Original Manuscript

Clinical Pharmacology
Pharmacokinetics, Safety, Tolerability, in Drug Development
2018, 00(0) 1–14
and Pharmacodynamics of Alicapistat, C 2018, The American College of

Clinical Pharmacology
DOI: 10.1002/cpdd.598
a Selective Inhibitor of Human Calpains
1 and 2 for the Treatment of Alzheimer
Disease: An Overview of Phase 1
Studies

Hoi-Kei Lon, Nuno Mendonca, Sandra Goss, Ahmed A. Othman, Charles Locke,
Ziyi Jin, and Beatrice Rendenbach-Mueller

Abstract
Alicapistat is an orally active selective inhibitor of calpain 1 and 2 whose overactivation has been linked to Alzheimer
disease (AD). Three studies were conducted in healthy subjects (18-55 years), 1 in healthy elderly subjects (65 years),
and 1 in patients with mild to moderate AD. Four studies assessed pharmacokinetics, 1 study in healthy subjects assessed
pharmacodynamics (sleep parameters, particularly rapid eye movement [REM], as a measure of central nervous system
[CNS] penetration and activity), and all studies assessed safety. Participants received single doses or multiple twice-daily
doses of alicapistat for up to 14 days. Maximum alicapistat plasma concentrations were reached in 2 to 5 hours; half-life
was 7 to 12 hours postdose. Alicapistat exposure was dose proportional in the alicapistat 50- to 1000-mg dose range.
Exposure of the alicapistat R,S diastereomer was approximately 2-fold greater than exposure of the R,R diastereomer in
healthy young and elderly subjects and patients with AD. Alicapistat at 400- or 800-mg twice-daily doses had no effect on
REM sleep parameters, whereas the active control, donepezil at 10 mg twice daily, affected sleep parameters. Across all
trials, the incidence of treatment-emergent adverse events was similar in the placebo and alicapistat groups. There were
no clinically significant changes in vital signs and laboratory measurements. The lack of an effect of alicapistat on sleep
suggests that concentrations in the CNS were inadequate or that preclinical studies do not predict alicapistat effects in
humans.

Keywords
calpain inhibitor, pharmacokinetics, pharmacodynamics, biopharmaceutics, neuroscience, clinical trials

Alzheimer disease (AD) is the most common form of
dementia1 ; with the aging of the population, AD will af-
fect an estimated 14 million people in the United States
by 2050.2 The main pathologic features of AD include
the accumulation of amyloid plaques, neurofibrillary
tangles, and diffuse loss of neurons and synapses.3,4 The
pathogenesis of AD is unclear, but many hypotheses
have been proposed.1,4
Currently approved treatment options include
acetylcholinesterase inhibitors (AChE-I’s) and the N-
methyl-D-aspartate receptor antagonist memantine,5
both of which have modest symptomatic effects.6–8
Therefore, efficacious treatment of AD, in particular
drugs that can halt or delay progression, is an unmet
medical need.8
The calpains are a family of proteases constitu-
tively expressed in an inactive form, some of which
are activated by Ca2+ .9 Calpain 1 and 2 are Ca2+ -
activated calpains and members of a subfamily of
cysteine proteinases. Calpain 1 and 2 differ in Ca2+
AbbVie Inc., North Chicago, IL, USA
Submitted for publication 18 January 2018; accepted 28 May 2018.
Corresponding Author:
Beatrice Rendenbach-Mueller, PhD, Senior Project Leader, Neuroscience
Development, AbbVie Deutschland GmbH & Co. KG, Knollstrasse,
Building 34, Room 205, 67061 Ludwigshafen, Germany
(e-mail: beatrice.rendenbach-mueller@abbvie.com)
Ahmed A. Othman is a fellow of the American College of Clinical
Pharmacology.
2 Clinical Pharmacology in Drug Development 2018, 00(0)
O
HN (R,S) N parameters, similar to effects observed with the AChE-
H
(R) O I donepezil. In short, alicapistat dose-dependently in-
O creased ACh levels in the medial prefrontal cortex and
N hippocampus, as well as glutamate levels in the hip-
O
Figure 1. Chemical structure of alicapistat.
sensitivities required for in vitro activation, have highly
conserved amino acid sequences, and are expressed
ubiquitously, including in the central nervous system
(CNS).9,10 Overactivation of calpain 1 and 2 has been
linked to neurodegenerative diseases including AD.10,11
In animal models of AD, inhibition of calpain 1
and 2 restores normal synaptic function and cognitive
behavior and decreases amyloid plaques.12,13
Alicapistat (ABT-957), an orally active selective
small-molecule inhibitor of human calpain 1 and
porcine calpain 2 that has high selectivity for calpain
1 and 2 versus other cysteine proteases such as cathep-
sins B, K, L, and S, was being investigated for the
treatment of AD in a program that has since been ter-
minated. Alicapistat is a 1:1 mixture of 2 interconvert-
ible diastereomers (alicapistat R,S and alicapistat R,R
diastereomers) with a molecular weight of 434 g/mol
(Figure 1). In preclinical studies, alicapistat exhibited
moderate permeability in human Caco-2 cells and was
a substrate for efflux via P-glycoprotein but not breast
cancer resistance protein in Madin-Darby canine kid-
ney II cells (unpublished observations). In vitro, the
fraction of alicapistat binding to human plasma pro-
tein was approximately 0.3; the fraction bound to
α1-acid glycoprotein was approximately 0.4. Alicapis-
tat was metabolized in vitro primarily by cytochrome
P450 (CYP) 3A4 (94%) and, to a far lesser extent,
by CYP2C198, CYP2D6, and CYP3A5; therefore, it
was expected that strong inhibitors of CYP3A4 would
raise exposure to alicapistat. Alicapistat was stable
in liver microsomes and hepatocytes across species
(mouse, rat, monkey, dog, and human) and demon-
strated low liability to cytosolic carbonyl reductase ac-
tivities, with low liver cytosol intrinsic clearance (0.005
and 0.07 L/h/kg in monkeys and humans, respectively).
In preclinical animal models (rats, dogs, and monkeys),
alicapistat penetrated the CNS with cerebrospinal fluid
(CSF)/plasma concentration ratios ranging from ap-
proximately 0.25 to 0.60 across species (unpublished
observations).
Alicapistat, like other selective calpain inhibitors,
has demonstrated efficacy in a broad range of an-
imal models relevant to AD, including cortical and
hippocampal acetylcholine (ACh) release, (age-related)
cognitive decline in rodents and monkeys, social recog-
nition in rodents, and rapid eye movement (REM) sleep
pocampus of rats, as assessed by microdialysis. The
same doses resulted in significant enhancement of
REM sleep time and the number of REM episodes.
The effects on both ACh release and REM sleep were
similar to those of donepezil. In addition to its effects
on biochemical and sleep parameters, alicapistat sig-
nificantly improved cognitive deficits, as seen in rodent
assays such as the social recognition task and the T-
maze alternation task, as well as in the delayed-match-
to-sample task in aged rhesus monkeys. In addition to
the effects on cognition, several experiments showed a
blockade of N-methyl-D-aspartate (NMDA)–induced
neurotoxicity in hippocampal slices and an attenuation
of in vivo NMDA-induced cholinergic neurodegenera-
tion after treatment with alicapistat. These findings sup-
port the hypothesis that selective calpain inhibition by
alicapistat may have the potential for both symptomatic
effects and attenuation of the neurodegeneration occur-
ring with AD. In the aforementioned multiple animal
models, efficacy was achieved at mean unbound alicapi-
stat plasma concentrations of 9 to 48 ng/mL and a geo-
metric mean of 13 ng/mL, corresponding to mean total
plasma concentrations of 290 to 690 ng/mL and a ge-
ometric mean of 418 ng/mL. Assuming that the most
conservative preclinical value for the CSF/plasma con-
centration (0.25) would apply to humans, it was esti-
mated that an efficacious concentration of alicapistat in
the CSF would be approximately 4 ng/mL (unpublished
observations).
This report summarizes the results of 5 phase 1 stud-
ies conducted to assess the pharmacokinetics, safety,
tolerability, and pharmacodynamics (ie, effect on sleep
parameters) of alicapistat in healthy subjects and in
patients with mild to moderate AD.
Methods
Study Designs
Studies 1, 2, and 3 were single-center, randomized,
double-blind, placebo-controlled trials in healthy men
and women designed primarily to test the pharma-
cokinetics, safety, and tolerability of single-escalating,
multiple-escalating, or multiple fixed oral doses of al-
icapistat (Table 1). Tested doses ranged from 50 to
1000 mg. Study 1 also included an open-label, 3-period,
randomized crossover substudy to test the effects of
food and ketoconazole (400 mg once daily) on the pro-
file of single doses of alicapistat 150 mg. Study 2 also
Lon et al 3

Table 1. Phase 1 Studies of Alicapistat

Pharmacokinetics Blood and CSF Sampling;

Study Participants Study Design Sample Size Determination

1 Healthy men and women aged Single-center study conducted at CPPM
18-55 years
n = 59
Part 1: randomized, double-blind,
Part 2: randomized, 3-period, open-label
2 Groups 1-4: healthy men and Single-center study conducted at
women aged 18-55 years
Group 5: healthy men and
women aged  65 years
n = 55
Groups 1-4: randomized, double-blind,
Research Unit, Grayslake, Illinois, between
March 2012 and August 2012 (IRB: Vista
Health System, Waukegan, Illinois).
placebo-controlled, single-dose study of
alicapistat (50, 100, 200, 400, 700, and
1000 mg). Six serial groups with up to 8
subjects/group (6 subjects received a
single dose of alicapistat [5 subjects in the
50-mg group], and 2 subjects received a
single dose of placebo).
crossover study of single doses of
alicapistat 150 mg (n = 12). In periods 1
and 2, subjects received alicapistat under
fasting or nonfasting conditions,
respectively. In period 3, subjects received
400 mg of ketoconazole once daily under
fasting conditions.
Northwestern Lake Forest Hospital,
Grayslake, Illinois, between June 2013 and
January 2014 (IRB: Vista Health System,
Waukegan, Illinois).
placebo-controlled, multiple-dose study
(each group: alicapistat, n = 9; placebo,
n = 3).
r Group 1: alicapistat 100 mg or
placebo twice daily for 7 days
r Group 2: alicapistat 200 mg or
placebo twice daily for 7 days
r Group 3: alicapistat 400 mg or
placebo twice daily for 14 days
r Group 4: alicapistat 800 mg or
placebo twice daily for 14 days
r Group 5: open-label single-dose
study of alicapistat 400 mg (n = 7)
Part 1: blood before dosing and 0.5, 1, 1.5, 2,
3, 4, 6, 9, 12, 16, 24, 36, 48, 60, and 72 hours
postdose.
Part 2: blood before dosing and 0.5, 1, 1.5, 2,
3, 4, 6, 9, 12, 16, 24, 36, 48, 60, 72, and 96
hours postdose.
Determination of sample size for
tolerability assessment in part 1: the
probability that a given AE would not be
observed in a group of 6 subjects was
0.004 for an AE with a true population
incidence rate of 0.5.
Groups 1 and 2: blood before the morning
dose on day 1 and 0.5, 1, 1.5, 2, 3, 4, 6, 9,
and 12 hours (before the evening dose),
before the morning dose on days 3, 5, and
6, and before the morning dose on day 7
and after 0.5, 1, 1.5, 2, 3, 4, 6, 9, 12 (before
the evening dose), 12.5, 13, 13.5, 14, 15, 16,
18, 21, 24, 36, 48, 60, and 72 hours.
Groups 3 and 4: blood before the morning
dose on day 1 and 0.5, 1, 1.5, 2, 3, 4, 6, 9,
and 12 hours (before the evening dose),
before the morning dose on days 3, 5, and
6, before the morning dose on day 7, and
after 0.5, 1, 1.5, 2, 3, 4, 6, 9, 12 (before the
evening dose), 12.5, 13, 13.5, 14, 15, 16, 18,
21, 24, 36, 48, 60, and 72 hours, before the
morning dose on days 10, 12, and 13, and
before the morning dose on day 14 and
after 0.5, 1, 1.5, 2, 3, 4, 6, 9, 12 (before the
evening dose), 12.5, 13, 13.5, 14, 15, 16, 18,
21, 24, 36, 48, 60, and 72 hours.
Group 5: blood before the dose on day 1
and after 0.5, 1, 1.5, 2, 3, 4, 6, 9, 12, 24, 36,
48, 60, and 72 hours.
Determination of sample size for
tolerability assessment: the probability
that a given AE would not be observed in
a group of 8 or 9 subjects was 0.004 and
0.002, respectively, for an AE with a true
population incidence rate of 0.5.

(Continued)
4 Clinical Pharmacology in Drug Development 2018, 00(0)
Table 1. Continued
Study Participants Study Design
3 Healthy men and women aged Single-center study conducted at Orlando
 65 years Clinical Research Center, Orlando, Florida, and at 0.5, 1, 1.5, 2, 3, 4, 6, 9, and 12 hours
n = 24 between May 2014 and July 2014 (IRB:
Aspire IRB, Santee, California).
Randomized, double-blind,
r Group 1: alicapistat 200 mg (n =
placebo-controlled, multiple-dose study:
8) or placebo (n = 4) twice daily
for 14 days
r Group 2: alicapistat 550 mg (n =
8) or placebo (n = 4) twice daily
for 14 days
4 Men and women aged Single-center study conducted by Compass Blood before the morning dose on day 1
55-90 years meeting Research, LLC, Orlando, Florida, between
NINCDS/ADRDA criteria September 2014 and March 2016 (IRBs,
for probable AD and on separate for each investigator: Schulman
stable dose of AChE-I Associates IRB, Cincinnati, Ohio; MidLands
n = 17 Independent Review Board, Overland
Park, Kansas; New England IRB, Newtown,
Massachusetts; IntegReview IRB, Austin,
Texas).
Randomized, double-blind,
r Group 1: alicapistat 200 mg (n =
placebo-controlled, multiple-dose study:
9) or placebo (n = 3) twice daily
for 7 days
r Group 2: alicapistat 550 mg (n =
3) or placebo (n = 2) twice daily
for 7 days
Pharmacokinetics Blood and CSF Sampling;
Sample Size Determination
Determination of sample size for the
comparison of single-dose AUC in the
elderly with that in the nonelderly in
study 1: it was anticipated that there
would be data for 8 elderly subjects in this
study and 6 nonelderly subjects at the
same dose in study 1. For these sample
sizes, the calculated power for a test at a
significance level of .05 on the difference
between the elderly and nonelderly with
respect to AUC is 0.895 if the ratio of
AUC central values is 2.00. The
corresponding power for Cmax is 0.74.
Blood before the morning dose on day 1
(before the evening dose), before the
morning dose on days 3, 5, and 6, before
the morning dose on day 7 and after 0.5,
1, 1.5, 2, 3, 4, 6, 9, 12 (before the evening
dose), 12.5, 13, 13.5, 14, 15, 16, 18, 21, and
24 hours, before the morning dose on
days 10, 12, and 13, and before the
morning dose on day 14 and after 0.5, 1,
1.5, 2, 3, 4, 6, 9, 12 (before the evening
dose), 12.5, 13, 13.5, 14, 15, 16, 18, 21, 24,
36, 48, 60, and 72 hours.
CSF: approximately 12 hours after the last
dose of alicapistat on day 14.
Determination of sample size for
tolerability assessment: the probability
that a given AE would not be observed in
a group of 8 subjects was 0.004 for an AE
with a true population incidence rate of
0.5.
and at 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 9, and 12
hours (before the evening dose), before
the morning dose on days 5 and 6, before
the morning dose on day 7 and at 0.5,
0.75, 1, 1.5, 2, 3, 4, 6, 9, 12 (before the
evening dose), 12.5, 12.75, 13, 13.5, 14, 15,
16, 18, 21, 24, 36, 48, 60, and 72 hours.
Determination of sample size for
tolerability assessment: the probability
that a given AE would not be observed in
a group of 7 patients was 0.008 for an AE
with a true population incidence rate of
0.5.
(Continued)
Lon et al 5

Table 1. Continued

Pharmacokinetics Blood and CSF Sampling;

Study Participants Study Design Sample Size Determination

5 Healthy men aged 25-45 years Single-center study conducted by Clinilabs,
with regular sleep habits Inc., New York, New York, between May
n = 17 2015 and September 2015 (IRB: New
England IRB, Newtown, Massachusetts).
Randomized, single-blind, placebo- and
active-controlled, 4-period crossover
study with 4 groups (n = 4 each, but 1
r Regimen A: alicapistat 400 mg
subject was replaced by another):
twice daily
r Regimen B: alicapistat 800 mg
twice daily
r Regimen C: placebo in the
morning and 10 mg donepezil in
the evening
r Regimen D: placebo twice daily
Blood approximately 1.75 hours after the
evening dose and at the end of
polysomnography.
Determination of sample size: it was
estimated that 16 subjects would allow
detection of a difference of 3%, 4%, and
5% versus placebo (power of 0.816, 0.968,
and 0.997, respectively, assuming an SD of
2.9%) in REM as a percentage of total
sleep time and a difference of 17, 23, and
31 minutes (power of 0.630, 0.873, and
0.987, respectively, assuming an SD of 20.5
minutes) in latency to REM sleep.

AChE-I, acetylcholinesterase inhibitor; AD, Alzheimer disease; AE, adverse event; AUC, area under the plasma concentration-versus-time curve; CSF,
cerebrospinal fluid; Cmax , maximum observed plasma concentration; ECG, electrocardiogram; IRB, institutional review board; NINCDS/ADRDA, Na-
tional Institute of Neurological and Communicative Disorders and Stroke/Alzheimer’s Disease and Related Disorders Association; REM, rapid eye
movement.

included an open-label substudy to test a single dose of
alicapistat 400 mg in subjects  65 years (the antici-
pated age range for most patients with AD), whereas
study 3 included only subjects  65 years. Study 4
was a multicenter, randomized, double-blind, placebo-
controlled, multiple-dose trial conducted in patients
with mild to moderate AD who were treated with stable
doses of AChE-I’s and assessed the pharmacokinetics,
safety, and tolerability of alicapistat. Study 5 was a ran-
domized, single-blind, placebo- and active-controlled,
4-period crossover trial that assessed sleep parameters
in healthy men to test the ability of alicapistat to pene-
trate and influence the CNS, compared with a positive
control that would improve sleep (donepezil).
The study protocols were approved by institutional
review boards (IRBs), and informed consent was ob-
tained from all participants; the specific IRBs are listed
in Table 1 for each study. Study 4 in patients with AD
was registered at ClinicalTrials.gov (NCT02220738).
Designs and the primary objectives of the phase 1 tri-
als are shown in Table 1. An additional phase 1 study
(study 6; NCT02573740) was designed to assess the
safety and tolerability of multiple doses of alicapistat
in patients with mild AD and patients with mild cogni-
tive impairment due to AD (Supplemental Appendix).
Subjects
For studies 1, 2, 3, and 5, healthy subjects were those
judged to be in general good health based on med-
ical history, physical examination, vital signs, labo-
ratory profile, and 12-lead electrocardiogram (ECG).
For study 4, patients had to meet the National In-
stitute of Neurological and Communicative Disorders
and Stroke/Alzheimer’s Disease and Related Disorders
Association criteria for probable AD.
For all studies, major exclusion criteria included
the use of any known inhibitors or inducers of
drug-metabolizing enzymes (eg, CYPs and uridine 5 -
diphospho-glucuronosyltransferase) within 30 days be-
fore the first dose of study drug and through the end
of the study. In older participants (group 5 of study 2
[all subjects were 65 years], study 3 [all subjects were
65 years], and study 4 [all patients were 55-90 years]),
psychoactive medications were not allowed within the
14-day period before receiving alicapistat; in study 5,
any sleep or CNS-active medications were not allowed
within 30 days before study drug administration.
Pharmacokinetics
Serial blood samples were taken at protocol-dependent
times following administration of alicapistat for all
studies except study 5 (Table 1), in which only 2
blood samples were collected, 1.75 hours following
the evening dose and at the end of polysomnography.
Alicapistat diastereomers were measured at AbbVie
(North Chicago, Illinois) using a validated stereoselec-
tive liquid chromatography/mass spectrometry method.
Alicapistat diastereomers were extracted from plasma
samples, previously treated with 2% potassium phos-
phoric acid, using 50%/50% v/v hexane/ethyl acetate
6 Clinical Pharmacology in Drug Development 2018, 00(0)
by a semiautomated 96-well plate method. Deuter-
ated internal standards for each diastereomer were
included. The samples were injected onto a C18 col-
umn (Acquity UPLC BEH, 1.7-μm bead size, 2.1
mm diameter × 50 mm length; Waters, Milford, Mas-
sachusetts) and eluted at 25°C and 0.2 mL/min with
a mobile phase consisting of 55%/45%/0.1% v/v/v
methanol/water/ammonium hydroxide. Eluted compo-
nents were quantified with an API 5500 mass spec-
trometer (Sciex, Framingham, Massachusetts) using
positive ion electrospray ionization in the multiple-
reaction monitoring mode and detecting the mass/
charge 434 to 174 transition for the unlabeled di-
astereomers and the 439 to 174 transition for the
deuterated internal standards. The chromatography/
mass spectrometry system was controlled by Analyst
software, version 1.5 or 1.6 (Applied Biosystems, Fos-
ter City, California). The validated range for the assay
was 0.0469 to 46.9 ng/mL for the alicapistat R,S di-
astereomer (r2  0.998) and 0.0551 to 55.1 ng/mL for
the alicapistat R,R diastereomer (r2  0.997). The in-
trarun and interrun coefficients of variation (CVs) were
3.9% and 4.0% for alicapistat R,S and R,R diastere-
omers, respectively; the mean intrarun bias ranged be-
tween -4.1% and 5.1%, whereas the mean interrun bias
was -0.6% to 2.6%. Across studies in this article, the fi-
nal lower limit of quantitation (LLOQ) for a 0.1-mL
plasma sample was 0.0442 to 0.0477 ng/mL for the ali-
capistat R,S diastereomer and 0.0519 to 0.0561 ng/mL
for the alicapistat R,R diastereomer.
In study 3, a CSF sample was also collected approx-
imately 12 hours after the last dose of alicapistat 200 or
550 mg twice daily on day 14. The CSF samples were
analyzed for the 2 diastereomers by a method similar
to that for the plasma samples. The validated range for
the assay was 0.0543 to 44.2 ng/mL for the alicapistat
R,S diastereomer (r2  0.995) and 0.0637 to 51.8 ng/mL
for the alicapistat R,R diastereomer (r2  0.999). The
intrarun and interrun CVs were 3.2% and 3.4% for
alicapistat R,S and R,R diastereomers, respectively; the
mean intrarun bias ranged between -2.3% and 12.1%,
whereas the mean interrun bias was -1.4% to 6.0%.
The LLOQ in study 3 for a 0.1-mL CSF sample was
0.0547 ng/mL for the alicapistat R,S diastereomer and
0.0642 ng/mL for the alicapistat R,R diastereomer.
Pharmacodynamics
Polysomnography was used in study 5 to assess the ef-
fects of alicapistat on various sleep parameters, partic-
ularly those characterizing REM, including number of
REM episodes, latency to REM sleep, REM density,
duration of REM, and REM as a proportion of total
sleep time, as described previously,14–17 using donepezil
as a positive control. Briefly, 3 nightly polysomnogra-
phy assessments were done in each of the 4 study peri-
ods: 1 session each on day -2 (for screening), day -1, and
day 1. The polysomnography assessments, taken via a
multichannel montage, included electroencephalogram
(EEG) activity, electrooculographic activity, and sub-
mental electromyographic activity. Subjects received 1
dose of study drug in the morning (approximately 30
minutes after starting breakfast) and 1 dose in the
evening (approximately 2 hours before the “lights-off”
period). Each of the 4 study periods was separated
from the others by at least 10 days. Subjects slept in
the sleep laboratory for 3 nights during their first study
period and for 2 nights in subsequent periods. Treat-
ment assignment was concealed from the subjects, and
they were blindfolded during administration of study
drug. Study staff who evaluated the polysomnographic
assessments also were blinded to treatment assignment.
Safety
Safety was evaluated throughout the studies based on
assessments of adverse events (AEs), vital signs, physi-
cal examination, brief neurological examination, ECG
measurements, laboratory tests, and Columbia-Suicide
Severity Rating Scale (C-SSRS).
Statistical Methods
Computation for the statistical tests was performed
with SAS software (Cary, North Carolina), version 8.2
or 9.2, on a Dell workstation using the Unix operat-
ing system. For the linear mixed-effects model anal-
ysis, SAS procedure PROC MIXED was used with
the Kenward-Roger option specified. SAS procedures
PROC UNIVARIATE and PROC MEANS were used
to obtain summary statistics. Determinations of sample
size from the perspective of the tolerability assessments
are reported in Table 1.
Pharmacokinetic Analyses
Pharmacokinetic parameters, including the maximum
observed plasma concentration (Cmax ), time to Cmax
(tmax ), terminal-phase elimination half-life (t½ ), and
area under the plasma concentration-versus-time curve
(AUC), were determined using noncompartmental
methods. Summary statistics of pharmacokinetic pa-
rameters were calculated by regimen for each study.
Study 1 (Healthy Subjects 18-55 years)
To address the issue of linear kinetics and dose pro-
portionality, an analysis of covariance (ANCOVA) was
performed for each pharmacokinetic parameter (tmax ,
dose-normalized Cmax , and dose-normalized AUC) in
turn. Logarithmic transformation was employed for
Cmax and AUC. For the investigation of the effect of
food, an analysis was performed on data from periods
1 and 2 (in one period, alicapistat was administered
Lon et al
under fasting conditions; in another period, alicapis-
tat was administered with a high-fat/high-calorie break-
fast). Analysis of variance was performed for tmax and
the natural logarithms of Cmax and AUC from time 0
to infinity (AUC0- ) of alicapistat diastereomers. The
model included fixed effects for sequence, period, and
regimen. The subjects were viewed as a random sam-
ple. For the assessment of the effect of ketoconazole
on the pharmacokinetics of alicapistat, an analysis was
performed on the same variables analyzed for the as-
sessment of the effect of food. A repeated-measures
analysis was performed on the data of the fasting regi-
men in periods 1 and 2 (alicapistat administered alone)
and the data from period 3 (alicapistat with ketocona-
zole). The model had effects for regimen (alicapistat
alone or alicapistat with ketoconazole), the period in
which the fasting regimen was administered, and the in-
teraction of regimen and the period factor.
Study 2 (Healthy Subjects 18-55 and 65 years)
To address the issue of linear kinetics and dose
proportionality, ANCOVA was performed on dose-
normalized Cmax and AUC of the morning-dose inter-
val of study day 7 (groups 1-2) and day 14 (groups 3-4).
Subjects were classified by dose level. Body weight was
included as a covariate in the model. A necessary con-
dition for including an explanatory variable in the final
model was that the regression coefficient be significant
at a level of 0.10.
Study 3 (Healthy Subjects  65 years)
To address the issue of linear kinetics and dose pro-
portionality, analyses were performed on each pharma-
cokinetic parameter of the morning-dose interval of
days 7 and 14 in turn using a linear mixed-effects model.
Observations were classified by dose level and day, and
there was an effect in the model for the interaction of
dose level and day. For measures of drug exposure, body
weight was a covariate.
Study 4 (Patients With Alzheimer Disease)
Pharmacokinetic parameters in group 1 (alicapistat
200-mg twice-daily dose) were determined using non-
compartmental methods.
Pharmacodynamic Analyses
Study 5 (Healthy Men 25-45 years). For the sleep
parameters, a linear mixed-effects model was used to
perform an analysis of the data obtained from the
night of day 1. The model had fixed effects for period,
regimen, and cohort. The subjects were viewed as
a random sample. An appropriate structure for the
covariance matrix for the observations from a subject
was selected for each sleep parameter, and in particular
7
the possibility that the regimens had unequal variances
was considered. Within the framework of the model,
the hypothesis of no difference between each active
drug regimen and placebo was tested. Each of these
tests was performed at a significance level of .05.
Safety Analyses
For all studies, the number and percentage of sub-
jects with treatment-emergent AEs (TEAEs) were tab-
ulated by Medical Dictionary for Regulatory Ac-
tivities preferred term and system organ class with
breakdowns by dose level, regimen, or treatment,
as useful. Also, potentially clinically significant val-
ues, according to predefined criteria, for labora-
tory parameters, blood pressure, pulse rate, and
weight were identified. ECG QT and QTc inter-
val values > 450 milliseconds and changes from
baseline > 30 milliseconds were noted.
Study 1 (Healthy Subjects 18-55 years). For the sched-
uled measurements of vital signs and ECG inter-
vals, repeated-measures analyses were performed. The
model had effects for baseline value, dose level (with
placebo considered a dose level), time of measure-
ment, and the interaction of dose level and time of
measurement.
Study 2 (Healthy Subjects 18-55 and 65 years). For
quantitative ECG variables, for each of study days 1,
7, and 14, a linear mixed-effects model analysis was
performed on the scheduled postdose measurements.
The mixed-effects model for these analyses had ef-
fects for baseline value, dose level (with placebo con-
sidered a dose level), time of measurement, and the
interaction of dose level with time of measurement.
One-way ANCOVA was performed on the clinical lab-
oratory variable data at the end of the regimen (study
day 7 for groups 1 and 2 and study day 14 for groups
3 and 4). The baseline value was the covariate. Subjects
were classified by regimen, with placebo considered a
regimen. For blood pressure and pulse rate and their
orthostatic changes, ANCOVA was performed on the
measurements of the last day of the regimen (prior to
dose and 4 hours postdose on study day 7 for groups 1
and 2 and on study day 14 for groups 3 and 4).
Study 3 (Healthy Subjects  65 years). For quanti-
tative ECG variables, blood pressure, and pulse rate,
a repeated-measures analysis was performed on the
scheduled postdose measurements on day 1. The
linear mixed-effects model for these analyses had
effects for baseline value, dose level (with placebo
considered a dose level), time of measurement, and the
interaction of dose level with time of measurement. A
second analysis for ECG variables, blood pressure, and
pulse rate was performed on the data from days 7 and
14 jointly. This analysis corresponded with that for day
1, but the observations from a subject were classified
8 Clinical Pharmacology in Drug Development 2018, 00(0)
Alicapistat R,S Diastereomer
1600
Concentraon, ng/mL
1400
1200
1000
800
600
400
200
0 0
0 8 16 24 32 40 48 56 64 72
Time, h
Figure 2. Alicapistat R,S and R,R diastereomer arithmetic mean plasma concentration-versus-time profiles after administration of
single oral doses of alicapistat to healthy subjects, in linear and log-linear (inset) scales. Concentration data of the first 6 groups
(50, 100, 200, 400, 700, and 1000 mg) are results from study 1. Concentration data of the 400-mg group in healthy elderly subjects are
results from study 2.
by day as well as by time relative to dosing. The model
had effects for baseline value, dose level, day, time
relative to dosing, and all the 2- and 3-way interactions
of dose level, day, and time relative to dosing. The
correlation among the repeated measurements from a
subject was accounted for by applying an appropriate
variance-covariance structure in the model. It was
intended that the structure allow for the possibility that
the correlation of a pair of measurements on the same
day would be higher than a pair of measurements from
different days. ANCOVA was performed on the clinical
laboratory variable data from day 14.
Results
Pharmacokinetics
Single Oral Doses of Alicapistat. After oral dosing in
healthy subjects, plasma concentrations of alicapistat
diastereomers reached peak levels within 2 to 4 hours
on average (Figure 2; Table 2). The t½ of alicapistat
diastereomers ranged from 7 to 10 hours. Exposures
(Cmax and AUC) of the alicapistat R,S diastereomer
were approximately 2-fold greater than those of the R,R
diastereomer for all alicapistat doses. The increase in al-
icapistat diastereomer Cmax values was less than dose
proportional (P < .05 for trend; especially notable in
the alicapistat 50- to 400-mg dose range), and the in-
crease in AUC0- was dose proportional (Figure 3A) in
the alicapistat 50- to 1000-mg dose range. Renal elimi-
nation of unchanged alicapistat was negligible (data not
shown).
Alicapistat diastereomer exposures were approxi-
mately 13% to 29% higher in healthy elderly subjects
 65 years than in young adult subjects following a sin-
gle 400-mg dose, but this difference was not statistically
significant (P  .1740, ANCOVA; Table 2).
Alicapistat R,R Diastereomer
1600
Concentraon, ng/mL
1400
1200
1000
800
600
400
200
0 8 16 24 32 40 48 56 64 72
Time, h
Overall, food did not significantly affect the alicapi-
stat exposure. Coadministration of alicapistat 150 mg
with the CYP3A inhibitor ketoconazole resulted in
an approximately 2.2-fold increase in alicapistat di-
astereomer Cmax values and a 3.7-fold increase in
AUC values relative to alicapistat administration alone
(Figure 4).
Multiple Twice-Daily Oral Doses of Alicapistat. In healthy
subjects  65 years, plasma exposure (Cmax and AUC
from time 0 to 12 hours [AUC0-12 ]) of alicapistat
R,S was approximately 1.9-fold greater than alicapis-
tat R,R for the 200-mg twice-daily dose and approxi-
mately 1.6-fold greater for the 550-mg twice-daily dose
(Figure 5; Table 3). In contrast, the concentration of
alicapistat diastereomers in CSF appeared to be simi-
lar; the mean alicapistat R,S and R,R concentrations
in CSF samples after 14 days of dosing were 3.76 and
4.26 ng/mL, respectively, for the 200-mg twice-daily
dose and 7.84 and 9.14 ng/mL, respectively, for the
550-mg twice-daily dose. Thus, the total targeted ali-
capistat CSF drug concentration as projected from pre-
clinical studies (4 ng/mL) was achieved or exceeded at
these doses in healthy subjects  65 years.
Comparison of alicapistat diastereomer values for
Cmax and AUC0-12 between subjects  65 years and
young subjects (Table 3) indicated that Cmax values were
21% to 33% lower and AUC0-12 values were 17% to 26%
higher in subjects  65 years than in young subjects at
the 200-mg twice-daily dose.
In patients 55 to 90 years with mild to moder-
ate AD, plasma exposures (Cmax and AUC0-12 ) of
alicapistat R,S were approximately 2-fold greater than
those of alicapistat R,R (Table 3), consistent with
results of the other presented studies. At the 200-
mg twice-daily dose, exposures (Cmax , AUC0-12 ) of
alicapistat R,S and R,R diastereomers in patients
Lon et al 9

Table 2. Pharmacokinetic Parameters of Alicapistat R,S and R,R Diastereomers in Plasma After a Single Oral Dose

Alicapistat Parameter, Mean ± SD

Healthy
Subjects
Healthy Subjects  65 years

Study 2
Study 1 (Group 5)

Dose Group 50 mg 100 mg 200 mg 400 mg 700 mg 1000 mg 400 mg

(Number of Subjects) (n = 5) (n = 6) (n = 6) (n = 6) (n = 6) (n = 6) (n = 7)

Alicapistat R,S diastereomer, mean ± SD

Cmax , ng/mL 210 ± 125 462 ± 221 633 ± 371 842 ± 275 1780 ± 937 2030 ± 679 1300 ± 519
tmax , h 1.8 ± 0.3 2.4 ± 1.0 2.6 ± 0.9 2.6 ± 1.7 3.6 ± 1.6 3.6 ± 2.0 2.6 ± 1.1
AUC0- , ng·h/mL 734 ± 411 1770 ± 538 3240 ± 1200 5530 ± 1700 10 100 ± 4000 15 200 ± 6020 8100 ± 4460
t½ , h
Harmonic mean ± 7.7 ± 2.0 9.5 ± 3.2 7.2 ± 1.4 7.8 ± 2.5 8.0 ± 1.6 7.4 ± 2.1 9.0 ± 2.9
pseudo SD
Mean ± SD 8.1 ± 2.0 10 ± 2.7 7.6 ± 2.2 8.3 ± 2.2 8.3 ± 1.9 7.9 ± 2.1 9.8 ± 3.2
Alicapistat R,R diastereomer, mean ± SD
Cmax , ng/mL 93 ± 49 206 ± 87 288 ± 145 452 ± 126 952 ± 484 1100 ± 295 636 ± 208
tmax , h 1.8 ± 0.3 2.4 ± 1.0 2.6 ± 0.9 2.7 ± 1.6 3.6 ± 1.6 3.8 ± 1.9 2.6 ± 1.1
AUC0– , ng·h/mL 385 ± 196 883 ± 235 1630 ± 473 3100 ± 732 5540 ± 1810 8370 ± 2200 4080 ± 1750
t½ , h
Harmonic mean ± 7.0 ± 1.4 9.1 ± 2.8 7.5 ± 1.5 8.3 ± 1.5 8.5 ± 1.8 7.6 ± 1.7 8.8 ± 2.2
pseudo SD
Mean ± SD 7.3 ± 1.7 9.6 ± 2.3 7.9 ± 2.2 8.6 ± 1.6 8.9 ± 2.0 8.0 ± 1.6 9.3 ± 2.4
AUC0– , area under the plasma concentration-versus-time curve from time 0 to infinity; Cmax , maximum observed plasma concentration; t½ , terminal-
phase elimination half-life; tmax , time to Cmax .

A B
(n g •h /m L /m g )
0 -1 2, ng•h/mL/mg
ng•h/mL/mg
g •h /m L /m g )

50 50

40 40
0 - , (n

UC 0–12
AU C0–∞

30 30 R,S
D o s e -No r m a liz e d AUC

AUC

R,R
R,S
R,S (Healthy Elderly)
o s e -No r m a liz e d A

20 R,R 20
Dose-Normalized

DDose-Normalized

R,R (Healthy Elderly)

R,S (Healthy Elderly)
R,R (Healthy Elderly) R,S (Mild AD)
R,R (Mild AD)
10 10

0 0
1 00

2 00

4 00

5 50

8 00
50
100
200

400

700

1000

Dose, mg
Dose (mg) Dose (mg)
Dose, mg
Figure 3. Mean ± SD alicapistat R,S and R,R diastereomer dose-normalized AUC0- after administration of single oral doses of
alicapistat (A) and mean ± SD alicapistat R,S and R,R diastereomer dose-normalized AUC0-12 after administration of multiple twice-
daily oral doses of alicapistat (B). AD, Alzheimer disease; AUC, area under the plasma concentration-versus-time curve; AUC0- , AUC
from time 0 to infinity; AUC0-12 , AUC from time 0 to 12 hours.

with mild to moderate AD were comparable with the In the sleep study (study 5), alicapistat R,S and R,R
exposures observed in healthy subjects  65 years diastereomer plasma concentrations following 400-
(Table 3). The increase in AUC0-12 was dose propor- and 800-mg twice-daily doses were comparable with
tional (Figure 3B) in the alicapistat 100- to 800-mg concentrations observed at corresponding doses from
twice-daily dose range. study 2.
10 Clinical Pharmacology in Drug Development 2018, 00(0)

1600 1600 R,R Fasting

R,S Fasting
R,S Non-fasting R,R Non-fasting

Alicapistat R,R Diastereomer

10000 10000
Alicapistat R,S Diastereomer
1400 1400
R,S Ketoconazole R,R Ketoconazole

Concentration, ng/mL
1000
Concentration, ng/mL
1000
1200 1200
100 100

1000 10
1000 10

800 1 800 1

0.1 0.1
600 600
0.01 0.01
400 0 8 16 24 32 40 48 56 64 72 80 88 96 400 0 8 16 24 32 40 48 56 64 72 80 88 96

200 200

0 0

0 8 16 24 32 40 48 56 64 72 80 88 96 0 8 16 24 32 40 48 56 64 72 80 88 96
Time, h Time, h

Figure 4. Arithmetic mean ± SD alicapistat R,S and R,R diastereomer plasma concentration-versus-time profiles after administration
of a single oral dose of alicapistat 150 mg to healthy subjects (n = 11) during part 2 of study 1, in linear and log-linear (inset) scales.
Subjects received alicapistat under fasting conditions (period 1), nonfasting conditions (period 2), and with 400 mg of ketoconazole
once daily (period 3).

Day 1 Day 7 Day 14

1800
Alicapistat R,S Diastereomer

1600
Concentraon, ng/mL

100 mg
1400 200 mg
1200 400 mg
800 mg
1000 200 mg (Healthy Elderly)
550 mg (Healthy Elderly)
800
200 mg (Mild AD)
600

400

200

0
2

4
0

12

8
31

31

32

33

33

34

34

35

36

36

37

37

38
14

15

15

16

16

Day 1 Day 7 Day 14

1000
Alicapistat R,R Diastereomer
Concentraon, ng/mL

800 100 mg
200 mg
400 mg
600 800 mg
200 mg (Healthy Elderly)
550 mg (Healthy Elderly)
400 200 mg (Mild AD)

200

0
2

4
0

12

8
31

31

32

33

33

34

34

35

36

36

37

37

38
14

15

15

16

16

Time, h
Figure 5. Alicapistat R,S and R,R diastereomer arithmetic mean plasma concentration-versus-time profiles after administration of
multiple twice-daily oral doses of alicapistat, in linear scale. Concentration data of the first 4 groups (100, 200, 400, and 800 mg)
are results from study 2. Concentration data of the 200- and 550-mg groups in healthy elderly subjects are results from study 3.
Concentration data of the 200-mg group in patients with mild to moderate AD are results from study 4. AD, Alzheimer disease.

Pharmacodynamics alicapistat in the morning and evening (Table 4). In sub-

Compared with placebo in study 5, there were no sig- jects receiving placebo in the morning and donepezil
nificant changes in any of the sleep-related EEG pa- 10 mg in the evening, expected changes in REM
rameters following administration of 400 or 800 mg sleep EEG parameters with donepezil were observed,
Lon et al 11

Table 3. Pharmacokinetic Parameters of Alicapistat R,S and R,R Diastereomers in Plasma on the Last Day of Twice-Daily Oral Dosing

Patients
With Mild to
Moderate
Healthy Subjects Healthy Subjects  65 years AD

Study 2 (Groups 1-4) Study 3 Study 4

100 mg 200 mg 400 mg 800 mg 200 mg 550 mg 200 mg

Dose Group Twice Daily Twice Daily Twice Daily Twice Daily Twice Daily Twice Daily Twice Daily
(Number of Subjects) (n = 9) (n = 9) (n = 9) (n = 9) (n = 7) (n = 7) (n = 8)
Last Day Day 7 Day 7 Day 14 Day 14 Day 14 Day 14 Day 7

Alicapistat R,S diastereomer, mean ± SD

Cmax , ng/mL 391 ± 134 1060 ± 725 1440 ± 796 2130 ± 841 711 ± 151 1880 ± 755 1120 ± 483
tmax , h 3.67 ± 1.58 2.83 ± 1.87 5.00 ± 3.32 3.44 ± 2.01 5.14 ± 1.07 3.64 ± 1.49 3.31 ± 1.83
AUC0-12 , ng·h/mL 1690 ± 344 3260 ± 1130 6510 ± 2280 10 600 ± 3160 3810 ± 602 8530 ± 2350 4400 ± 1390
t½ , h
Harmonic mean ± 9.38 ± 1.95 11.3 ± 1.97 9.60 ± 2.82 11.0 ± 4.32 9.48 ± 4.16 11.7 ± 2.94 Not
pseudo SD determined
Mean ± SD 9.78 ± 2.17 11.7 ± 2.42 10.7 ± 4.35 12.5 ± 4.69 11.2 ± 4.99 12.4 ± 3.36 Not
determined
Alicapistat R,R diastereomer, mean ± SD
Cmax , ng/mL 167 ± 55.8 458 ± 274 772 ± 335 1160 ± 472 360 ± 74.1 1210 ± 568 524 ± 197
tmax , h 3.67 ± 1.58 2.94 ± 1.91 5.11 ± 3.22 3.56 ± 1.94 5.43 ± 0.98 3.64 ± 1.49 3.25 ± 1.91
AUC0–12 , ng·h/mL 804 ± 125 1620 ± 543 3670 ± 1040 6110 ± 1940 2040 ± 420 5420 ± 1520 2300 ± 664
t½ , h
Harmonic mean ± 8.48 ± 1.54 9.37 ± 1.37 7.99 ± 2.16 9.63 ± 4.20 8.23 ± 2.80 10.8 ± 2.75 Not
pseudo SD determined
Mean ± SD 8.71 ± 1.46 9.58 ± 1.65 8.46 ± 2.10 11.2 ± 4.36 9.34 ± 3.93 11.4 ± 2.88 Not
determined
AD, Alzheimer disease; AUC0-12 , area under the plasma concentration-versus-time curve from time 0 to 12 hours; Cmax , maximum observed plasma
concentration; t½ , terminal-phase elimination half-life; tmax , time to Cmax .

Table 4. Study 5: Summary of Sleep-EEG Parameters at Baseline and After Single AM and PM Oral Doses of PBO, Donepezil, or
Alicapistat

Treatment AM / PM

PBO/PBO PBO/Donepezil 10 mg Alicapistat 400 mg/400 mg Alicapistat 800 mg/800 mg

(n = 17) (n = 16) (n = 17) (n = 14)
Parameter, mean
± SD Baseline Day 1 Baseline Day 1 Baseline Day 1 Baseline Day 1

REM episodes, n 4 ± 1 4 ± 1 4 ± 1 4 ± 1a 4 ± 1 4 ± 1 5 ± 2 4 ± 1
Latency to REM, 57.1 ± 38.2 64.0 ± 26.9 68.3 ± 19.8 52.2 ± 31.0 59.9 ± 21.2 69.6 ± 41.3 53.8 ± 25.9b 78.7 ± 24.6
min
REM density, 7.2 ± 4.5 7.6 ± 4.7 6.6 ± 4.1 10.7 ± 7.1a 8.5 ± 6.2 9.1 ± 5.9 6.4 ± 4.0b 8.4 ± 4.8
number/min
REM duration, min 82.1 ± 33.8 71.7 ± 23.2 68.3 ± 24.7 74.3 ± 24.0 79.7 ± 34.5 73.4 ± 29.7 78.0 ± 32.5 72.6 ± 21.9
REM as percent of 20.5 ± 6.5 18.0 ± 5.2 17.5 ± 5.3 21.7 ± 5.1a 19.6 ± 7.3 18.4 ± 6.5 18.9 ± 7.4 19.1 ± 5.3
total sleep
EEG, electroencephalogram; PBO, placebo; REM, rapid eye movement.
a P < .05 versus PBO/PBO.
b n = 13.
12
Table 5. Summary of TEAEs
TEAE, n/N (%)
Study Placebo
1 2/12 (16.7)
2 3/12 (25.0)
3 4/8 (50.0)
4 2/5 (40.0)
5 3/17 (17.6)a
TEAE, treatment-emergent adverse event.
a Subjects received placebo in the morning and donepezil 10 mg in the
evening or placebo at both times; no subjects (0/4) who received placebo
alone experienced an AE.
including increases in the number of REM episodes
during sleep, REM density, and REM time as a per-
centage of total sleep time.
Safety
In general, the incidence of TEAEs was similar in the
placebo and alicapistat treatment groups (Table 5). Ex-
cept for minor and inconsistent effects on blood pres-
sure, pulse rate, PR interval, and QTcF interval at
higher doses, there were no clinically significant changes
in other vital signs and laboratory measurements across
doses of alicapistat. All subjects were negative for sui-
cidal ideation or behavior as assessed by the C-SSRS.18
Study 1 (Healthy Subjects 18-55 years)
Most TEAEs were mild in severity. One subject who
received alicapistat reported 2 TEAEs (hypotension
and presyncope) that were moderate in severity as as-
sessed by the investigator. TEAEs reported by subjects
who received alicapistat were abdominal pain, diarrhea,
nausea, pharyngitis, dizziness, headache, paresthesia,
confused state, and dysmenorrhea. One subject who re-
ceived alicapistat reported 2 TEAEs (dizziness and con-
fused state) that were assessed by the investigator as
possibly related to the study drug. One subject who re-
ceived alicapistat discontinued from the study owing to
a TEAE of neutropenia that was assessed by the inves-
tigator as not related to alicapistat and mild in severity.
No deaths, serious AEs, or severe AEs occurred during
the study.
Study 2 (Healthy Subjects 18-55 and 65 years)
The majority of TEAEs were mild in severity and
considered unrelated to alicapistat. The most common
TEAEs were discomfort after lumbar puncture (11.1%),
constipation (5.6%), nausea (5.6%), and headache
(5.6%). No subjects discontinued the study prematurely
because of a TEAE.
There were no serious TEAEs or deaths.
Clinical Pharmacology in Drug Development 2018, 00(0)
Study 3 (Healthy Subjects 65 years)
One subject receiving alicapistat 550 mg had a seri-
ous and severe TEAE of acute myocardial infarction
All Alicapistat on treatment day 13 and was discontinued from the
study. The TEAE was not considered to be related to
6/35 (17.1) alicapistat.
11/43 (25.6)
8/16 (50.0)
4/12 (33.3)
Study 4 (Patients With Alzheimer Disease)
2/17 (11.8)
The proportions of patients who reported 1 TEAE
were similar in patients who received alicapistat 200 mg
twice daily (44%) and those who received placebo twice
daily (40%); no patients who received 550 mg alicapistat
twice daily reported a TEAE. There were no deaths or
serious TEAEs reported in treated patients. One patient
who was screened had a serious AE (severe syncope)
and was discontinued from the study before beginning
a study drug regimen.
Study 5 (Healthy Men 25-45 years)
There was no pattern to the nature or frequency of
TEAEs following administration of alicapistat, and all
events were mild in severity. There were no deaths or
serious TEAEs.
Discussion
Alicapistat is a potent and selective inhibitor of calpain
and has demonstrated efficacy in a broad range of
animal models relevant to AD. To our knowledge, this
is the first report of a comprehensive phase 1 program
for an orally active, selective, small-molecule calpain in-
hibitor. The pharmacokinetic and safety characteristics
of alicapistat were favorable for a clinical develop-
ment candidate. Exposure was dose proportional
and without a food effect or dramatic perturbations
related to age. There were no patterns that could have
indicated safety concerns. However, the pharmaco-
dynamic results were not supportive of continued
development.
The pharmacokinetic characteristics of alicapistat
were generally consistent across the 5 phase 1 stud-
ies discussed in this report. Cmax was reached in 2 to
5 hours, and t½ ranged from 7 to 12 hours, depend-
ing on dose and study. AUC was dose proportional in
the alicapistat 50- to 1000-mg dose range. Exposure
to the alicapistat R,S diastereomer in plasma was ap-
proximately 2-fold greater than exposure to the R,R
diastereomer in both healthy young subjects and sub-
jects 65 years, as well as in patients with mild to
moderate AD. The greater exposure to the R,S diastere-
omer was favorable, as it is a far more potent inhibitor
of calpain (inhibitor constant [Ki ], 56 nM) than the
R,R diastereomer (Ki , 1330 nM) in vitro. Overall, food
did not significantly affect the alicapistat pharmacoki-
netic profile, but coadministration of alicapistat with
Lon et al
ketoconazole resulted in an approximately 2.2-fold in-
crease in alicapistat diastereomer Cmax values and a
3.7-fold increase in AUC values relative to alicapistat
administration alone. Because alicapistat is a substrate
of CYP3A4, the increases in alicapistat exposure when
CYP3A4 was inhibited by ketoconazole were consistent
with expectations. Pharmacokinetics was comparable
in young and elderly subjects and patients with mild to
moderate AD.
No safety signals of concern were identified across
the 5 alicapistat phase 1 trials, and the incidence of
TEAEs was similar in placebo and alicapistat groups.
There were no deaths or serious TEAEs related to
alicapistat, and all subjects were negative for suicidal
ideation or behavior. Safety was similar regardless of
age.
The pharmacodynamic potential of alicapistat was
probed by measuring REM sleep parameters in healthy
men. To potentially exert an effect in patients with AD,
a therapeutic agent must be able to reach the CNS.
Studies have shown that donepezil reaches therapeutic
concentrations in CSF in humans19 and that it mod-
ulates brain function as reflected by changes in REM
sleep parameters in patients with AD, probably via its
cholinergic actions. In animal models, both donepezil
and alicapistat had robust effects (eg, ACh release and
EEG REM sleep), indicating effects within the CNS,
which will be described in detail in a separate publica-
tion. Therefore, we assessed the pharmacodynamic ef-
fects of alicapistat 400 or 800 mg twice daily on sleep
behavior, including REM, in healthy subjects as an indi-
cation of CNS penetration and activity (study 5). Com-
pared with placebo, alicapistat had no effect on REM
sleep parameters, whereas the active control, donepezil,
had the expected effects on REM sleep,20 suggesting
that the study was appropriately designed, powered,
and conducted. The lack of an effect on REM sleep
was unexpected because the CSF total alicapistat con-
centration at the doses used was predicted to be ap-
proximately 8 ng/mL (based on study 3). Although
the estimated levels of alicapistat stereoisomers in the
CSF (range, 3.76-9.14 ng/mL, corresponding to 8.66-
21.1 nM) did not reach the in vitro Ki values (R,S di-
astereomer, 56 nM; R,R diastereomer, 1330 nM), this
CSF concentration of alicapistat exceeded the concen-
tration of 4 ng/mL, suggested to be efficacious based
on animal studies. However, it is known that animal
models have limited predictive power for estimating the
achieved concentrations of drugs in human CSF.21 The
use of higher doses of alicapistat in the sleep study
was not considered because, although earlier preclin-
ical toxicology studies had suggested that they could
be tolerated (up to 250-500 mg/kg in mice; up to 800-
1000 mg/kg in dogs), there was a ceiling effect on drug
exposure with greater doses (ie, levels of alicapistat did
13
not increase further). Of note, alicapistat is a substrate
of P-glycoprotein, an efflux transporter, which might
have contributed to its limited CNS levels.22 Therefore,
the lack of a pharmacodynamic effect of alicapistat
in the sleep study suggests that inadequate concentra-
tions of alicapistat might have been achieved in the
CNS in that population, that higher concentrations are
needed in humans than in animals to observe pharma-
codynamic effects, or that animal studies are not at all
predictive of the effects of alicapistat in humans. This
failure to confirm a central pharmacodynamic effect
represents a major risk for further development of
alicapistat for AD.
Acknowledgments
Medical writing support was provided by Richard M. Ed-
wards, PhD, and Michael J. Theisen, PhD, of Complete Pub-
lication Solutions, LLC (North Wales, Pennsylvania), which
was funded by AbbVie. AbbVie and the authors thank the
participants in the clinical trials and all study investigators
for their contributions. The authors thank all colleagues in
AbbVie preclinical functions who provided preclinical data on
ABT-957 and its diastereomers.
Declaration of Conflicting Interests
All authors are employees of AbbVie and may own AbbVie
stock or options.
Funding
AbbVie Inc. funded these studies. AbbVie participated in the
study design, research, data collection, analysis and interpre-
tation of data, writing, reviewing, and approving the publica-
tion.
This clinical trial data can be requested by any qualified
researchers who engage in rigorous, independent scientific re-
search, and will be provided following review and approval of
a research proposal and Statistical Analysis Plan (SAP) and
execution of a Data Sharing Agreement (DSA). Data requests
can be submitted at any time and the data will be accessible
for 12 months, with possible extensions considered. For more
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References
1. Querfurth HW, LaFerla FM. Alzheimer’s disease. N Engl
J Med. 2010;362(4):329–344.
2. Hebert LE, Weuve J, Scherr PA, Evans DA. Alzheimer
disease in the United States (2010-2050) estimated using
the 2010 census. Neurology. 2013;80(19):1778–1783.
14
3. Getz GS. Calpain inhibition as a potential treatment of
Alzheimer’s disease. Am J Pathol. 2012;181(2):388–391.
4. Reitz C, Brayne C, Mayeux R. Epidemiology of
Alzheimer disease. Nat Rev Neurol. 2011;7(3):137–152.
5. Yiannopoulou KG, Papageorgiou SG. Current and fu-
ture treatments for Alzheimer’s disease. Ther Adv Neurol
Disord. 2013;6(1):19–33.
6. Birks J. Cholinesterase inhibitors for Alzheimer’s disease.
Cochrane Database Syst Rev. 2006(1):CD005593.
7. McShane R, Areosa Sastre A, Minakaran N. Me-
mantine for dementia. Cochrane Database Syst Rev.
2006(2):CD003154.
8. Cummings J, Aisen PS, DuBois B, et al. Drug develop-
ment in Alzheimer’s disease: the path to 2025. Alzheimers
Res Ther. 2016;8:39.
9. Goll DE, Thompson VF, Li H, Wei W, Cong J. The cal-
pain system. Physiol Rev. 2003;83(3):731–801.
10. Vosler PS, Brennan CS, Chen J. Calpain-mediated sig-
naling mechanisms in neuronal injury and neurodegen-
eration. Mol Neurobiol. 2008;38(1):78–100.
11. Ferreira A. Calpain dysregulation in Alzheimer’s disease.
ISRN Biochem. 2012;2012:728571.
12. Trinchese F, Fa M, Liu S, et al. Inhibition of cal-
pains improves memory and synaptic transmission in
a mouse model of Alzheimer disease. J Clin Invest.
2008;118(8):2796–2807.
13. Medeiros R, Kitazawa M, Chabrier MA, et al. Cal-
pain inhibitor A-705253 mitigates Alzheimer’s disease-
like pathology and cognitive decline in aged 3xTgAD
mice. Am J Pathol. 2012;181(2):616–625.
14. Hornung OP, Regen F, Dorn H, et al. The effects of
donepezil on postlearning sleep EEG of healthy older
adults. Pharmacopsychiatry. 2009;42(1):9–13.
15. Kanbayashi T, Sugiyama T, Aizawa R, et al. Effects
of donepezil (Aricept) on the rapid eye movement sleep
Clinical Pharmacology in Drug Development 2018, 00(0)
of normal subjects. Psychiatry Clin Neurosci. 2002;
56(3):307–308.
16. Riemann D, Gann H, Dressing H, Muller WE, Alden-
hoff JB. Influence of the cholinesterase inhibitor galan-
thamine hydrobromide on normal sleep. Psychiatry Res.
1994;51(3):253–267.
17. Schredl M, Weber B, Leins ML, Heuser I. Donepezil-
induced REM sleep augmentation enhances memory
performance in elderly, healthy persons. Exp Gerontol.
2001;36(2):353–361.
18. Posner K, Brown GK, Stanley B, et al. The Columbia-
Suicide Severity Rating Scale: initial validity and inter-
nal consistency findings from three multisite studies with
adolescents and adults. Am J Psychiatry. 2011;168(12):
1266–1277.
19. Litvan I, Phipps M, Pharr VL, et al. Randomized
placebo-controlled trial of donepezil in patients with
progressive supranuclear palsy. Neurology. 2001;57(3):
467–473.
20. Jackson S, Ham RJ, Wilkinson D. The safety and toler-
ability of donepezil in patients with Alzheimer’s disease.
Br J Clin Pharmacol. 2004;58(suppl 1):1–8.
21. Nau R, Sorgel F, Eiffert H. Penetration of drugs
through the blood-cerebrospinal fluid/blood-brain bar-
rier for treatment of central nervous system infections.
Clin Microbiol Rev. 2010;23(4):858–883.
22. Miller DS, Bauer B, Hartz AM. Modulation of P-
glycoprotein at the blood-brain barrier: opportunities
to improve central nervous system pharmacotherapy.
Pharmacol Rev. 2008;60(2):196–209.
Supporting Information
Additional supporting information may be found on-
line in the Supporting Information section at the end
of the article.