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



ABSTRACT.INTRODUCTION:
The β-secretase enzyme, β-site amyloid precursor protein-cleaving
enzyme 1 (BACE1), cleaves amyloid precursor protein (APP) in the first step in
β-amyloid (Aβ) peptide production. Thus, BACE1 is a key target for candidate
disease-modifying treatment of Alzheimer’s disease. In a previous exploratory Aβ
biomarker study, we found that BACE1 inhibitor treatment resulted in decreased
levels of Aβ1-34 together with increased Aβ5-40, suggesting that these Aβ species
may be novel pharmacodynamic biomarkers in clinical trials. We have now examined
whether the same holds true in humans.

ABSTRACT.METHODS:
In an investigator-blind, placebo-controlled and randomized study,
healthy subjects (n =18) 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). We used hybrid
immunoaffinity-mass spectrometry (HI-MS) and enzyme-linked immunosorbent assays to
monitor a variety of Aβ peptides.

ABSTRACT.RESULTS:
Here, we demonstrate dose-dependent changes in cerebrospinal fluid
(CSF) Aβ1-34, Aβ5-40 and Aβ5-X after treatment with the BACE1-inhibitor LY2811376.
Aβ5-40 and Aβ5-X increased dose-dependently, as reflected by two independent
methods, while Aβ1-34 dose-dependently decreased.

ABSTRACT.CONCLUSION:
Using HI-MS for the first time in a study where subjects have been
treated with a BACE inhibitor, we confirm that CSF Aβ1-34 may be useful in
clinical trials on BACE1 inhibitors to monitor target engagement. Since it is less
hydrophobic than longer Aβ species, it is less susceptible to preanalytical
confounding factors and may thus be a more stable marker. By independent
measurement techniques, we also show that BACE1 inhibition in humans is associated
with APP-processing into N-terminally truncated Aβ peptides via a
BACE1-independent pathway.

ABSTRACT.TRIAL REGISTRATION:
ClinicalTrials.gov NCT00838084. Registered: First received: January 23, 2009, Last updated: July
14, 2009, Last verified: July 2009.

BODY.INTRODUCTION:
Alzheimer’s disease (AD) is a slowly progressing brain disease
manifesting several neuropathological characteristics including accumulation of
extracellular plaques, mainly composed of amyloid-β (Aβ) peptides of various lengths
[1,2]. Aβ is derived via two-step enzymatic cleavage of the
transmembrane amyloid precursor protein (APP) catalyzed by the β-site APP-cleaving
enzyme 1 (BACE1, β-secretase) [3] and
γ-secretase [4]. BACE1 cleaves APP at
the first amino acid of the Aβ domain and is crucial for the production of Aβ
peptides starting at position 1, including Aβ1-42. Thus, BACE1 is a key target for
disease-modifying AD treatments, since one focus for such therapies is to minimize
Aβ production [5].
To evaluate the biochemical effects of novel BACE1 inhibitor
candidates, biomarkers that reflect target engagement are needed [6]. Analyzing a wide range of Aβ species in
cerebrospinal fluid (CSF) gives useful information on APP metabolism in humans
[7,8]. In a recent preclinical study, we showed that APP-transfected
cells and dogs treated with several different BACE1-inhibitors expressed decreased
levels of Aβ1-34 and concurrently increased the levels of Aβ5-40 in cell media and
CSF, suggesting that these peptides may be pharmacodynamic markers of BACE1
inhibition in the central nervous system (CNS) [9]. Inhibition of γ-secretase, another AD drug candidate approach,
increased APP processing via the α-secretase-mediated pathway [10-13] and decreased CSF levels of Aβ1-34 in humans, even at dosages
when Aβ1-42 was unchanged, further supporting the use of novel CSF biomarkers to
monitor target engagement of anti-Aβ drugs [14-16].
Here, for the first time with a peptidomics approach, we have
demonstrated changes in CSF levels of Aβ1-34 and Aβ5-40 in humans treated with the
BACE1 inihibitor LY2811376 (Eli Lilly and Company, Indianapolis, IN, USA). The
translation of these findings from preclinical models to man indicates that CSF
Aβ1-34 and Aβ5-40 have potential utility as markers of BACE1 inhibition in clinical
research. Furthermore, the results strongly suggest that Aβ peptides starting at
amino acid 5 are produced through a non BACE1-dependent pathway in humans.

BODY.METHODS:
BODY.SUBJECTS:
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.

BODY.HYBRID IMMUNOAFFINITY-MASS SPECTROMETRY:
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.

BODY.CELL EXPERIMENTS:
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.

BODY.ENZYME-LINKED IMMUNOSORBENT ASSAY:
For quantification of Aβ5-40 and
Aβ5-x using ELISA, microtiter plates were coated with
10 μg/mL 2G3 [21]
(anti-Aβx-40; epitope including valine at position 40,
Eli Lilly & Company, Indianapolis, IN, USA) or 266 [22] (anti-Aβ1-x; epitope
13-28, Eli Lilly & Company) overnight at 4°C. After blocking plates in 2%
bovine serum albumin (BSA), dilutions of Aβ5-40 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-Aβ5-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
Aβ5-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 Aβ5-42 as compared
to full-length Aβ1-42. 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 Aβ5-42 standard. Note, the
migration of the Aβ peptides in this gel system completely separates the
Aβ5-42 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 Aβ5-x
epitope versus the full-length peptide (Aβ1-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].

BODY.STATISTICAL ANALYSIS:
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).


BODY.RESULTS:
BODY.LY2811376 INDUCES A CHARACTERISTIC AΒ PEPTIDE PATTERN IN A HUMAN-DERIVED
NEUROBLASTOMA CELL LINE:
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.Figure 1
Mass spectra displaying the effect of treatment on
multiple Aβ species in cell media. (A) DMSO (vehicle),
(B) 1.25 μM of the BACE1-inhibitor
LY2811376, (C) 2.5 μM of the
BACE1-inhibitor LY2811376, (D) 1.25 μM of
the BACE-inhibitor BACE IV and (E) 2.5 μM
of the BACE-inhibitor BACE IV in media from SH-SY5Y cells. *Represent
unidentified peaks. Aβ, β-amyloid; BACE, β-site APP-cleaving enzyme; DMSO,
dimethyl sulfoide.


BODY.THE BACE1 INHIBITOR LY2811376 CAUSES A RELATIVE REDUCTION IN CSF AΒ1-34 AND
AN INCREASE IN CSF AΒ5-40 IN HUMANS AS REFLECTED BY MASS SPECTROMETRY:
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.Figure 2
Mass spectra displaying multiple Aβ species
recovered from human CSF specimens by immunoprecipitation with the
anti-Aβ antibodies 6E10 and 4G8. (A) Pre-treatment, (B) 12, (C) 24,
and (D) 36 hours post treatment with
90 mg of LY2811376. The right-hand panels are magnified spectra displaying
the increase in Aβ5-40 and decrease in Aβ1-34 in response to treatment.
Aβ, β-amyloid; CSF, cerebrospinal fluid.

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.Figure 3
The relative mass spectrometric change from baseline
and ELISA-derived concentrations in response to a single dose of 30 mg
or 90 mg of the BACE inhibitor LY2811376. (A) Mass
spectrometric change in the CSF Aβ1-34 time course after LY2811376
treatment and (B) mass spectrometric
change in the CSF Aβ5-40 time course after LY2811376 treatment. (C) ELISA-derived concentrations of the CSF
Aβ5-40 time course after LY2811376 treatment and (D) ELISA-derived concentrations of the CSF Aβ5-X time
course after LY2811376 treatment. Open circles represent placebo, grey
squares represent treatment with 30 mg LY2811376 and closed triangles
represent treatment with 90 mg LY2811376. Data are presented as mean ± SD
and n =6 for both graphs. Aβ,
β-amyloid; BACE, β-site APP-cleaving enzyme; CSF, cerebrospinal fluid; n,
number; SD, standard deviation.


BODY.THE BACE1-INHIBITOR LY2811376 CAUSES AN ABSOLUTE INCREASE IN BOTH CSF
AΒ5-40 AND AΒ5-X IN HUMANS AS REFLECTED BY ELISA:
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.Figure 4
Correlation between Aβ5-X and (A) sAPPα and (B)
sAPPβ in the 90-mg treatment group. Aβ, β-amyloid; APP,
amyloid precursor protein.



BODY.DISCUSSION:
In the present study, we show marked effects on CSF Aβ5-40 (which
increases) and Aβ1-34 (which decreases) in response to BACE1 inhibitor treatment.
These findings confirm earlier pre-clinical data [9] and suggest that CSF Aβ5-40 and Aβ1-34 may be useful
pharmacodynamic markers for assessing the biochemical effects of BACE-1 inhibitors
in the CNS in clinical trials. The relatively low concentrations of both Aβ5-40 and
Aβ5-X fit previous findings with comparable percentage reductions in Aβ1-40 versus
AβX-40 and Aβ1-42 versus AβX-42 in dog CSF following oral administration of
LY2811376 [17].
Since the discovery and molecular cloning of BACE1 in 1999 by several
independent groups, this enzyme has been a tempting target for pharmacological
lowering of cerebral Aβ levels with the intent of treating or preventing AD. To
date, there are only a few reports of BACE1 inhibitors that have demonstrated
sufficient access to the brain. In a recent paper, oral administration of the
non-peptidic BACE1 inhibitor LY2811376 to healthy subjects (same patients as
included in the present study) dose-dependently lowered CSF Aβ1-40, Aβ1-42 and sAPPβ
levels and dose-dependently increased CSF sAPPα, providing evidence of desirable
central pharmacodynamic effects on APP processing [17]. In another study, a therapeutic antibody that reduces BACE1
activity was used, resulting in lowered CNS Aβ concentrations in preclinical models
[23]. Whether this approach can be
translated to humans and if other Aβ species besides Aβ1-40 are affected in response
to treatment remain to be elucidated.
LY2811376 treatment consistently increased CSF levels of Aβ5-40. The
increase of Aβ5-40 in response to BACE1 inhibition clearly suggests that production
of Aβ peptides starting at position 5 is formed via a BACE1-independent
APP-processing pathway [9]. In agreement
with this, it has been suggested that inhibition of BACE1 might be linked to a
distinct processing of APP between Phe4 and Arg5 mediated by α-secretase-like
proteases [24]. Other enzymes which
might cleave in this region of Aβ include α-chymotrypsin, myelin basic protein and
protease IV [25]. However, while these
enzymes have been shown to cleave Aβ in vitro,
data from the CNS showing which enzyme that cleaves between Phe4 and Arg5 inhibition
of BACE1 is lacking.
Recently, we showed in pre-clinical models that CSF Aβ1-34 is a
sensitive marker for BACE1 inhibition [9]. We have previously shown, in two independent clinical trials,
that CSF Aβ1-34 is a pharmacodynamic marker of γ-secretase inhibition in humans
[14,15] and here we show for the first time that it is also a marker of
BACE1 inhibition in humans. It has been shown that the cleavage between Leu34 and
Met35 depends on both BACE1 and γ-secretase [26,27]. Thus, Aβ1-34
is an intriguing peptide to follow in clinical trials of BACE1 inhibitors since
cleavages at position 1 and position 34 both depend on BACE. It is also possible
that Aβ1-34 is more stable than Aβ1-42, as it is less hydrophobic and may thereby be
less prone to preanalytical confounding factors.
Aβ5-40 has been found in AD brains [28], but the exact role of this Aβ species in AD pathogenesis (and
normal physiology), if any, is unknown and we propose that further studies of
biological functions and how the peptide might be relevant to AD pathophysiology are
warranted.
We found a positive correlation between sAPPα and Aβ5-X. This
correlation may reflect a compensatory increase in APP cleavage at the α-site and
between amino acid 4/5, or that there might be more substrate for these enzymes due
to inhibition of BACE. We also found a negative correlation between sAPPβ and Aβ5-X,
clearly showing that while the amyloidogenic pathway is affected, the (as yet)
unknown enzyme generating Aβ5-X cleaves its substrate more.
There are several non-quantitative aspects of HI-MS. The relative
quantification using mass spectrometry cannot be interpreted as a direct reflection
of an absolute or relative abundance. However, in the present study we have verified
the mass spectrometric data showing increased relative levels of Aβ5-40 with a
proprietary ELISA showing increased concentrations of both Aβ5-40 and Aβ5-X in
response to inhibition of BACE1. What also should be noted is that the ELISA
measures an absolute concentration while MS reports the relative change of Aβ5-40
relative to all other Aβ peptides detected in the same spectra. A previous study on
the same patients as those included in the present study showed a marked decrease in
CSF Aβ1-40 in response to LY2811376 treatment [17]. Due to the relative quantification used in the present study,
we were not able to measure the expected decrease. However, by implementing
isotopically-labelled Aβ peptides for each peptide of interest, relative small
changes in response to treatment should be possible to detect with HI-MS.

BODY.CONCLUSIONS:
In summary, our results confirm that CSF Aβ1-34 may be useful in
clinical trials on BACE1 inhibitors to monitor target engagement. By independent
measurement techniques, we show that BACE1 inhibition in humans is associated with
APP-processing into N-terminally truncated Aβ peptides via a BACE1-independent
pathway. The data presented also provide evidence for CSF Aβ1-34 and Aβ5-40 as
translatable pharmacodynamic markers for BACE1-inhibition from cell and animal
models to humans.

Question: Compared to placebo what was the result of 90 mg of LY2811376 on Aβ5-X and Aβ5-40 levels?
Evidence: 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).
Answer: significantly increased