The use of physiology-based pharmacokinetic and pharmacodynamic modeling in the discovery of the dual orexin receptor antagonist ACT-541468

Article abstract of DOI:10.1124/jpet.117.241596

The identification of new sleep drugs poses particular challenges in drug discovery owing to disease-specific requirements such as rapid onset of action, sleep maintenance throughout major parts of the night, and absence of residual next-day effects. Robust tools to estimate drug levels in human brain are therefore key for a successful discovery program. Animal models constitute an appropriate choice for drugs without species differences in receptor pharmacology or pharmacokinetics. Translation to man becomes more challenging when interspecies differences are prominent. This report describes the discovery of the dual orexin receptor 1 and 2 (OX1 andOX2) antagonist ACT-541468 out of a class of structurally related compounds, by use of physiology-based pharmacokinetic and pharmacodynamic (PBPK-PD) modeling applied early in drug discovery. Although all drug candidates exhibited similar target receptor potencies and efficacy in a rat sleep model, they exhibited large interspecies differences in key factors determining their pharmacokinetic profile. Human PK models were built on the basis of in vitro metabolism and physicochemical data and were then used to predict the time course of OX2 receptor occupancy in brain. An active ACT-541468 dose of 25 mg was estimated on the basis of OX2 receptor occupancy thresholds of about 65% derived from clinical data for two other orexin antagonists, almorexant and suvorexant. Modeling predictions for ACT-541468 in man were largely confirmed in a single-Ascending dose trial in healthy subjects. PBPK-PD modeling applied early in drug discovery, therefore, has great potential to assist in the identification of drug molecules when specific pharmacokinetic and pharmacodynamic requirements need to be met.

Full text of DOI:10.1124/jpet.117.241596

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The use of physiology-based PK and PD modeling in the discovery of
the dual orexin receptor antagonist ACT-541468
Alexander Treiber, Ruben de Kanter, Catherine Roch, John Gatfield, Christoph Boss, Markus
von Raumer, Benno Schindelholz, Clemens Mühlan, Joop van Gerven, Francois Jenck
Departments of Preclinical Drug Metabolism and Pharmacokinetics (A.T., R.d.K.), Preclinical
Pharmacology (C.R, F.J.), Biology (J.G.), Chemistry (C.B.), Clinical Pharmacology (C.M.)
and Preclinical Development (B.S., M.v.R.), Idorsia Pharmaceuticals Ltd, Allschwil,
Switzerland. Center for Human Drug Research, Leiden, The Netherlands (J.v.G).
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Running title: Role of physiology-based modeling in ACT-541468 discovery
Corresponding author: Alexander Treiber
Idorsia Pharmaceuticals Ltd
Hegenheimermattweg 91
4
123 Allschwil
Switzerland
+
41 61 565 65 92 (phone)
41 61 565 89 03 (fax)
+
alexander.treiber@idorsia.com
Text pages: 46
Number of tables: 8
Number of figures: 10
Number of references: 67
Abstract: 250 words
Introduction: 817 words
Discussion: 1630 words
Recommended section assignment: Drug Discovery and Translational Medicine,
Neuropharmacology
Abbreviations: ADAM, advanced dissolution, absorption, and metabolism; ADME,
absorption, distribution, metabolism, and excretion; AMS, accelerator mass spectrometry;
ANOVA, analysis of variance; AUC, area under the plasma concentration vs. time curve;
AUC0-inf, area under the plasma concentration vs. time curve extrapolated to infinity; AUC0-
last, area under the plasma concentration vs. time curve up to the last measurable
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concentration; CHO, Chinese hamster ovary; CL, total plasma clearance; CL , intrinsic
int
clearance; Cmax, peak observed plasma concentration; CRC, concentration-response curve;
DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DORA, dual orexin receptor
antagonist; EEG, electroencephalographic; EMG, electromyographic; F, oral bioavailability;
FaSSIF, fasted state stimulated intestinal fluid; FCS, fetal calf serum; FeSSIF, fed state
stimulated intestinal fluid; fu,brain, fraction unbound in the brain; fu,gut, fraction unbound in the
gut; fu,mic, fraction unbound in liver microsomes; fu,OX2assay, fraction unbound in OX binding
2
assay; fu,plasma, fraction unbound in plasma; HATU, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium-hexafluorphosphate; HBSS, Hank´s buffered saline solution; HPLC,
high performance liquid chromatography; HPβCD, 2-hydroxypropyl-β-cyclodextrin; Kb,
inhibition constant as determined by a functional assay; K , tissue to plasma partition
p
coefficient; LC-MS/MS, liquid chromatography coupled to mass spectrometry; logD,
distribution coefficient; MDCK-II, Madin-Darby canine kidney (cell line); OAT, organic
anion transporter, OX , orexin-1 receptor; OX , orexin-2 receptor; OxA, orexin A; PBPK-PD,
1
2
physiology-based
pharmacokinetic
and
pharmacodynamic
(modeling);
PD,
pharmacodynamic; PK, pharmacokinetic; (non-)REM, (non-)rapid eye movement; RFU,
relative fluorescence units; ROt , receptor occupancy half-life; RT, room temperature; T ,
1/2
1/2
half-life; Tmax, time to reach peak plasma concentration; V , apparent volume of distribution
ss
at steady-state; σ , geometric standard deviation.
g
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ABSTRACT
The identification of new sleep drugs poses particular challenges in drug discovery due to
disease-specific requirements such as rapid onset of action, sleep maintenance throughout
major parts of the night, and absence of residual next-day effects. Robust tools to estimate
drug levels in human brain are therefore key for a successful discovery program. Animal
models constitute an appropriate choice for drugs without species differences in receptor
pharmacology or pharmacokinetics. Translation to man becomes more challenging in case
inter-species differences are prominent. This report describes the discovery of the dual OX1
and OX receptor antagonist ACT-541468 out of a class of structurally related compounds by
2
use of physiology-based pharmacokinetic and pharmacodynamic (PBPK-PD) modeling
applied already early in drug discovery. While all drug candidates exhibited similar target
receptor potencies and efficacy in a rat sleep model, they exhibited large inter-species
differences in key factors determining their pharmacokinetic profile. Human PK models were
built based on in vitro metabolism and physico-chemical data, and were then used to predict
the time course of OX2 receptor occupancy in brain. An active ACT-541468 dose of 25 mg
was estimated based on OX receptor occupancy thresholds of about 65% derived from
2
clinical data of two other orexin antagonists, almorexant and suvorexant. Modeling
predictions for ACT-541468 in man were largely confirmed in a single-ascending dose trial
in healthy subjects. PBPK-PD modeling applied early in drug discovery therefore has great
potential to assist in the identification of drug molecules when specific pharmacokinetic and
pharmacodynamic requirements need to be met.
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INTRODUCTION
Insomnia, characterized by difficulties with sleep onset and/or sleep maintenance, affects 10–
5% of the population chronically, with additional 15–20% suffering from it occasionally.
1
Insomnia has a wide range of effects on quality of life and is associated with increased
accident risk and chronic health problems (Zammit, 2007). Historically, insomnia treatments
have targeted GABA-A, histamine, serotonin or melatonin receptors. GABA-A receptor
modulators such as zolpidem, which currently dominate the market, tend to increase non-
rapid eye movement (non-REM) sleep and decrease REM sleep compared with normal sleep
architecture (Bettica et al., 2012b). This may be the reason for the observed decrease in
cognitive performance and locomotor skills when walking during nighttime awakening
(
Otmani et al., 2008; Frey et al., 2011), the latter being of particular concern for the elderly
Glass et al., 2005).
(
The orexin system was discovered in the late 1990s (de Lecea et al., 1998; Sakurai, 1999) and
is comprised of two peptides, orexin A (OxA) and orexin B, produced in a small number of
neurons in the lateral hypothalamus, and two G protein-coupled receptors, orexin-1 (OX1)
and orexin-2 (OX ), widely expressed throughout the brain. Orexin deficiency has been
2
linked to human narcolepsy/cataplexy, a neurological disorder in which patients suffer from
an uncontrolled sleep-wake cycle (Peyron et al., 2000; Thannickal et al., 2000). This indicates
that the orexin system plays an essential role in promoting alertness and maintaining
wakefulness under motivational circumstances. It supports behavioral and physiological
adaptation to relevant internal and external environmental stimuli such as physiological need
state, exposure to threats or reward opportunities (Sakurai, 2007; Carter et al., 2009; Tsujino
and Sakurai, 2009; Mahler et al., 2014).
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With the discovery of potent low molecular weight compounds, orexin receptor antagonism
has emerged as a novel concept for the treatment of insomnia disorders. Unlike classical
hypnotics targeting GABA-A, histamine or serotonin receptors, orexin receptor antagonists
promote sleep in a distinct manner as they maintain a natural sleep architecture and do not
impair cognitive function or locomotor skills (Hoever et al., 2012b; Ramirez et al., 2013;
Tannenbaum et al., 2016).
Both, dual orexin receptor antagonists (DORAs) including almorexant (Hoever et al., 2012b),
suvorexant (Herring et al., 2012), filorexant (Connor et al., 2016), lemborexant (Yoshida et
al., 2015) and SB-649868 (Bettica et al., 2012a), as well as the OX -selective antagonist
2
seltorexant have been tested in insomnia patients (Bonaventure et al., 2015a).
Polysomnography in animals and healthy human subjects suggest a qualitatively comparable
sleep-promoting profile for both DORAs and OX -selective compounds, although relative
2
impact on REM vs. non-REM sleep may differ (Dugovic et al., 2014; Jacobson et al., 2016).
Comparative studies with DORAs and OX -selective compounds indicate that higher OX
2
2
receptor occupancies are required by OX -selective compounds (Mieda et al., 2011; Gotter et
2
al., 2016), as recently shown with the DORA filorexant and the OX -selective MK-1064. As
2
OX -selective antagonists alone lack sleep-promoting efficacy (Gozzi et al., 2011), the
1
overall data suggest a complementary role of OX antagonism in the sleep-promoting
1
efficacy of DORAs. OX receptors also appear to play an essential role in anxiety states
1
(
Merlo Pich and Melotto, 2014), and OX -selective antagonists additionally attenuate
1
behavioral and cardiovascular responses to stress without altering baseline motor or
autonomic functions (Gozzi et al., 2011; Johnson et al., 2012; Bonaventure et al., 2015b).
In 2014, suvorexant was approved in the USA under the trade name Belsomra for the
treatment of insomnia. During regulatory review, the Food and Drug Administration
expressed concerns about potential next-day residual effects (Citrome, 2014; Vermeeren et
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al., 2015) associated with higher Belsomra doses as a consequence of its extended plasma
half-life. This episode highlights the importance of identifying compounds with an optimal
pharmacokinetic and pharmacodynamic (PK/PD) profile in man already during the discovery
process of an insomnia drug.
From a drug discovery perspective, the identification of candidate molecules for the treatment
of insomnia poses particular challenges. Beyond pharmacological target potency and brain
penetration as basic requirements, rapid onset and adequate sleep duration are key features.
For compounds with little inter-species differences in receptor pharmacology and
pharmacokinetics, appropriate drug candidates might be readily identified on the basis of
animal pharmacology data. Translational aspects become more important for molecules
exhibiting pronounced inter-species differences in drug disposition. For example, plasma
protein binding affects drug distribution into tissues and total blood clearance, and thus
influences the pharmacokinetic half-life of a drug. Physiology-based pharmacokinetic and
pharmacodynamic (PBPK-PD) modeling has been established as an appropriate tool to
translate animal PK and PD data into man early in drug development (Jones et al., 2009). The
present report describes the central and early role of PBPK-PD modeling in drug discovery as
a key tool in the selection process of ACT-541468, a DORA that is currently in phase 2
clinical development for the treatment of insomnia disorders. While this report focuses on
only three prototypical compounds, the approach was in fact applied to a set of about 20
structural analogs from the same chemical class.
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MATERIALS AND METHODS
Chemicals and reagents
S)-(2-(5-chloro-4-methyl-1H-benzo[d]imidazol-2-yl)-2-methylpyrrolidin-1-yl)(5-methoxy-
(
2
1
-(2H-1,2,3-triazol-2-yl)phenyl)methanone (ACT-541468), (S)-(2-(5-chloro-1,4-dimethyl-
H-benzo[d]imidazol-2-yl)-2-methylpyrrolidin-1-yl)(5-methyl-2-(2H-1,2,3-triazol-2-yl)
phenyl)methanone (ACT-605143), (S)-(2-(6-bromo-4-methyl-1H-benzo[d]imidazol-2-yl)-2-
methylpyrrolidin-1-yl)(5-methyl-2-(pyrimidin-2-yl)phenyl)methanone (ACT-658090),
almorexant, suvorexant (Figure 1), and their corresponding salts were synthesized by the
Medicinal Chemistry department of Actelion Pharmaceuticals Ltd (Allschwil, Switzerland) as
described in Supplemental Figure S1, or according to published methods (Mangion et al.,
2
012; Boss et al., 2013; Verzijl et al., 2013; Boss et al., 2015a; Boss et al., 2015b). Chemical
purity of all compounds was in excess of 97%.
Orexin receptor antagonist assays
OxA curve shift experiments were performed to determine the surmountability of antagonism
and to calculate the antagonistic potency (apparent K ) of the three benzimidazole
b
compounds. Chinese hamster ovary (CHO) cells expressing the human or rat OX or OX
1
2
receptors were grown in culture medium (Ham's F-12 with L-glutamine, Life Technologies)
containing 300 µg/mL (for human receptor-expressing cells) or 1000 µg/mL (for rat receptor-
expressing cells) G418, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% heat-
inactivated fetal calf serum (FCS). Prior to the experiment, the cells were seeded at
2
0,000 cells/well into 384-well black clear-bottom sterile plates (Greiner) and incubated
overnight at 37°C in 5% CO . On the day of the assay, culture medium in each well was
2
replaced with 50 µL of staining buffer (1× Hank´s buffered saline solution [HBSS], 1% FCS,
2
0 mM HEPES, 0.375 g/L NaHCO , 5 mM of the OAT inhibitor probenecid [Sigma], 3 µM
3
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of the fluorogenic calcium indicator fluo-4 AM [Life Technologies], and 10% pluronic® F-
1
27 [Life Technologies]). The cell plates were incubated for 1 h at 37°C in 5% CO followed
2
by equilibration at room temperature (RT) for ≥ 30 min. Fluo-4-stained cells were
supplemented with 10 µL of 1:10 serially diluted 6-fold concentrated antagonist solution in
the assay buffer (1× HBSS buffer, 0.1% bovine serum albumin, 20 mM HEPES, 0.375 g/L
NaHCO , pH 7.4) containing 0.6% DMSO. After 120 min, cells were stimulated with 10 µL
3
of 1:5 serially diluted 7× OxA solution in assay buffer containing ≤ 0.1% methanol, resulting
in final concentrations between 0.01 and 1000 nM. Calcium mobilization (proportional to OX
receptor activation by OxA) was measured using the fluorescence imaging plate reader
®
(
FLIPR ) assay (Tetra, Molecular Devices; excitation: 470–495 nm; emission: 515–575 nm).
To calculate the apparent K values, FLIPR traces were subjected to spatial uniformity
b
correction and normalized by trace alignment at the last time point before agonist addition
(
ScreenWorks software, Molecular Devices). The maximum fluorescence signals per well
were used to generate OxA concentration-response curves (CRCs) in GraphPad Prism
GraphPad Software). The EC values and Hill slopes (n) for the OxA CRCs, and the IC
50
(
5
0
values of the antagonists at approximately EC of OxA (1.6 nM for human OX and OX , 8
7
0
1
2
nM for rat OX and OX ) were calculated using the proprietary IC Witch software (curve-
1
2
50
intrinsic minima and maxima were used). The apparent K values were calculated via the
b
generalized Cheng-Prusoff equation, using the on-day OxA EC value (EC50OxA) and the
5
0
OxA concentration used for stimulation ([OxAstim]), with the following formula (Cheng and
Prusoff, 1973; Miller et al., 1999).
IC50
퐾푏 =
1
푛
푛
[
OxAstim
]
EC50OxA
�2 + �
ꢀ ꢀ − 1
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Approximate receptor occupancy half-lives (ROt ) of the antagonists were determined using
1
/2
calcium release assays after antagonist wash-out. To this end, fluo-4-stained CHO cells were
supplemented with 10 µL of 6× concentrated antagonists (serially diluted 1:10 in assay buffer
with 0.6% DMSO). After 120 min, cells were washed extensively with assay buffer and then
stimulated at several time points (0 to 63 min) with 10 µL of a 7× OxA solution, giving a
final OxA concentration of EC . Compound incubations and wash-out phase were performed
70
o
at 37 C. Calcium mobilization was measured, and IC values were calculated and converted
5
0
to the apparent K values via the generalized Cheng-Prusoff equation, using the OxA CRC
b
slope determined at every time point, as described above (software settings: curve-intrinsic
maximum; fixed minimum: full block by 10 µM suvorexant). The geometric means of Kb
values generated in three independent experiments were then plotted on a semi-logarithmic
scale against time. The ROt1/2 was calculated from the K values obtained at 0 and 33 min
b
after wash-out, assuming first-order dissociation kinetics, with the following formula:
3
3 푚푖ꢁ
퐾푏 33 ꢃꢄ푛
퐾푏 0 ꢃꢄ푛
ROt1/2
=
log ꢂ
ꢅ
2
Animal housing
Male, adult Wistar rats were used for pharmacokinetic (Crl:WI(Han) [Charles River,
Sulzfeld, Germany]) and pharmacology experiments (RccHan:WIST [Harlan, Horst, The
Netherlands] or Crl:WI(Han)). All rats were maintained under standard lab conditions
(temperature 20 ± 2°C, relative humidity 55–70%, standard Provimi Kliba diet 3336
[
Kaiseraugst, Switzerland, www.kliba-nafag.ch], and domestic mains tap water ad libitum)
on a regular 12-h light–dark cycle (lights on 06:00 AM). After arrival, rats were allowed at
least one week of acclimatization to Actelion’s animal facility and were carefully monitored
to ensure good health and suitability for inclusion in the study. Unless noted otherwise, rats
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were socially housed in groups of four in standard plastic rodent cages, and all tests were
conducted during the light phase (08:00 AM to 6:00 PM) under illumination of >600 lx.
Pharmacokinetic experiments in the Beagle dog were performed in the Shanghai animal
facilities of Actelion Pharmaceuticals Ltd in accordance with the Swiss animal protection
law. The dogs were group-housed during the study and only separated for feeding and
monitoring of clinical signs. With the exception of a period of fasting from the night before
dose administration until 4 h after dose administration, animals were given a daily allowance
of 350 g of a standard laboratory diet and had free access to tap water.
All animals were housed in accordance with the National Institute of Health guidelines and
experimental procedures were approved by the Basel-Landschaft Veterinary Office and
strictly adhered to Swiss federal regulations on animal experimentation. Well-being of
animals was monitored during the day by trained technical stuff. Animals were checked on
body weight loss, abnormal breathing, pilo-erection, grooming and locomotion as criteria of
distress according to the company's animal welfare policy and guideline on humane
endpoints.
Pharmacokinetic experiments in rat and dog
Male Wistar rats (n = 3) with a body weight of ca. 200–250 g were used for pharmacokinetic
experiments under license no. 169. For intravenous sampling, a jugular vein catheter was
implanted 2 days prior to drug dosing under aseptic conditions. After recovery from general
isoflurane anesthesia, animals were housed individually with free access to water and food
during the recovery period and the entire duration of the experiment. Compounds for
intravenous use were formulated as aqueous solution in 30% 2-hydroxypropyl-beta-
cyclodextrin (HPβCD) starting from a 5% stock solution in DMSO. For oral gavage dosing,
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compounds were formulated as either a suspension in 0.5% methyl cellulose or as solution in
PEG400.
Male Beagle dogs (n = 3) with body weights of 14.3–15.6 kg at the start of treatment were
used in a cross-over design with a wash-out period of 7 days under license number 2016-06-
QCB-32. All experiments were performed in fasted state, and gastric pH was controlled by
giving intramuscular pentagastrin at a dose of 6 µg/kg 20 min before and 30 min after oral
dosing. For intravenous dosing, compounds were formulated as aqueous solution in 30%
HPβCD starting from a 5% stock solution in DMSO. For the oral route, compounds were
either suspended in 0.5% methyl cellulose, dissolved in PEG400, or given as capsules
containing either mannitol or a 4:1 mixture of Cremophor RH40 and lauroglycol.
Serial blood samples of 0.25 mL (rats) or 2 mL (dogs) were taken over a period of 24 h and
transferred into vials fortified with EDTA as anticoagulant. Blood samples after oral dosing
to rats were taken under light isoflurane anesthesia. Plasma was generated by centrifugation
and stored at –20°C pending analysis.
Pharmacokinetic parameters were estimated using non-compartmental analysis within the
Phoenix software package (version 6.4, Pharsight Corporation, Cary, USA). AUC0-inf was
accepted if the percentage of area extrapolated to infinity (AUC%extp) did not exceed 20%,
otherwise AUC0-last was reported.
Analytical methods
Samples from in vitro and in vivo experiments were fortified with 3–6 volume equivalents of
methanol containing a close structural analog as internal standard, and proteins were removed
by centrifugation at 3220 g for 20 min at 4°C. If required, supernatants were diluted with 1%
aqueous formic acid or a 1:1 mixture of acetonitrile and 1% aqueous formic acid prior to
quantification by liquid chromatography coupled to mass spectrometry (LC-MS/MS) on
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API5000 or API5500 mass spectrometers (AB SCIEX, Concord, Ontario, Canada) in selected
reaction monitoring mode. Chromatographic separation was achieved on Phenomenex Luna
C8, C18, Gemini C18, Synergy Polar RP columns (4–5 µm, 2.0 × 20 mm ID) operated at
room temperature with a flow rate of 0.6 mL/min, and using a linear gradient starting from 5–
1
0% mobile phase B and a run time of 0.95 min. Mobile phases were 0.1% aqueous formic
acid or 5 mM ammonium formate (pH 9, phase A) and acetonitrile or methanol (phase B).
Physiologically-based pharmacokinetic modeling
Simcyp® Population-Based absorption, distribution, metabolism, and excretion (ADME)
Simulator (version 15; Sheffield, UK) was used for PBPK modeling and simulation PBPK
models were built based on physicochemical, binding, permeability and metabolic stability
data (Table 7). Oral absorption was modeled using the advanced dissolution, absorption, and
metabolism (ADAM) model within Simcyp. Compounds were modeled as a solid
formulation in immediate-release capsules for dog and human or as suspension for rat, using
the diffusion-layer model of Wang and Flanagan (Jamei et al., 2009) to calculate the rate of
dissolution from fine particles with an assumed diameter of 1 µm. The effect of bile salts
upon dissolution and solubility was considered, matching the measured solubility in fasted
state (FaSSIF) and fed state (FeSSIF) simulated intestinal fluids. Default values for particle
density of 1.2 g/mL, a supersaturation ratio of 10, a precipitation rate constant of 15 min, and
a diffusion layer thickness of 30 µm were assumed. A full PBPK, perfusion-limited
distribution model was applied using method 2 within Simcyp, with an assumed tissue-to-
plasma partition coefficient (K ) scalar of 0.5 (Rodgers and Rowland, 2007; de Kanter et al.,
p
2
016). Elimination was assumed to be driven by CYP3A4-mediated metabolism, based on
evidence for almorexant (Dingemanse et al., 2014), suvorexant (Cui et al., 2016), and ACT-
41468 (AT, unpublished observation) using the well-stirred liver and ADAM gut model
with fu,gut assumed to be 0.01. A factor of 2 was used to correct for the underprediction of in
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vitro to in vivo CL. Thus, the model was populated with measured CL values from rat, dog
int
or human liver microsomes, set to occur by hepatic and intestinal cyp3a (rat), cyp3a12 (dog)
or CYP3A4 (human), multiplied by 2, and corrected for non-specific assay binding (fu,mic
,
Table 7). OX occupancy was estimated using predicted unbound brain concentration vs.
2
time profiles, mean unbound K on the OX receptor (Table 1 and Table 7) and a sigmoid
b
2
Emax model with an Emax of 100% and a Hill slope of 1.
퐸ꢃ푎푥 [unbound brain concentration]
퐾퐵 + [unbound brain concentration]
OX occupancy =
2
Free distribution of unbound drug between plasma and brain was assumed, as supported by
the physico-chemical properties of all compounds and the absence of relevant P-gp and
BCRP-mediated efflux (AT, unpublished observation).
Ten virtual trials of 10 subjects each were simulated in a male, healthy volunteer population
with an age range of 18–45 years. Variation of all physiological parameters was based on the
default variation in the healthy volunteer population database used in Simcyp.
Brain partitioning in the rat
Brain and plasma concentrations were determined 3 h following oral administration at 30 and
1
00 mg/kg to rats (n = 3–4 per group) under license no KV-BL-426. All compounds were
formulated as solutions in PEG400. Blood was collected from the vena cava caudalis into
plastic tubes containing EDTA as anticoagulant and centrifuged to yield plasma. Brain was
collected after cardiac perfusion of 10 mL NaCl 0.9%. Brain tissue was then homogenized
with one volume equivalent of cold phosphate buffer (pH 7.4), and compounds were
extracted with methanol. Compound concentrations in plasma and brain were determined
using LC-MS/MS as described above.
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Telemetric sleep/wake cycle evaluation in rats
Sleep/wake cycles were evaluated based on electroencephalography/electromyography
(EEG/EMG) and home cage activity recorded in individually housed Wistar rats under free-
moving conditions under license no KV-BL-205. Rats (250-350 g, 6-8 weeks of age) were
equipped with telemetric transmitters (TL11M2-F20-EET; Data Science International, St
Paul, MN, USA) that allowed the noninvasive detection of EEG/EMG and activity via signal
transmission to a receiver. The surgical transmitter implantation was performed under aseptic
conditions. A pre-operative analgesia (buprenorphine 0.015 mg/kg s.c.) was administered 30
min before anesthesia. The entire surgical implantation was performed under general
anesthesia (isoflurane inhalation [1–5% vol.] initiated in an anesthetic chamber). The rat was
placed and secured in a stereotaxic apparatus. The body of the transmitter was placed
subcutaneously along the dorsal flank of the rat with the leads routed subcutaneously to an
incision accessing the cranium. For EEG recordings, two trepanations were placed in the
skull, 2 mm from either side of the midline and 2 mm anterior to the lambda suture for
placement of one differential pair of electrodes. Two other superficial trepanations were
drilled for screws as support for cementing the electrodes. The EMG leads were inserted in
either side of the muscles of the neck and sutured into place. After surgery, rats recovered
o
from anesthesia in a chamber equipped with a heating pad at 37 C. They were then housed
individually and received analgesia with 0.015 mg/kg s.c. of buprenorphine b.i.d. for the first
2
days of the 2-week post-surgery recovery period. Before the experiment, rats were
acclimatized for 3 days in their home cages placed in ventilated sound attenuating boxes, on a
regular 12-h light/dark cycle. Experiments used a 'crossover' design, i.e., each animal was
alternatively treated with a test compound and the vehicle, with at least 72 h between
administrations. Treatments were administered orally (gavage) at the transition between the
day and the night phase. Each telemetric recording covered a 24-h 'baseline' period preceding
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the treatment, the 12-h night period immediately following the administration of treatment,
and a 36-h recovery (wash-out) period. The recordings were split into a sequence of equal
intervals of user-defined length (10 s). Sleep and wake stages (active wake, quiet wake, non-
REM sleep, or REM sleep) were assigned to each interval automatically using the
Somnologica Science software (Medcare, Embla, USA) based on frequency estimation for
EEG, amplitude discrimination for the EMG, and the locomotor activity, as follows. Wake
was characterized by low-amplitude EEG activity with relatively greater power in the higher
frequency bands, such as alpha-band (10–13 Hz), accompanied by moderate to high levels of
EMG activity. Active and quiet wake were distinguished based on the locomotor activity and
the amplitude of the EMG. Non-REM sleep was defined by high-amplitude EEG activity
with greater power in the delta frequency band (0.5–5 Hz) and low EMG activity. REM sleep
was characterized by low-amplitude EEG activity focused in the theta frequency band (6–9
Hz) and no EMG activity. Data were analyzed by two-tailed paired Student’s t test or one-
way analysis of variance (ANOVA) followed by the post-hoc Tukey’s multiple comparisons
test, using GraphPad Prism (GraphPad Software). Differences were considered statistically
significant at p < 0.05.
PK and PD assessments of ACT-541468 in healthy subjects
The single-dose pharmacokinetic profile of ACT-541468 after daytime and nighttime
administration was assessed in the two single-center, double-blind, placebo-controlled,
randomized studies AC-078-101 (NCT02919319) and AC-078-102 (NCT02571855). Both
trials were conducted in full compliance with the principles of the Declaration of Helsinki,
and study protocols were approved by independent Ethics Committees. Detailed information
about study endpoints, inclusion and exclusion criteria are available at
www.clinicaltrials.gov. For daytime pharmacokinetic assessments, 8 healthy male subjects
received either placebo (n = 2) or ACT-541468 (n = 6) at doses of 5, 25, 50, 100, and 200
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mg, given in the morning after overnight fasting. Blood samples were collected over a period
of 96 h post-dose, plasma was separated by centrifugation, and ACT-541468 concentrations
1
4
therein determined by LC-MS/MS. CL and V were determined using an intravenous C-
ss
ACT-541468 microdose given on top of 100 mg oral ACT-541468 (n=4). Plasma levels of
radiolabeled ACT-541468 were quantified by accelerator mass spectrometry (AMS)
technique after chromatographic separation. Pharmacokinetic parameters after nighttime
administration were assessed in 6 healthy subjects who received either 25 mg ACT-541468
(n = 4) or placebo (n = 2). PD effects were assessed in study AC-078-101 at all doses after
daytime administration of ACT-541468 during the first 10 h after dosing using a battery of
objective tests including saccadic eye movements, adaptive tracking and body sway.
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RESULTS
Inhibition of orexin receptor signaling in cellular assays
Almorexant and suvorexant have shown efficacy in inducing sleep in animals and humans,
and, therefore, served as benchmark for the present benzimidazole series. Table 1 and
Figure 2 summarize the characterization of the three benzimidazoles ACT-541468, ACT-
6
05143 and ACT-658090 in calcium release assays in comparison with almorexant and
suvorexant. The IC values measured at approximately EC of Ox (1.6 nM) were used to
5
0
70
A
calculate the apparent K values. All five compounds behaved as potent dual antagonists, and
b
displayed an insurmountable profile at both receptors. K values ranged from 0.52 to 8.3 nM
b
at human OX receptors, and from 0.42 nM to 5.9 nM at human OX receptors. K values on
1
2
b
the corresponding rat and human receptors were comparable (Supplemental Table S1).
Using calcium release assays after antagonist wash-out, ROt on OX was estimated to be 6–
1
/2
1
9
min for suvorexant and all benzimidazoles, and 22 min for almorexant (Table 2). On OX2,
suvorexant, ACT-541468 and ACT-605143 showed ROt1/2 in the range of 4–6 min, while
ROt1/2 was 13–14 min for ACT-658090 and almorexant. In summary, the three
benzimidazoles displayed in vitro receptor interaction profiles similar to those of suvorexant
and almorexant. ACT-541468 was a selective OX and OX receptor antagonist in a panel
1
2
screen of more than 130 established central and peripheral pharmacological targets (AT,
unpublished observation).
Pharmacology in the rat
Brain penetration
Brain penetration of ACT-541468, ACT-658090 and ACT-605143 was determined 3 h after
administration of oral doses of 30 mg/kg and 100 mg/kg. At 30 mg/kg, mean total brain
concentrations of ACT-541468 and ACT-658090 reached 665 nM and 614 nM but was only
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1
54 nM for ACT-605143 (Table 3). Total brain levels increased in a more than dose-
proportional manner at 100 mg/kg, reaching 2247–12,000 nM. Correction for in vitro brain
binding (fu,brain; Table 7) yielded unbound brain concentrations around or above the K values
b
on rat OX (Supplemental Table S1), warranting further investigations in a rat sleep model.
2
Effect on sleep/wake cycles in rats
All three benzimidazole DORAs were tested in male Wistar rats implanted with telemetric
transmitters to allow for continuous EEG/EMG-based evaluation of sleep/wake cycles in
freely moving animals in their home cages. ACT-541468, ACT-605143 and ACT-658090
were dosed at 30 mg/kg at the beginning of the night-active phase, when endogenous orexin
levels are rising.
All three compounds showed robust effects on sleep/wake stages over the 6 h period post-
dose (Figure 3). ACT-541468 decreased the time spent in active wake by 22% compared to
vehicle-treated rats (−45 min; p = 0.0068, paired t test) and increased the time spent in non-
REM and REM sleep by 29% and 84% (+26 and +10 min; p = 0.0009 and p = 0.0228),
respectively. Latency to persistent non-REM sleep significantly decreased by 59% from 44 to
1
8 min (p = 0.0002), and to REM sleep by 58% from 71 to 30 min (p = 0.0006, Figure 4). A
similar effect was observed with ACT-605143, which decreased the time spent in active wake
by 21% compared to vehicle treated rats (−45 min; p = 0.0089), and increased the time spent
in non-REM and REM sleep by 34% and 146% (+24 and +16 min; p = 0.0092 and p <
0
.001), respectively. Latency to persistent non-REM sleep significantly decreased by 49%
from 35 to 18 min (p = 0.0033, Figure 4), and to REM sleep by 44% from 62 to 35 min
p = 0.007). Finally, ACT-658090 decreased the time spent in active wake by 25% (−46 min;
(
p < 0.001) and increased the time spent in quiet wake by 21% (+10 min; p = 0.0099). It
increased the time spent in both non-REM and REM sleep by 27% and 57% (+26 min,
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p = 0.0001; and +10 min, p = 0.0127), respectively. Latency to persistent non-REM sleep
significantly decreased by 50% from 42 to 21 min (p = 0.030), and to REM sleep by 54%
from 84 to 39 min (p = 0.032). Efficacy of the 3 benzimidazole DORAs over the 6 h period
following a 30 mg/kg dose was similar (Figure 4). The increase in total sleep time over the 6
h period was between 36 and 40 min for all compounds and differences between compounds
were not statistically significant (p = 0.9087, one-way ANOVA). Drug exposure, expressed
as AUC0-last, was also similar, within a 2.5-fold range from 2950–7510 nM∙h (Table 4).
The dose response of ACT-541468 in the rat sleep model was characterized at 10, 30 and
1
00 mg/kg (Figure 5). ACT-541468 dose-dependently impacted sleep/wake parameters.
Over the 6-h time period following administration, time spent in active wake decreased by 6
to 50 min, and time spent in non-REM and REM sleep increased by 2 to 29 min and 5 to 15
min, respectively. The lowest dose giving significant effects was 30 mg/kg. In the vehicle
group, latency to persistent non-REM sleep varied from 40 to 47 min, depending on the
group. Compared to vehicle, ACT-541468 significantly decreased the latency to persistent
non-REM sleep to 18 min at 30 (p = 0.0002) or 100 mg/kg (p = 0.0079), and the latency to
persistent REM sleep down to 30 min and 37 min at 30 mg/kg (p = 0.0006) and 100 mg/kg (p
=
0.014), respectively.
In the vehicle-treated animals, the proportion of non-REM and REM sleep relative to total
sleep time varied between 79–86% for non-REM sleep, and between 14–21% for REM sleep
(Table 5). The proportion of non-REM and REM sleep did not change in a statistically
significant manner for ACT-541468 at 30 mg/kg (p = 0.15) or 100 mg/kg (p = 0.59), or for
ACT-658090 at 30 mg/kg (p = 0.27). In contrast, the time spent in non-REM sleep
significantly decreased from 86% to 78% (p = 0.00074) for ACT-605143 at 30 mg/kg.
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Pharmacokinetics in rat and dog
Pharmacokinetics of all 3 benzimidazole compounds were characterized in male rats and
dogs in order to generate a database for validation of the respective PBPK models.
Pharmacokinetic results after oral and intravenous administration are summarized in Table 4
and Table 6, respectively. Plasma clearance (CL) of ACT-605143 and ACT-658090 in the rat
was 32–36 mL/min/kg, and 85 mL/min/kg for ACT-541468. After correction for
blood/plasma partitioning (Table 7), blood clearance of all three compounds was in a narrow
range of 36–45 mL/min kg, i.e., ca. 51–64% of liver blood flow in the rat. Plasma and blood
/
clearances in the dog were 3.9–9.5 mL/min/kg and 6.5–15 mL/min/kg, respectively, the latter
corresponding to 44–48% of liver blood flow for ACT-541468 and ACT-605143, but only
2
1% for ACT-658090. V in the rat was in large excess of total body water, indicating
ss
significant drug distribution into tissues: 3.2 and 3.9 L/kg for ACT-541468 and ACT-658090,
but only 0.8 L/kg for ACT-605143. V in the dog was in a more narrow range, between 1.1–
ss
2
.4 L/kg.
Oral absorption of all compounds was rapid in rat and dog, with peak plasma concentrations
Cmax) being reached within 0.25–2.3 h (Table 4). Oral exposures (AUC0-last) in the rat at
0 mg/kg varied by about 10-fold, from 412 nM⋅h for ACT-541468 to 4060 nM⋅h for
(
1
ACT-658090, which was also reflected in their different oral bioavailability (F) of 9 vs. 43%.
Smaller differences in AUC0-last were observed in the dog, with 2020 nM⋅h for ACT-541468
and 5610 nM⋅h for ACT-658090. Similar trends were evident for Cmax in both species
(Table 4).
PBPK model development for predicting human PK profiles
PBPK models for the benzimidazole DORAs ACT-541468, ACT-605143 and ACT-658090
were built based on in silico and biochemical data (Table 7), as outlined in Figure 6. PBPK
models were initially developed for rat and dog and validated against observed PK data in
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both species. All three PBPK models consistently overpredicted observed V . Therefore, a
ss
K scalar of 0.5 was applied to empirically correct the predicted tissue-to-plasma ratios in all
p
simulated tissues. In contrast, CL predictions underestimated observed values by
approximately 2-fold, and microsomal CLint was therefore multiplied by 2 in the PBPK
models. The simulated pharmacokinetic parameters of these refined PBPK models after
intravenous and oral dosing in rat and dog were considered similar enough to the observed
values to construct the corresponding human PBPK models (Table 4 and Table 6). The
simulated plasma concentration vs. time profiles and derived pharmacokinetic parameters of
the three benzimidazoles in healthy volunteers at a dose of 25 mg are shown in Figure 7A. At
this dose, the predicted fraction absorbed was 96–100% and not sensitive to the
supersaturation ratio (1–100) or precipitation constant (0.4–40). Among the three compounds,
ACT-541468 showed the fastest simulated oral absorption with a Tmax of 2 h, the shortest
initial half-life of 4.3 h, and the lowest Cmax and AUC0-24h of 814 nM and 6500 nM⋅h,
respectively (Table 8). ACT-658090 and ACT-605143 exhibited 5.9- to 7.3-fold higher
AUC0-24h, and 2.9–3.3 times higher Cmax, and reached peak plasma concentrations later than
ACT-541468, with Tmax of 3.6–3.9 h. Most important for a sleep agent, plasma levels of
ACT-658090 and ACT-605143 declined with relatively long half-lives of 10.4 and 26.8 h,
resulting in significant residual drug levels at the end of the 24 h dosing interval (Figure 7A).
PBPK models for suvorexant and almorexant were developed using physicochemical and
biochemical data (Supplemental Table S2) together with published clinical data for both
compounds (Hoch et al., 2012; Sun et al., 2013; Dingemanse et al., 2014; TGA, 2014). For
both, suvorexant and almorexant, oral absorption was modeled using ADAM and cellular
permeability determined in MDCKII cells. Tissue distribution of suvorexant and almorexant
was modeled as described above, while elimination was modeled based on published plasma
clearances of 5 L/h and 43 L/h, respectively (Hoch et al., 2012; TGA, 2014). Final model
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parameters after intravenous and oral dosing are presented in Table 6 (CL and V ) and
ss
Table 8 (AUC0-24h, Cmax, Tmax, initial T ). The observed and simulated mean plasma
1/2
concentration–time profiles of 40/50 mg suvorexant and 200 mg almorexant are shown in
Figure 8.
Human dose estimates
PK/PD analysis of almorexant and suvorexant in rats as basis for human PD threshold
concentrations
An attempt to estimate drug exposures needed to induce sleep in humans was done based on a
PK/PD analysis in rats. The sleep-wake pattern in rats was analyzed after treatment with
almorexant and suvorexant at doses of 10, 30 and 100 mg/kg given at the beginning of the
active period (lights off), together with exposure data from PK experiments in a satellite
group of rats. Both compounds were inactive at 10 mg/kg (Supplemental Figure S2), with
almorexant and suvorexant plasma exposures in the first hours reaching 97 nM and 443 nM,
respectively. At 30 mg/kg, total sleep time increased significantly with both compounds to
about 3–7 h for almorexant, and about 2 h for suvorexant (Supplemental Figure S2 and
Figure S3) at total and unbound plasma concentrations of ca. 195 nM and 1.2 nM for
almorexant, and ca. 890 nM and 3.5 nM for suvorexant, which were regarded as PD threshold
plasma concentrations in rat. Considering human plasma protein binding and the absence of
potency differences at target receptors (Table 1 and Supplemental Table S1), and assuming
the need for similar degrees of OX blockade in rats and humans, the predicted human PD
2
threshold plasma concentrations would be 195 nM for almorexant and 3550 nM for
suvorexant. These projected values were about 5- to 6-fold above the exposures observed in
man with sleep-inducing doses of almorexant (200 mg; 8-h residual exposure of 20 ng/mL)
and suvorexant (50 mg; 8-h residual exposure of 240 ng/mL; Figure 8). It is noteworthy, that
an exact determination of sleep duration in rats is technically difficult and is a methodological
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limitation of this approach. Moreover, the fragmented sleep pattern in rats, i.e. alternating
sleep and wake periods both in the active night and the inactive day period might render a
direct translation of PD threshold concentrations from rat to humans difficult. Due to these
uncertainties, human dose predictions were performed based on OX receptor occupancy
2
estimates.
Orexin 2 receptor occupancy estimates of almorexant and suvorexant in humans
Based on PBPK models of almorexant and suvorexant (Table 8 and Figure 8) and assuming
OX blockade as the driving force for sleep maintenance, PBPK-PD models, i.e. OX
2
2
receptor occupancy vs. time profiles, were constructed using a sigmoid Emax model, unbound
drug concentrations in brain, and the unbound K values for OX inhibition shown in
b
2
Table 1. OX receptor occupancies were then estimated at 8 h post-dose as the presumed end
2
of the sleep period (Table 8). According to these PBPK-PD models, 200 mg almorexant
resulted in a peak OX receptor occupancy of 81% at 2.5 h post-dose which declined to 63%
2
at 8 h post-dose. Similarly, a 50 mg dose of suvorexant yielded a maximal OX receptor
2
occupancy of 73% reached at 3.2 h after drug intake which slowly declined to 66% at 8 h
post-dose.
Human dose estimates for benzimidazole DORAs
The OX receptor occupancy vs. time profiles of the three benzimidazole DORAs in healthy
2
male subjects were derived starting from the respective PBPK models (Figure 7A). OX2
receptor occupancy vs. time profiles in brain (Figure 7B) were then predicted based on
unbound brain concentrations, assuming that unbound drug in plasma readily equilibrates
with brain and using the unbound K constants on OX in a sigmoid E model with a Hill
b
2
max
factor of 1. Using this approach, ACT-541468 doses were identified targeting an OX2
receptor blockade of about 65% over a period of 8 h. As shown in Table 8, an ACT-541468
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dose of 25 mg resulted in a predicted OX receptor occupancy of 58% at 8 h post-dose. OX
2
2
receptor occupancies estimated for ACT-605143 and ACT-658090 at 8 h post-dose at the
same 25 mg dose yielded values of 53% and 14%, respectively (Table 8).
ACT-541468 pharmacokinetics and pharmacodynamics in healthy subjects
The pharmacokinetic and pharmacodynamic profile of ACT-541468 was assessed in healthy
subjects (n=6 on active, n= 2 on placebo) after daytime administration at doses ranging from
5
-200 mg. Nighttime pharmacokinetic data were only generated for the 25 mg dose (n=4).
Only data on the 25 mg dose are reported here. Geometric mean pharmacokinetic parameters
median for Tmax) after daytime and nighttime are summarized in Table 8 and are graphically
(
depicted in Figure 9. After daytime administration, ACT-541468 was rapidly absorbed as
judged from the median Tmax of 1 h. Peak plasma concentrations and AUC0-24h were 1400 nM
and 5700 nM∙h, respectively. Oral absorption was slightly delayed to 1.5 h after nighttime
dosing and peak plasma concentrations were reduced to 1050 nM. AUC0-24h was 8270 nM∙h,
i.e. about 45% higher compared to daytime administration. The pharmacodynamic effects of
ACT-541468 were assessed during the first 10 h after dosing using a battery of objective tests
including saccadic eye movements, adaptive tracking and body sway. The effect of 25 mg
ACT-541468 on adaptive tracking as one of the most sensitive assessments of vigilance and
attention is depicted in Figure 10.
1
4
CL and V were determined from an intravenous C-ACT-541468 microdose given on top
ss
of 100 mg oral ACT-541468 (n=4), followed by quantification of ACT-541468 using
accelerator mass spectrometry (AMS) technique after chromatographic separation. Results
are summarized in Table 6 and were used for the validation of the human PBPK model.
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DISCUSSION
An ideal sleep drug should mediate rapid sleep onset and sleep maintenance through major
parts of the night and be devoid of next-day effects. While sleep onset may be optimized by
appropriate formulation strategies including particle size control or use of solubility
enhancers, sleep maintenance is dependent on intrinsic factors such as the time-course of
target receptor blockade in the brain. Tools allowing for robust estimates of drug levels in
human brain are therefore key for a successful sleep drug discovery program. Sleep models in
rodent and non-rodent animal species have been conventionally used for this purpose and
constitute an appropriate choice for compounds with little inter-species differences in
receptor pharmacology and pharmacokinetics. Translation of animal data to man becomes
challenging with prominent inter-species differences.
All three benzimidazole DORAs described here fulfill the basic requirements of a sleep drug.
They are potent and insurmountable antagonists of both orexin receptors showing comparable
K values on OX and OX (Table 1). In line with their physico-chemical properties and the
b
1
2
absence of relevant P-gp efflux, they readily cross the blood-brain barrier, as evidenced by
the similarity of unbound brain and plasma concentrations in rats (Table 3). A 30 mg/kg dose
increased non-REM, REM, and total sleep time in telemetrized rats, and decreased the
latency to the first episode of persistent non-REM and REM sleep compared to vehicle-
treated animals. The pharmacological profiles in the rat were similar to each other and
comparable to other DORAs described in literature (Aissaoui et al., 2008; Boss et al., 2008;
Cox et al., 2010; Sifferlen et al., 2010; Winrow et al., 2012; Sifferlen et al., 2013; Boss et al.,
2
014; Sifferlen et al., 2015; Heidmann et al., 2016). No clear differentiation between
compounds was therefore possible on the basis of drug efficacy in rats.
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In contrast, the benzimidazoles exhibited significant inter-species differences in key
parameters driving drug distribution and clearance (Table 7). Metabolic stability in liver
microsomes differed by 7.2- to 14-fold between rat and human, while plasma protein binding,
the major determinant for general tissue and specifically brain penetration, differed by 9- to
3
0-fold. Ratios of unbound brain and plasma concentrations in the rat were below unity and
increased with dose (Table 3). Non-linear plasma protein binding was demonstrated for
ACT-541468 at concentrations relevant for rat pharmacology assessments but not clinical
testing (Supplemental Table S3), which might, at least in part, contribute to the dose-
dependent brain/plasma ratios. Other factors, such as P-gp or bcrp-mediated efflux might also
play a role in rats but have been excluded for man. Pharmacokinetic behavior was therefore
expected to significantly differ between rat and man, calling for a tailored approach for the
identification of a candidate drug exhibiting, beyond rapid sleep onset and efficacy, a sleep
maintenance in man not longer than 8 h.
The pharmacokinetic differences predicted for these benzimidazoles are mostly a reflection
of differences in drug CL and tissue distribution (Table 6 and Figure 7A). ACT-541468 had
a 6–12 times higher simulated CL compared to ACT-605143 and ACT-658090 (Table 6),
resulting from its intrinsic clearance (CL ) in liver microsomes and the lowest binding to
int
plasma and liver microsomal proteins (Table 7). On the other hand, ACT-541468 exhibited a
larger simulated volume of distribution, i.e. 0.5 L/kg vs. 0.2 L/kg, in line with its lower
plasma protein binding and distribution coefficient (logD). The combination of higher
clearance and larger volume of distribution resulted in a simulated initial ACT-541468
plasma half-life of only 4.3 h, which favorably compared to the 10.4 h and 26.8 h of ACT-
6
05143 or ACT-658090, respectively, and also to suvorexant with a simulated half-life of 8.7
h. It is noteworthy, that these differences in pharmacokinetic profiles were not obvious from
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simple inspection of the individual modeling input data such as CL , plasma or microsomal
int
binding, pK and logD, which were all within a 2- to 3-fold range.
a
Published data on human pharmacodynamics and pharmacokinetics of 200 mg almorexant
(Hoever et al., 2012a) and 50 mg suvorexant (Sun et al., 2013) after nighttime administration
were used as a basis to estimate active doses of the benzimidazoles in man. Initial inspection
of the plasma concentration vs. time profiles of both compounds at the end of the presumed
8
2
-h sleep period indicated residual almorexant and suvorexant plasma concentrations of
0 ng/mL and 240 ng/mL, respectively (Figure 8). This 12-fold difference in total plasma
concentrations between both compounds appeared somewhat surprising in light of apparently
identical OX receptor potency in cellular assays (Table 1). Introducing corrections for
2
molecular weight, plasma protein binding, and non-specific binding in the orexin binding
assays (Table 7) yielded comparable unbound almorexant and suvorexant concentrations of
1
.5 and 2.3, when expressed as multiples of their respective unbound K values at OX .
b 2
PBPK-PD models constructed for both antagonists yielded consistent levels of OX receptor
2
occupancy of 63–66% at the end of an 8-h period (Table 8). A very similar threshold of 65–
8
0% was recently reported for other DORAs in rat and dog (Gotter et al., 2013). While
almorexant and suvorexant both exhibited the same degree of OX receptor blockade at 8 h
2
post-dose, only suvorexant was associated with impaired next-day performance (Citrome,
2
014; Vermeeren et al., 2015). In addition to the absolute values in OX blockade, their rate
2
of decline at the end of the sleep period was identified as a second key factor determining the
absence of next day effects. The 65% OX threshold was then used as guidance for predicting
2
active doses of the benzimidazoles (Figure 7B). The time course of OX receptor blockade
2
generally followed plasma concentrations for all three compounds. Major differences were,
however, observed in the magnitude of OX blockade. Despite the lowest simulated total
2
plasma exposure, ACT-541468 elicited the highest peak OX receptor blockade of 79%,
2
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while only 59% were reached with ACT-605143. Differences in plasma protein binding
compensated for the 2.9-fold higher total plasma concentrations of ACT-605143, resulting in
almost equal unbound peak concentrations of ACT-541468 in brain, i.e., 1.1 nM vs. 0.96 nM.
At 8 h post-dose, OX receptor occupancies were 58% for ACT-541468 and 53% for ACT-
2
6
05143, indicating that both compounds met the requirement to maintain sleep over an 8-h
period. Slightly lower OX receptor occupancies than those deduced from clinical data on
2
almorexant and suvorexant were targeted on purpose to compensate for the higher drug
concentrations expected after nighttime dosing (Hoever et al., 2012a; Sun et al., 2013), which
PBPK-PD modeling was technically unable to capture. Compared to ACT-605143, the
decline in OX receptor occupancy was faster with ACT-541468, in line with the higher CL,
2
lower V and resulting shorter half-life (Table 6). Based on PBPK-PD modeling, ACT-
ss
6
58090 was predicted to elicit a maximum OX receptor blockade of only 15%. This
2
somewhat surprising outcome in light of the excellent pharmacokinetic profile (Figure 7A) is
best explained by the lower OX receptor potency combined with very high plasma binding
2
(Table 7), resulting in insufficient unbound drug levels in brain to elicit relevant OX2
receptor blockade. Based on above analysis and its overall favorable profile in terms of
animal pharmacology, early safety and drug-drug interaction potential (AT, unpublished
observation), ACT-541468 was selected for clinical development.
The pharmacokinetic profile of ACT-541468 in healthy subjects was characterized after
daytime and nighttime administration in two Phase 1 studies, while its pharmacodynamics
were assessed during the first 10 h after daytime administration using a battery of objective
and subjective assessments. Observed PK parameters at a dose of 25 mg, (Table 8 and
Figure 9), indicated rapid oral absorption after daytime administration with peak plasma
concentrations of 1400 nM being reached within 1 h. Plasma AUC0-24h was well predicted
within a 14% range. The observed initial half-life was 3.0 h and thus slightly shorter than the
2
9

JPET Fast Forward. Published on June 29, 2017 as DOI: 10.1124/jpet.117.241596
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JPET #241596
simulated value of 4.3 h, well in accordance with the higher observed CL, i.e., 1.0 mL/min/kg
vs. predicted 1.2 mL/min/kg, and lower V , 0.4 L/kg vs. 0.5 L/kg (Table 6). The terminal
ss
half-life of ACT-541468 was about 6 h (Mühlan et al., 2017). After nighttime dosing of
ACT-541468, mean peak plasma concentrations only reached 1050 nM while AUC0-24h
increased by 27% to 8270 nM∙h. Oral absorption was slightly delayed, with a Tmax of 1.5 h,
and plasma half-life increased from 3.0 h to 5.1 h. The differences between ACT-541468
daytime and nighttime pharmacokinetics may result from diurnal differences in physiology
between night and day such as intestinal motility and perfusion, or liver blood flow. It is
important to note, that the observed differences in ACT-541468 PK were rather small, in
contrast to the significantly delayed oral absorption and reduced plasma levels after nighttime
administration of almorexant (Table 8 and Figure 8) (Hoever et al., 2012a). From a
methodological point of view it is noteworthy, that PK simulations were run with a total of
1
00 subjects, while the observed PK data are means of only six and four healthy subjects,
which might not properly represent a larger population. Pharmacodynamically, 25 mg ACT-
41468 revealed significantly reduced vigilance and attention (Figure 10), and had clear
5
effects on visuomotor coordination and postural stability. Onset of effects was observed
within 1 h, and returned to baseline within 3–6 h. These early assessments in healthy subjects
after daytime dosing suggest that a 25 mg dose has the potential to elicit the desired sleep
effects in insomnia patients.
In conclusion, we describe the discovery of the dual orexin receptor antagonist ACT-541468
based on early use of PBPK and PD modeling as part of the lead optimization process. ACT-
5
41468 was identified out of a larger set of structurally related derivatives based on its
simulated OX receptor occupancy, targeting for an effect duration of about 8 h at a 25 mg
2
dose, and a sufficiently short half-life to minimize next-day effects. Phase I clinical trials with
ACT-541468 in healthy subjects confirmed PBPK model-based predictions, as ACT-541468
3
0