N = 5 patients) by CT1812 in a dose‐dependent manner normalized to vehicle treated brain sections (dashed line) from the same individual with the mean of all the points represented by the horizontal lines (*P < 0.05, ANOVA with Tukey's post hoc test for each CT1812‐treated vs. vehicle‐treated controls). H, Aβ intensity within plaques does not change. Scale bar = 20 μM. I, J, Microimmunoelectrodes coated with oligomer‐specific antibody A11 (I) or pan‐Aβ antibody HJ2 (J) in 12‐month‐old transgenic hAPP/PS1 mice detect soluble Aβ in the lateral ventricle CSF. I, After intravenous injection of 0.3 (n = 7) or 3 (n = 5) mg/kg CT1812 (vertical dashed line), soluble Aβ oligomers are significantly elevated in lateral ventricle CSF compared to vehicle‐treated mice (n = 5, means ± SEM). J, CT1812 treatment (n = 5) did not cause a change in Aβ monomer levels in the CSF compared to vehicle treatment (n = 4 means ± SEM). *** P < 0.001, **** P < 0.0001, two‐way ANOVA Sidak's post hoc test for effect of drug vs. vehicle

FIGURE 4

Treatment with CT1812 improves learning and memory deficits in transgenic Alzheimer's mice. A, Transgenic Thy‐1 huAPPSwe/Ldn+ male mice treated with CT1812 (Tg + CT1812) learn the Morris water maze task significantly better than do transgenic vehicle‐treated mice (Tg + vehicle; P = 0.016, two‐way repeated measures analysis of variance, Bonferroni post hoc *P < 0.5; mean ± standard error of the mean). CT1812 treatment does not affect non‐transgenic animal performance (nTg + CT1812). B, Transgenic mice treated with CT1812 remember previous arms entered in the Y maze task significantly better (P = 0.013, Student's t test) than chance (dashed line), but transgenic vehicle‐treated animals do not (nTg + vehicle, 62.7 ± standard deviation [SD] 12.2%; Tg + vehicle, 56.1 ± SD 9.2%; Tg + CT1812, 58.5 ± SD 9.4%; nTg + CT1812, 65.3 ± SD 6.0%). C, Transgenic mice show deficits in the Contextual Fear Conditioning test (P = 0.037, Student's t test), while transgenic and nontransgenic mice treated with CT1812 do not (nTg + vehicle, 52.5 ± SD 5.4%; Tg + vehicle, 37.9 ± SD 6.4%; Tg + CT1812, 44.6 ± SD 6.5%; nTg + CT1812, 50.9 ± SD 5.1%). Each data point in (B) and (C) represents an individual mouse with the mean of all the points represented by the horizontal lines. D, Plasma and brain concentration of CT1812 after a single p.o. dose of 10 mg/kg (n = 3 mice per time point, mean ± SD)

FIGURE 5

CT1812 treatment significantly impacts cerebrospinal fluid (CSF) biomarkers of target engagement and disease‐related biology. Protein concentrations in CSF samples from Alzheimer's disease (AD) patients in a 28‐day phase 1b/2a clinical trial of CT1812 vs. placebo were measured via enzyme‐linked immunosorbent assay (ELISA), liquid chromatography tandem mass spectrometry (LC‐MS/MS), or gel electrophoresis. A, After 28 days the concentration of amyloid beta (Aβ) oligomers measured via western blot in CT1812‐treated AD patient CSF increases compared to the patient's own baseline and vs. placebo (P = 0.014, Student's t test, n = 3 placebo, 10 CT1812‐treated), providing supporting evidence of clinical target engagement. At day 28 of the study, the concentration of synaptic damage proteins neurogranin (B, measured by ELISA) and synaptotagmin‐1 (C, measured by LC‐MS/MS) decreases compared to the patient's own baseline and vs. placebo (P = 0.050 analysis of covariance, n = 5 placebo, 11 CT1812‐treated; and F1,12 = 8.8, P = 0.011, n = 4 placebo, 9 CT1812‐treated, respectively) providing evidence of a positive effect on synapses in patients with AD. A, B, and C, Data from individual patients are displayed with the treatment group mean represented by the horizontal lines. These changes are corroborated (D) by significant expression changes (P < 0.05) in multiple synaptic proteome proteins in the CSF measured by unbiased LC‐MS/MS in AD patients treated for 28 days with CT1812 vs. placebo. E, CSF proteomics identifies a subset of proteins significantly altered with CT1812 that have been reported to be significantly regulated in AD. Data are expressed as mean % change in protein concentration in CSF from CT1812 treated (n = 11) vs. placebo (n = 4; blue) and AD vs. control to illustrate how these proteins are altered in AD relative to cognitively normal age‐matched controls. All analytes shown are significantly regulated (P < 0.05) in AD patients vs. control and in the present study. F, After 28 days of treatment, the abundance of six tau phosphorylation peptides decreased by 30% (the threshold for noise distribution), or more after treatment with CT1812 compared to placebo (T52, T205, S262, S263, S285, S305) while one site increased more than 30% (S191), but the concentration of unphosphorylated tau did not change, providing evidence of CT1812 impact on pathological disease signaling. Colored bars represent amino acid sites within tau that are phosphorylated by GSK3β with priming (blue bars) or without priming (green bars),

FIGURE 6

Hypothesized mechanism of action of CT1812: synaptoprotective displacement of toxic amyloid beta (Aβ) oligomers from neurons. A, In the absence of Aβ oligomers, pre‐ and post‐synaptic proteins such as synaptotagmin‐1 and neurogranin are expressed at normal levels. B, The sigma‐2 receptor complex tightly regulates the oligomer receptor complex by stabilizing plasma membrane expression of oligomer receptor component proteins. C, Oligomer binding results in changing expression/localization of synapse associated proteins, spine loss, and memory failure, as well as upregulation of the sigma‐2 receptor complex. D, Fragmented pre‐ and post‐synaptic proteins such as synaptotagmin‐1 and neurogranin move into interstitial fluid and then cerebrospinal fluid. E, CT1812 binds to the sigma‐2 receptor ligand binding site within the sigma‐2 receptor complex, acting as a negative allosteric modulator of Aβ oligomer binding. This destabilizes the oligomer binding site and displaces oligomers from synapses without affecting normal synapse protein function. F, Synapse number and memory are restored to normal levels