Discovery of APD334: Design of a clinical stage functional antagonist of the sphingosine-1-phosphate-1 receptor

Article abstract of DOI:10.1021/ml500389m

APD334 was discovered as part of our internal effort to identify potent, centrally available, functional antagonists of the S1P₁ receptor for use as next generation therapeutics for treating multiple sclerosis (MS) and other autoimmune diseases

Full text of DOI:10.1021/ml500389m

Letter
pubs.acs.org/acsmedchemlett
Discovery of APD334: Design of a Clinical Stage Functional
Antagonist of the Sphingosine-1-phosphate‑1 Receptor
Daniel J. Buzard,* Sun Hee Kim, Luis Lopez, Andrew Kawasaki, Xiuwen Zhu, Jeanne Moody,
Lars Thoresen, Imelda Calderon, Brett Ullman, Sangdon Han, Juerg Lehmann, Tawfik Gharbaoui,
Dipanjan Sengupta, Lorene Calvano, Antonio Garrido Montalban, You-An Ma, Carleton Sage,
Yinghong Gao, Graeme Semple, Jeff Edwards, Jeremy Barden, Michael Morgan, Weichao Chen,
Khawja Usmani, Chuan Chen, Abu Sadeque, Ronald J. Christopher, Jayant Thatte, Lixia Fu,
Michelle Solomon, David Mills, Kevin Whelan, Hussien Al-Shamma, Joel Gatlin, Minh Le,
Ibragim Gaidarov, Todd Anthony, David J. Unett, Anthony Blackburn, Jaimie Rueter, Scott Stirn,
Dominic P. Behan, and Robert M. Jones
Arena Pharmaceuticals, Inc., 6154 Nancy Ridge Drive, San Diego, California 92121, United States
S
* Supporting Information
ABSTRACT: APD334 was discovered as part of our internal effort to identify potent,
centrally available, functional antagonists of the S1P1 receptor for use as next generation
therapeutics for treating multiple sclerosis (MS) and other autoimmune diseases.
APD334 is a potent functional antagonist of S1P1 and has a favorable PK/PD profile,
producing robust lymphocyte lowering at relatively low plasma concentrations in several
preclinical species. This new agent was efficacious in a mouse experimental autoimmune
encephalomyelitis (EAE) model of MS and a rat collagen induced arthritis (CIA) model
and was found to have appreciable central exposure.
KEYWORDS: Sphingosine-1-phosphate, S1P1, APD334, gilenya, FTY720, fingolimod
in humans is unclear. S1P5 is expressed on oligodendrocytes
phingosine-1-phosphate-1 (S1P1) is a Class A G-protein
Scoupled receptor (GPCR) expressed on lymphocytes, and its activation on human brain endothelial cells improves
neural cells, and the endothelium.1 This receptor belongs to a
barrier integrity and reduces transendothelial monocyte
migration.10 S1P4 is expressed on hematopoietic, lymphocytic,
family of five GPCRs (S1P1−5), which recognize the
and dendritic cells and the loss of this receptor has been shown
to reduce TH17 differentiation of TH cells.11 TH17 cells are
associated with MS pathogenesis, and populations of this cell
type in MS patients are typically increased.12 Reduced
interaction with S1P3 has been a consistent theme in the
literature, as this receptor was believed to be responsible for the
transient bradycardia observed in humans treated with
fingolimod. It is now understood that in humans at least, this
cardiovascular event more likely results from S1P1-dependent G
protein gated inwardly rectifying potassium (GIRK) channel
activation on human atrial myocytes.13,14 Regardless, S1P3-
sparing agonists may still be desirable, as nonselective S1P1
agonists are associated with pro-fibrotic responses via S1P2- and
S1P3-dependent signaling.15,16
sphingolipid sphingosine-1-phosphate (S1P) and collectively
perform a variety of functions, with S1P1 regulating vascular
development and lymphocyte trafficking.2 Gilenya (1, fingoli-
mod) (Figure 1) is an oral medication that targets the S1P
receptors and is indicated for the treatment of relapsing forms
of multiple sclerosis (MS).3 In vivo, fingolimod is phosphory-
lated by sphingosine kinase to the (S)-monophosphate (2),
which is an agonist of four of the five S1P receptors (S1P1,
S1P3, S1P4, and S1P5).4 Sustained activation (agonism) of
lymphocyte expressed S1P1 results in internalization and
proteasomal degradation of this receptor, thus resulting in
“functional antagonism” of S1P1.5 Reduced S1P1 expression
interrupts the normal trafficking of lymphocytes and retains
autoreactive lymphocytes in the lymph nodes and thymus,6
thus preventing their accumulation in the brain and spinal cord
of MS patients. Additionally, fingolimod readily passes through
the blood−brain barrier and is believed to reduce neuro-
inflammation through direct action on S1P1 expressed on
astrocytes.7,8 The unique biology associated with the S1P1
receptor has encouraged others to identify alternative agents for
treatment of autoimmune disease.9
Identification of new agents with reduced elimination half-life
has been sought after, as full lymphocyte recovery following
fingolimod treatment requires >5 weeks and could be a
complicating factor in the event of opportunistic infection.17 In
addition, teratogenicity has been observed in rodents, and the
Received: September 23, 2014
Accepted: November 3, 2014
Monophosphate 2 is also a potent agonist of S1P3, S1P4, and
S1P5, but the relative contribution of these subtypes to efficacy
© XXXX American Chemical Society
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Letter
Figure 1. Structures of gilenya, monophosphate 2, 5-phenyl-1,2,4-oxadiazole 3, and APD334.
long half-life in humans (8 days) requires contraception for two
months after stopping treatment.18 A further challenge is
identifying therapeutics with appreciable central exposure as
this is believed to be necessary for success in treating MS.19
Lastly, new agents with reduced hepatotoxicity in humans
would be highly beneficial. Herein we report the discovery of
APD334 (4, Figure 1), a centrally available, selective, functional
antagonist of the S1P1 receptor currently in development for
the treatment of autoimmune disease.
Our early efforts to identify a functional antagonist with a
competitive pharmacodynamic profile focused on optimization
of 5-phenyl-1,2,4-oxadiazoles (e.g., 3, Figure 1).20,21 Despite
discovery of several potent and selective analogues, we failed to
identify a compound with good activity in the mouse
lymphocyte lowering (LL) experiment at an acceptable plasma
concentration (e.g., 3 LL IC50 = 1.62 μM). Furthermore, 3 was
undetectable in rat brain and CSF after 24 h intravenous
infusion. To improve these parameters, we investigated a
related series of 7-benzyloxy analogues (e.g., 7−21). These
racemic mixtures were prepared from 5 utilizing the synthetic
sequence outlined in Scheme 1.
Table 1. Racemic 7-Benzyloxy SAR (human S1P1, HTRF
cAMP)
a
compd
R1
R2
R3
EC50, nM (%)
7
CN
CF3
CN
CN
CF3
CF3
CN
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
OCF3
CF3
16.0 (90%)
26.0 (98%)
1.7 (109%)
120 (136%)
141 (104%)
159 (117%)
0.087 (98%)
0.077 (96%)
0.073(104%)
0.45 (109%)
3.68 (118%)
0.49 (107%)
55.8 (112%)
0.44 (103%)
4.58 (111%)
8
9
OCF3
OCH3
CN
10
11
12
13
14
15
16
17
18
19
20
21
Cl
cyclohexyl
cyclohexyl
cyclopentyl
cyclobutyl
cyclopropyl
n-propyl
neopentyl
iso-butyl
methylcyclohexyl
a
EC50 values are the mean of three or more replicates. Value in
parentheses is the % agonism relative to S1P. S1P EC50 = 0.062 nM; 2
EC50 = 0.006 nM (101%).
a
Scheme 1
of these analogues were examined in an acute mouse
lymphocyte lowering experiment measuring lymphocyte
count and plasma concentration at 24 h following a single 1.0
mg/kg oral dose. This abbreviated pharmacokinetic/pharma-
codynamics (PK/PD) analysis suggested 15 may offer a
significant improvement in in vivo potency relative to 3 and
prompted characterization of each enantiomer independently.
An enantiomeric separation was performed, and both
isomers were tested in the S1P1 HTRF assay. Each isomer
was very active in vitro, with one offering a moderate potency
advantage (i.e., hS1P1 EC50 = 0.04 nM, Emax = 102% versus 0.09
nM, Emax = 103%). A single crystal of the more potent
enantiomer was grown from a saturated acetone/acetonitrile
solution, and the absolute stereochemistry was determined by
X-ray to be S. Correspondingly, an X-ray structure proof was
also obtained for the R-isomer (Figure 2, 4, APD334). The R-
isomer was ultimately selected as the development candidate in
a
Reagents and conditions: (a) 2-iodo-4-methoxyaniline, Si(OEt)4,
PPTS, Pd(OAc)2, DIEA, DMF; (b) BBr3, CH2Cl2, 31% (2 steps); (c)
DIAD, PPh3, ArCH2OH or ArCH2Cl, Cs2CO3, 41% (15, Table 1); (d)
LiOH, THF, 70% (15, Table 1).
Internalization of S1P1 is preceded by activation of the
receptor (agonism); thus, EC50 values were generated in a
homogeneous time-resolved fluorescence (HTRF) cyclase
assay to assess engagement of the receptor (Table 1).
Compound 7 was the first analogue prepared and possesses
the CN/OCF3 substitution pattern previously identified as a
potency driver in the 1,2,4-oxadiazole structure−activity
relationship (SAR) (e.g., 3, S1P1 EC50 = 1.1 nM). This aryl
motif, however, was less well tolerated in the benzyloxy series
(7, S1P1 EC50 = 16 nM). Instead, the analogous R1 and R2
disubstituted benzene was preferred (9, S1P1 EC50 = 1.7 nM).
Subsequent SAR focused on the R2 group and revealed a
preference for larger alkyl groups. In particular, cyclopentyl and
cyclohexyl were optimal in terms of potency (13−15). Several
Figure 2. Mercury 3.1 Development (Build RC5) ellipsoid rendition22
of X-ray crystal structure of (R)-2-(7-((4-cyclo-pentyl-3-
(trifluoromethyl)benzyl)oxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-
yl)acetic acid [4, APD334].
B
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Table 2. APD334 in Vitro S1P1 Preclinical Species EC50 Data and Human S1P1‑5 EC50 Values
preclinical species S1P1 EC50 cAMP, nM (Emax %)
human S1P1−5 EC50 β-arrestin, nM (Emax %)
human
mouse
rat
dog
monkey
S1P1
S1P2
S1P3
S1P4
S1P5
0.093 (103)
0.44 (95)
0.32 (99)
0.34 (87)
0.32 (95)
6.10 (110)
>10,000
>10,000
147 (63)
24.4 (73)
a
Table 3. APD334 Preclinical Species Pharmacokinetics and in Vivo Lymphocyte Lowering (LL) IC50 Values
Vss
(L/kg)
b
b
c
species
dose iv/po (mg/kg) Clsys (L/h/kg)
%LBF
Cmax (μg/mL) tmax (h) AUC0‑inf (μg·h/mL) t1/2 po (h)
%F
LL IC50 (μM)
mouse
rat
1/1
1/1
1/3
1/3
0.110
2.00
3.45
0.746
2.23
1.93
0.780
0.218
3.71
8.00
7.33
7.33
3.33
10.3
3.79
164.0
23.1
12.3
9.64
28.8
6.37
100
0.101
0.051
0.058
0.098
0.145
1.61
54.0
73.2
43.8
dog
0.0138
0.0568
0.609
0.411
monkey
2.87
a
b
Amorphous APD334 (vide infra) was dosed as a suspension in 0.5% methyl cellulose. Clsys and Vss were determined by intraveneous
c
administration. Values were generated from a full 24 h time course of drug effect on lymphocyte lowering (LL).
part due to superior stability in rat liver microsomes (t1/2 > 60
min versus t1/2 = 9 min for the S-isomer). Instability of the S-
isomer was anticipated to complicate testing in rat disease
models, PK experiments, and toxicity studies. Of note, both
enantiomers were observed to be stable in human liver
microsomes (t1/2 > 60 min). In addition to improved rat
microsomal stability, the R-isomer (APD334) forms a
crystalline, high melting, nonhygroscopic, anhydrous L-arginine
salt, which was regarded as a significant development advantage
given the difficulty in identifying other suitable salt forms for
either enantiomer. Interestingly, the crystalline free acid
prepared for the X-ray determination was observed to possess
similarly good physical properties; however, this crystal form
was not orally bioavailable in rats when dosed as an aqueous
suspension. This contrasts with the amorphous free acid form
of 4, which demonstrated good bioavailability when dosed as a
suspension (%F = 54). Importantly, the L-arginine salt achieved
good exposure levels in SD rat when dosed orally as a solution
in sterile water (Cmax = 0.361 μg/mL, 1.0 mg/kg equivalent).
S1P1 functional data for five species was obtained, as well as
EC50 values for the five human S1P subtypes (Table 2). S1P1
activity was maintained in all preclinical species examined, and
APD334 was devoid of any agonism or antagonism at human
S1P2 and S1P3. Furthermore, moderate agonism at human S1P4
and S1P5 was observed but is reduced relative to S1P1, both in
terms of potency and efficacy. The extent to which 4
internalizes human S1P1 was evaluated in CHO cells expressing
HA tagged S1P1, and 4 was found to have an IC50 value of 1.88
nM.
produce substantial lymphopenia in all species (Table 3).
Notably, the calculated LL IC50 values reflect total plasma
concentration wherein APD334 is highly protein bound (97.8%
human, 98.0% rat).
Constant infusion CNS distribution studies in male
Sprague−Dawley (SD) rats were performed and APD334 was
observed to have improved central exposure relative to its
predecessor 3. After a 48 h constant infusion (14.5 μg/h/kg),
APD334 reached a brain concentration of 0.21 μM, which is
approximately twice that of the plasma concentration (0.11
μM) and translates to a brain-to-plasma (B/P) ratio of 1.9.
Drug plasma concentration at the 48 h time point is 2-fold
higher than the estimated rat lymphocyte lowering IC50 value
(0.051 μM).
APD334 was evaluated in a murine experimental auto-
immune encephalomyelitis (EAE) model.24 This model mimics
MS by inducing a demyelinating autoimmune response by
utilizing myelin/oligodendrocyte glycoprotein (MOG35−55) as
autoantigen. Prophylactically, APD334 prevented the onset and
severity of disease relative to vehicle up to day 25, at which time
dosing was discontinued. All treatment arms went on to
develop severe disease as shown by their clinical scores (Figure
3). Therapeutic administration of APD334 was also examined
(Figure 4). Treatment began at day 18, by which time all
animals had developed severe disease, and APD334 was
administered out to day 37. APD334 reversed disease relative
to vehicle and was similar to the efficacy observed with
APD334 is stable in mouse, rat, dog, monkey, and human
liver microsomes (t1/2 > 60 min), and iv/po PK studies were
performed in all preclinical species (Table 3). Consistent with
the in vitro data, APD334 has a relatively low systemic clearance
(<4% of hepatic blood flow) and high Cmax across all species. In
both dog and monkey a significant decrease in volume of
distribution (Vss) was observed relative to rodent. Oral
bioavailability was in the range of 40−100%, and the terminal
phase half-life varied from 6 h in monkey, to as long as 29 h in
dog. Rat and monkey t1/2 values for siponimod (another S1P1
modulator currently in human trials) have been disclosed and
are 6 and 19 h, respectively.23
Peripheral lymphocyte counts were measured over a 24 h
time course following oral administration of APD334, in order
to establish a PK/PD relationship. Lymphocyte lowering (LL)
IC50 values were calculated, which illustrated that relatively low
blood concentrations (0.051 to 0.101 μM) are required to
Figure 3. Prophylactic mouse experimental autoimmune encephalo-
myelitis (EAE): female C57Bl/6 mice, vehicle = 0.5% methylcellulose
(MC); q.d. dosing began on day 3 and continued to day 25.
C
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ACS Medicinal Chemistry Letters
Letter
expressed on human atrial myocytes was subsequently
examined in vitro. APD334 and S1P were tested and were
found to have EC50 values of 29.9 and 2.1 nM, respectively.
Genotoxic potential was evaluated in an Ames bacterial
reverse mutation assay, and APD334 was nonmutagenic both
with and without rat liver S9 activation. [3H]-Astemizole
binding and cardiac repolarization (patch clamp) assays ruled
out any hERG channel interaction (IC50 > 30 μM,
respectively), and APD334 was subsequently evaluated in
telemeterized dog. No changes in pulse pressure, heart rate,
body temperature, or ECG parameter were observed at dose
levels up to 40 mg/kg dose.
CNS and respiratory safety studies were performed in SD
rats. No neurobehavioral effects or changes in respiratory
function were observed up to the top dose of 350 mg/kg in
both studies. Finally, a 14 day repeat dose rat toxicity assay was
conducted using 30, 100, 300, and 1000 mg/kg does groups.
APD334 was well tolerated up to 300 mg, with mortality and
CNS clinical signs observed at the 1000 mg/kg dose. In a
separate study, APD334 was found to be dose proportional up
to 300 mg/kg in SD rat with high levels of exposure at the top
doses (Cmax = 135.7 μM at 300 mg/kg, and Cmax = 182.7 μM at
1000 mg/kg) suggesting a comfortable safety window versus
the pharmacologically effective plasma concentration of 0.051
μM in rat. Lastly, no safety related findings with respect to liver
function were observed in rat.
Figure 4. Therapeutic mouse experimental autoimmune encephalo-
myelitis (EAE): female C57Bl/6 mice, vehicle = 0.5% methylcellulose
(MC); q.d. beginning on day 18 and the arrow indicates the start of
drug treatment.
fingolimod (1). Furthermore, histological examination of spinal
cord and brain collected on day 37 revealed significantly fewer
inflammatory foci compared to vehicle control, and a marked
decrease in number of infiltrating lymphocytes was also
observed (Figure 5).
In summary, we identified APD334, a structurally novel,
selective, functional antagonist of S1P1, which met our
preclinical safety and efficacy criteria. Importantly, APD334
achieves good central exposure following oral dosing and
possesses a favorable pharmacokinetic profile in multiple
preclinical species. APD334 is currently in development for a
number of indications related to autoimmune disease. The
outcome of these studies and plans for future development will
be reported in due course.
Figure 5. Immunofluorescent staining of cerebellum of therapeutically
treated mice at 37 days postimmunization. Pink color indicates CD45+
T-cells, green CD3+ T-cells, and blue 4′,6-diamidino-2-phenylindole
fluorescent stain (DAPI): (a) 0.5% methylcellulose; (b) 1.0 mg/kg 1;
(c) 3.0 mg/kg APD334.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis and analytical data for compounds 7−21 and
APD334. This material is available free of charge via the
Internet at http://pubs.acs.org.
APD334 was similarly efficacious in a collagen induced
arthritis (CIA) model. Prophylactic oral administration in
female Lewis rats resulted in a significant reduction in ankle
diameters (mean SEM) on the day 17 end point following a
daily oral dose of 1.0 and 3.0 mg/kg (0.2803 0.00570 and
0.2690 0.0031 in., versus 0.3352 0.0045 for vehicle) and
weight regain (178.6 4.1 and 182.3 1.9 g, versus 160.3
2.3 for vehicle) and was similar to that observed in rats treated
with 1.0 mg/kg of fingolimod (0.2694 0.0039 in., 181.9 2.2
g) or 0.075 mg/kg of methotrexate (0.2795 0.0066 in., 181.7
2.9 g). Improvement in histological parameters in the knees
and ankles of CIA rats was also observed, suggesting that
inhibiting lymphocyte entry into arthritic joints with APD334
treatment suppresses CIA in rodents.
AUTHOR INFORMATION
Corresponding Author
*E-mail: dbuzard@arenapharm.com.
■
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
PPTS, pyridinium p-toluenesulfonate; DIEA, diisopropylethyl-
amine; DMF, dimethylformamide; DIAD, diisopropyl azodi-
carboxylate; THF, tetrahydrofuran; CNS, central nervous
CYP inhibition IC50 values were determined, and APD334
has a very low inhibitory potential (i.e., 3A4, 2D6, 2C9, 1A2,
2C19; IC50 >50 μM), thus ruling out the likelihood of a drug−
drug interaction. Selectivity was further examined in a broad
panel of receptors, ion channels, and enzymes. APD334
exhibited weak or no activity, in more than 100 different
binding and functional assays. The most potent off target
interaction observed was for the human adenosine A3 receptor,
which afforded an IC50 value of 3.3 μM in the A3 binding assay.
Activation of inward-rectifying potassium channel (IKAch)
̀
system; hERG, human ether-a-go-go-related gene; ECG,
electrocardiogram; CHO, chinese hamster ovary; CYP,
cytochrome P450; CSF, cerebrospinal fluid
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