Inhibitor GRL-8234 enters the brain and inhibits the level of soluble Aβ. A) Amount of GRL-8234 (μg inhibitor/g tissue) in the plasma and brain of rats over a 24-h period following a single intravenous administration of ¹⁴C-GRL-8234 (4 mg/kg). At each time point (n=3), plasma and brain were collected for scintillation counting. Level of inhibitor in brain tissue was corrected (open squares) for the presence of 4% plasma (23). Brain penetration of the inhibitor was estimated by AUC to be 16.3% over 24 h. B) Effect of GRL-8234 on interstitial Aβ in Tg2576 mice. Mice (n=4) were intraperitoneally injected with GRL-8234 (33.4 μg/g body weight in vehicle solvent, a 1:1 mixture of 0.5% PEG-300 and 5% glucose), and interstitial Aβ was measured by in vivo microdialysis for 14 h (n=4/group). ISF Aβ levels were reduced by 46.6 ± 1.5% at 3 h after treatment compared to vehicle-treated mice and reduced by 52.3 ± 12.1% at 14 h (P=0.0019 and P=0062, respectively; means±se). C) Plasma Aβ₄₀ of Tg2576 after a single intraperitoneal injection of 8 mg/kg of GRL-8234. Statistical differences were observed at 5 time points from 3 to 11 h (P<0.02, n=6).

Effect of GRL-8234 treatment on cognitive performance of Tg2576 mice. A–D) Effect of β-secretase inhibitor GRL-8234 on cognitive performance of Tg2576 mice. Mice from experiment 1 (n=7; Fig. 3A) were treated with either inhibitor or vehicle solvent and tested for spatial reference memory in MWM at time point in treatment indicated at top. A) Escape latency time for control (open circles) and treated mice (solid circles). *P < 0.05. B) Mean ACI from probe trials. *P < 0.001. C) Mean percentage TQ from probe trials. *P < 0.001. D) Mean ACI from probe trials of experiments 2 (n=4) and 3 (n=9). Data were obtained at the end of each experiment, as shown in A. Probe trials were administered at the beginning of d 3, 5, and 6 of training for experiment 2 and d 3, 5, 7, and 8 of training for experiment 3. *P < 0.001 (left panel); *P = 0.005 (right panel). E) Effect of β-secretase inhibitor GRL-8234 on Y-maze cognitive performance of Tg2576 mice. Mice from experiment 3 (n=9) were tested for spatial working memory in Y maze followed by MWM. Latency to exit the start arm and sequence and number of arm visits were recorded. Total arm entry N and spontaneous alternation, % alternation/(N − 2), were calculated. *P = 0.04.

Summary of the properties of GRL-8234. ^aUnpublished results. ^bTested in rotarod, wire hanging, open field, and Irwin tests, respectively.

Effect of GRL-8234 treatment on plasma Aβ levels. A) Experimental schemes. Infusion of inhibitor or vehicle solvent was initiated at surgical implantation of osmotic pump (open arrows) in mice at the age indicated by the scale at top and was continued by a series of pumps, each lasting 21 d, throughout the experimental period (horizontal bar). Cognitive performance of mice was evaluated with Morris water maze at time points indicated (solid arrows). Brain tissues were collected at end of the last cognitive tests. Number of mice (n) used in each control and inhibitor-treatment group is listed for each experiment. B) Effect of GRL-8234 inhibitor treatment on plasma Aβ₄₀ and Aβ₄₂ concentrations in Tg2576 mice. Open arrow indicates time of initial inhibitor or vehicle solvent delivery. Blood samples were collected weekly; only 1/4 of data points are shown. Data are from experiment 1 (A); n = 7/group. *P < 0.05.

Effect of GRL-8234 on Aβ and APP in brains of Tg2576 mice. A) Effect of inhibitor treatment on amyloid-β plaques. Brains from mice in experiment 1 were fixed, sliced, and immunohistochemically stained with anti-Aβ_39–43 monoclonal antibody. Left panels: representative stainings for control (vehicle; left) and treated mice (GRL-8234, right). Low-power (×40) and high-power (×200) views show a reduction in amyloid plaque deposits. Right panels: average plaque numbers (left) and average plaque occupied area in percentage of total area (right) in cortex of the brain in vehicle- and GRL-8234-treated mice (n=7). *P = 0.017 (left); *P = 0.036 (right). B) Amount of Aβ₄₀ (left graphs) and Aβ₄₂ (right graphs) in extract of Tg2576 mouse brains at end of experiment 1. One hemisphere of each mouse was extracted for soluble Aβ using a buffer containing CHAPS (left panels) and for insoluble Aβ using a buffer containing guanidine (right panels). Amounts of Aβ₄₀ and Aβ₄₂ from both homogenates were determined by sandwich ELISA. Open and solid bars represent data from control and inhibitor-treated mice, respectively (n=7). *P = 0.05. C) Brain Aβ oligomer species from extracellular-enriched fractions in control (open bars) and treated Tg2576 mice (solid bars). One brain hemisphere from mice in experiment 3 was homogenized; extracellular-enriched (left panel) and intracellular-enriched (right panel) fractions were prepared, and Aβ oligomer species were determined by densitometry reading of bands in Western blots. Only the decrease of the 12-mer in the inhibitor-treated group was significant (P=0.007, n=9). D) Brain full-length APP in vehicle- and GRL-8234-treated Tg2576 mice from experiment 3. Membrane fraction of brain homogenate was prepared and extracted with 3% SDS in 50 mM Tris-HCl buffer (pH 7.4) and analyzed by Western blot. Top panel: representative pattern from each group; M and I indicate bands of mature and immature APP, respectively. Bands at bottom are from a separate Western blot of the membrane marker: ε2 subunit of N-methyl d-aspartate (NMDA) receptor. Bottom panel: average density of APP species and NMDA receptor, as determined from Western blot bands. Amounts of APP from the two groups were not statistically different (n=9). E) Brain β-secretase in intracellular-enriched fractions of vehicle- and GRL-8234-treated Tg2576 mice from experiment 3. Top panel: representative pattern of β-secretase from each group; M and I indicate bands of mature and immature β-secretase, respectively, with a single band for recombinant mature β-secretase. Bands at bottom are from a separate Western blot of the control β-actin blot. Bottom panel: average density of β-secretase as determined from Western blot bands. Amounts of mature or immature β-secretase from vehicle- and GRL-8234-treated mice are not statistically different (P=0.214 and P=0.497, respectively; n = 9).