β-site amyloid precursor protein-cleaving enzyme 1(BACE1) inhibitor treatment induces Aβ5-X peptides through alternative amyloid precursor protein cleavage

The study, conducted at PAREXEL International Early Phase Los Angeles, CA, USA, from February to June 2009, was previously reported in detail [17]. In brief, the study was a subject- and investigator-blind, placebo-controlled, randomized, single-dose design. The California Institutional Review Board approved the study. All subjects provided written informed consent before the beginning of the study. The trial was conducted in compliance with the Declaration of Helsinki and International Conference on Harmonisation/Good Clinical Practice guidelines. Eighteen healthy subjects (21 to 49 years old, seventeen men and one woman) participated in the study and were randomly assigned to receive a single dose of 30 mg of LY2811376 (n =6), 90 mg of LY2811376 (n =6) or placebo (n =6). An indwelling lumbar catheter was placed four hours before administration of the study drug and subjects remained supine for the duration of the CSF sample collection period. CSF samples were collected prior to and at regular intervals over 36 hours after drug administration and analyzed by immunoprecipitation in combination with mass spectrometry (MS). All CSF samples were collected in polypropylene tubes and stored at -80°C.

Immunoaffinity capture of Aβ species was combined with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for analyzing a variety of Aβ peptides in a single analysis as described in detail elsewhere [18]. In brief, the anti-Aβ antibodies 6E10 and 4G8 were separately coupled to magnetic beads. After washing of the beads, the 4G8 and 6E10 coated beads were used in combination for immunoprecipitation. After elution of the immune-purified Aβ peptides, analyte detection was performed on an UltraFlextreme MALDI TOF/TOF instrument (Bruker Daltonics, Bremen, Germany). For relative quantification of Aβ peptides, an in-house developed MATLAB (Mathworks Inc. Natick, MA, USA) program was used. For each peak the sum of the intensities for the three strongest isotopic signals was calculated and normalized against the sum for all the Aβ peaks in the spectrum, followed by averaging of results for separately determined duplicate samples. In the 30-mg group, one sample, six hours post treatment, was omitted from further analysis due to blood in the CSF.

SH-SY5Y cells [19] obtained from the European Collection of Cell Cultures (ECACC 94030304), stably expressing human APP, were maintained in Dulbecco’s modified Eagle’s medium F-12 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, L-glutamine and antibiotics. SH-SY5Y cells were treated with the BACE1-inhibitor β-secretase inhibitor IV (Calbiochem, Merck, compound 3, Darmstadt, Germany) [20], LY2811376, or dimethyl sulfoxide (DMSO) and incubated for 20 hours.

For quantification of Aβ_5-40 and Aβ_5-x using ELISA, microtiter plates were coated with 10 μg/mL 2G3 [21] (anti-Aβ_x-40; epitope including valine at position 40, Eli Lilly & Company, Indianapolis, IN, USA) or 266 [22] (anti-Aβ_1-x; epitope 13-28, Eli Lilly & Company) overnight at 4°C. After blocking plates in 2% bovine serum albumin (BSA), dilutions of Aβ_5-40 standards (Anaspec) and CSF samples were incubated on plates in 1% BSA, 0.55 M guanidine-HCL, 5 mM Tris in phosphate buffered saline (PBS) with complete ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor (Roche, Mannheim, Germany) overnight at 4°C. After washing in PBS-0.05% Tween 20, biotinylated 5H5 (anti-Aβ_5-x; epitope including arginine at position 5, Eli Lilly & Company) was used to detect the truncated Aβ beginning at the arginine at position 5. The 5H5 monoclonal antibody was developed in mice following standard methods and the specificity for the truncated Aβ_5-x was investigated by acid urea gel (a technique that separates Aβ peptides by mass and charge) and ELISA methods. Acid urea gel separation of synthetic Aβ peptides followed by Western blotting with 5H5 revealed complete selectivity for the truncated Aβ_5-42 as compared to full-length Aβ_1-42. Additionally, acid urea gel/5H5 Western blotting analysis of human cortical tissue from multiple Alzheimer’s subjects resulted in a single identifiable band that co-migrated at the same position as the synthetic Aβ_5-42 standard. Note, the migration of the Aβ peptides in this gel system completely separates the Aβ_5-42 from all other Aβ peptides (truncated or full-length). ELISA analyses to investigate the 5H5 epitope selectivity demonstrated a 20,000-fold selectivity for the Aβ_5-x epitope versus the full-length peptide (Aβ_1-x). Following additional washes in PBS-0.05% Tween 20, plates were incubated with streptavidin-horseradish peroxidase (HRP) (Biosource, San Diego, CA, USA) and subsequently, 3,3´,5,5´-Tetramethylbenzzidine (TMB) (Sigma, St. Louis, MO, USA) color development was monitored at 650 nm in a spectrophotometer.

Quantification of CSF sAPPα and sAPPβ was conducted as described previously and the results from these analyses have already published [17].

The time series for each treatment were analyzed using Friedman’s test (SPSS v13, Chicago, IL, USA). A dose-dependent effect was considered significant if P <0.05 and if the P-value decreased with increasing dose. Association analyses were performed by Spearman’s rank correlation and the correlation coefficient is presented by spearman’s rho (rs).

As expected, SH-SY5Y cells treated with the BACE1-inhibitor LY2811376 or BACE IV secreted less Aβ1-40 and Aβ1-42 to the cell medium while the relative levels of Aβ5-40 (relative to the other Aβ peptides detected) increased, as compared to vehicle-treated cells (Figure 1). These data clearly demonstrate that LY2811376 inhibits BACE1 activity and that the generation of Aβ5-40 is BACE independent.

To evaluate if the BACE1-mediated changes described in exploratory Aβ biomarker studies were translatable to humans, the CSF mass spectrometric Aβ peptide pattern from untreated subjects was compared to the pattern from subjects treated with different concentrations of the BACE1-inhibitor LY2811376. Representative CSF Aβ peptide mass spectra from a subject before treatment and 36 hours after drug administration are shown in Figure 2A-D. Although barely detectable versus background before treatment, BACE1 inhibition increased the mass spectrometric signal for Aβ5-40 while the signal corresponding to Aβ1-34 decreased. In total, 13 Aβ species ranging from Aβ1-15 up to Aβ1-42 were reproducibly detected.

The BACE1 inhibitor LY2811376 dose-dependently reduced Aβ1-34 relative to baseline with a nadir of 42% in the 30-mg group (P =0.002) and 57% in the 90-mg group (P <0.001) respectively, 24 hours after drug administration (Figure 3A). By contrast, LY2811376 dose-dependently increased Aβ5-40 to a maximum relative to baseline after 18 hours in the 30-mg (P =0.213) and the 90-mg (P <0.001) groups, respectively (Figure 3B). The mass spectrometric signal for Aβ5-40 in the placebo group was below the limit of detection while in the 90-mg treatment group the signal-to-noise ratio was 4 to 5. At 36 hours post-treatment, both Aβ5-40 and Aβ1-34 had started to return towards baseline levels in both treatment groups.

The increase in Aβ5-40 detected by mass spectrometry in response to treatment with the BACE1 inhibitor LY2811376 was further confirmed by a proprietary ELISA. While the placebo concentrations were low, in the range of approximately 100 pg/mL and approximately 50 pg/mL for Aβ5-X and Aβ5-40, respectively, there were clear increases in the LY2811376 high dose (90 mg) group over time for both Aβ5-X and Aβ5-40 (Figure 3C-D) of which the increase in Aβ5-X was statistically significant (P =0.02). The ELISA-determined concentrations of Aβ5-42 were too low to yield an accurate assessment, which is in agreement with the mass spectrometric data where Aβ5-42 could not be detected in any treatment group.

In the 90-mg dose group, there was a compensatory increase in the concentrations of both Aβ5-X and sAPPα (rs =0.94, P =0.02) while Aβ5-X was negatively correlated with sAPPβ (rs = -0.89, P =0.03) as presented in Figure 4A,B. There were no correlations between the two peptides starting at amino acid five and sAPPα or sAPPβ in the 30-mg and placebo groups.