Discovery of a novel class of potent and orally bioavailable sphingosine 1-phosphate receptor 1 antagonists

Article abstract of DOI:10.1021/jm201533b

A series of subtype selective sphingosine 1-phosphate receptor 1 (S1P ₁) antagonists are disclosed. Our high-throughput screening campaign revealed hit 1 for which an increase in potency and mouse oral exposure was achieved with minor modifications to the chemical scaffold. In vivo efficacy revealed that at high doses compounds 12 and 15 inhibited tumor growth. Further optimization of our lead series led to the discovery of proline derivatives 37 (XL541) and 38 which had similar efficacy as our first generation analogues at significantly lower doses. Analogue 37 displayed excellent pharmacokinetics and oral exposure in multiple species.

Full text of DOI:10.1021/jm201533b

Supporting Information
The Discovery of a Novel Class of Potent and
Orally Bioavailable Sphingosine-1-Phosphate
Receptor 1 Antagonists
Mohamed A. Ibrahim, Henry W. B. Johnson*, Joon Won Jeong, Gary L. Lewis, Xian Shi,
Robin T. Noguchi, Matthew Williams, James W. Leahy, John M. Nuss, John Woolfrey,
Monica Banica, Frauke Bentzien, Yu-Chien Chou, Anna Gibson, Nathan Heald , Peter
Lamb, Larry Mattheakis, David Matthews, Aaron Shipway, Xiang Wu, WenTao Zhang,
Sihong Zhou and Geetha Shankar
Exelixis, Department of Drug Discovery, 169 Harbor Way, South San Francisco CA,
94083, USA.
Table of Contents
S2 – Pharmacokinetics
S5 – Efficacy Studies
S10 – Cytochrome P450 and MLM Assays
1
S

Pharmacokinetics
Experimental Animals
The animals were housed at Exelixis vivarium facilities according to the guidelines
outlined by the Exelixis Institutional Animal Care and Use Committee (IACUC). Prior to
initiation of a study, the animals were allowed to acclimate for a minimum of 48 h.
During these studies, animals were provided food and water ad libitum and housed in a
room conditioned at 70-75 °F and 60% relative humidity. A 12 h light and 12 h dark
cycle was maintained with automatic timers. All animals were examined daily for
compound induced or tumor related deaths. Moribund animals were euthanized when
necessary and animal carcasses were disposed of according to IACUC guidelines.
Bioanalytical Analysis: The concentration of compounds in plasma samples was
determined by HPLC/MS/MS analysis using sample preparation and analytical
conditions described on page S2. A non-compartmental model was applied to calculate
pharmacokinetic (PK) parameters for all routes of administration using WinNonlin 4.2
software
Mouse HTS Data: Female Nu/Nu homozygous mice approximately 8 weeks of age and
weight approximately 20-22 grams were purchased from Taconic (Hudson, NY).
Compounds were administered orally at a dose of 100 mpk as a solution in 10% EtOH
45% PEG400 / Water plus 1 molar equivalent of HCl, 10% EtOH 40% PEG400 / Water
plus 1 molar equivalent of HCl, 10% ethanol / 40%PEG400 / 20% Captisol plus 1 molar
equivalent of HCl, or 10% NMP / 90% corn oil at a dose volume of 10 mL/kg. Whole
blood was collected at 1 and 4 hours post dose into heparinized tubes, plasma prepared
and stored at -80 ºC until analysis.
2
S

Study of 37 in Female CD Rats: After IV and PO Administration: Female CD rats
(Sprague Dawley) approximately 9-10 weeks of age and weighing approximately 201-
225 grams were purchased from Charles River Laboratories (Hollister, CA). The female
CD rats were fasted overnight prior to dosing and returned to food 4 h post-dose
administration. Compound 37 was administered at 3 mpk orally as a solution in Ethanol /
PEG400 / water (EPW, 5:45:50) to groups of 4 female CD Rats. Blood was collected
(100 µL) in heparinized tubes via a jugular catheter at 0.08, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12,
24, 32, 48, 72, and 144 h post-dosing. The plasma obtained was stored at -80 °C and a
volume of 50 µL was used for analysis.
Study of 37 in Male Beagle Dogs: After PO Administration: The in life portion of the
pharmacokinetic studies were conducted by Covance Laboratories (Madison, WI)
according to the terms and conditions outlined by Study Number 7359-436 (3 mpk IV
and PO). Male Beagle dogs were fasted overnight prior to dosing and returned to food
four h post-dose. Compound 37 was administered by oral gavage at 3 mpk as a solution
formulated in an EtOH:PEG400:Water (EPW, 5:45:50). Plasma concentrations of 37
were evaluated over a 48-hour period in experimental groups comprised of three animals.
Blood (1000 µL) was sampled in potassium EDTA tubes at the following time points:
pre-dose, 0.08, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, 24, and 48 h post-dose. Plasma was prepared
and the samples were frozen and shipped on dry ice to Exelixis for analysis.
Study of 37 in Male Cynomolgus Monkeys: After IV and PO Administration: The in
life portion of the pharmacokinetic studies was conducted by Maccine Laboratories
(Singapore) according to the terms and conditions outlined by Study Number JPL-004 (3
mpk IV and PO). Male Cynomolgus monkeys were fasted overnight prior to dosing and
returned to food four h post-dose. Compound 37 was administered intravenously and by
3
S

oral gavage at 3 mpk as a solution formulated in an EtOH:PEG400:Water (EPW,
5:45:50). Plasma concentrations of 37 were evaluated over a 48-hour period in
experimental groups comprised of three animals. Blood (1000 µL) was sampled in
potassium EDTA tubes at the following time points: pre-dose, 0.08, 0.25, 0.5, 1, 2, 3, 4,
6, 8, 12, 24, and 48 h post-dose. Plasma was prepared and the samples were frozen and
shipped on dry ice to Exelixis for analysis.
4
S

Efficacy Studies
Experimental Animals
The animals were housed at Exelixis vivarium facilities according to the guidelines
outlined by the Exelixis Institutional Animal Care and Use Committee (IACUC). Prior to
initiation of a study, the animals were allowed to acclimate for a minimum of 48 h.
During these studies, animals were provided food and water ad libitum and housed in a
room conditioned at 70-75 °F and 60% relative humidity. A 12 h light and 12 h dark
cycle was maintained with automatic timers. All animals were examined daily for
compound induced or tumor related deaths. Moribund animals were euthanized when
necessary and animal carcasses were disposed of according to IACUC guidelines. Female
athymic nude mice (NCr) 5-8 weeks of age and weighing approximately 20 g were
purchased from Taconic (Germantown, NY).
Cancer Cell lines
All cell lines were tested by the University of Missouri Research Animal Diagnostic
Animal Laboratory for all known species of mycoplasma and for a panel of murine
viruses, which included: sendai virus, parvovirus, murine hepatitis virus, Theiler’s murine
encephalomyelitis
virus,
Reovirus3,
Rotavirus,
Extramelia,
Lymphocytic
choriomeningitis virus, Polyoma virus, K virus, adenovirus, cytomeglavirus, lactate
dehydrogenase elevating virus, thymic virus, and Hanta virus.
Solid Tumor Models - Tumor Cell Implantation and Measurement
The MDA-MB-231T human breast cancer cell line was chosen as a highly vascularized
orthotopic xenograft model, with an average of 90 tumor vessels per mm2 in control
tumors at the end of a 14 dosing period (Exelixis in-house data). The MDA-MB-231T
cell line harbors a homozygous activating mutation in B-RafG464V, and a heterozygous
5
S

activating mutation in K-RasG13D. MDA-MB-231T cells also harbor homozygous
mutations in tumor suppressor genes TP53 and CDKN2A known to regulate cell cycle
progression. MDA-MB-231T cells were cultured in vitro in DMEM (Mediatech)
supplemented with 10% Fetal Bovine Serum (Hyclone), Penicillin-Streptomycin and
non-essential amino acids at 37 °C in a humidified, 5% CO2 atmosphere. On day 0, cells
were harvested by trypsinization, and 1 x 106 cells (passage #8-12, > 98% viability) in 0.1
mL ice-cold Hank’s balanced salt solution were implanted subcutaneously into the
mammary fat pad of 5-8 week old female athymic nude mice.
Analysis of Efficacy Data
In mammary fat pad implanted tumors, the mean tumor weight of each animal in the
respective control and treatment groups was determined twice-weekly during the duration
of the study. Tumor weight (TW) was determined by measuring perpendicular diameters
with a caliper, using the following formula:
tumor weight (mg) = [tumor volume = length (mm) x width2 (mm2)]/2
These data were recorded and plotted on a tumor weight vs. days post-implantation line
graph and presented graphically as an indication of tumor growth rates. Percent inhibition
of tumor growth (TGI) is determined with the following formula:
1 – ( Xf – X0 )
( Yf – X0 )
* 100
where
X0 = average TW of all tumors on staging day
Xf = TW of treated group on Day f
Yf = TW of vehicle control group on Day f
6
S

If tumors regress below their starting sizes, then the percent tumor regression is
determined with the following formula:
( X0 – Xf ) * 100
X0
Tumor size is calculated individually for each tumor to obtain a mean SEM value for
each experimental group. Statistical significance is determined using the 2-tailed
Student’s t-test (significance defined as p < 0.05).
In addition to tumor weight determination, body weights of all animals on study were
recorded daily. A transponder was implanted in each mouse for identification, and
animals were monitored daily for clinical symptoms and survival.
Formulation
The compound was formulated for oral (PO) administration in efficacy studies by
dissolution of the dry powder in 10% EtOH 40% PEG400 / Water + 1:1 HCl (12 and 15),
Water (25, ZD6474), 0.75% CMC 0.1% Tween80 / Water (26, Merck Receptor Agonist),
10% NMP / corn oil (37 and 38). Merck Receptor Agonist was synthesized in-house. All
dosing solutions were prepared fresh daily and administered at 10 mL/kg of body weight.
7
S

25.0
20.0
15.0
Vehicle, 10 ml/kg, PO qd
32, 30 mpk, PO qd
33, 60 mpk, PO qd
26
25
16, 300 mpk, PO qd
12
19, 300 mpk, PO qd
15
0
5
10
15
Dose Day
Figure 1: Mean Body Weights in the MDA-MB-231T Xenograft Model.
8
S

25.0
20.0
15.0
Vehicle:1, 10 ml/kg + Vehicle:1, 10 ml/kg
53, 100 mpk, PO qd
38
52, 100 mpk, PO qd
37
0
5
10
15
Dose Day
Figure 2: Mean Body Weights in the MDA-MB-231T Xenograft Model.
9
S

Cytochrome P450 Assay
CYP450 inhibition profiling was evaluated by quantification of drug substrate
metabolism to specific products known to be catalyzed by CYP450 isozymes present as
pooled human liver microsomal fractions. Tolbutamide methyl-hydroxylation is
catalyzed by CYP2C9 and bufuralol-1-hydroxylation is catalyzed by CYP2D6. The
assays were performed in 96-well microtiter plates using a final volume of 500 µL in 100
mM potassium phosphate buffer (50 mM potassium phosphate buffer for CYP2C8), pH
7.4 at 37 °C. Reaction and analysis conditions were optimized for all CYP450 isozymes,
and are summarized Table 1. At the end of the incubation period, reactions were
quenched by addition of 100 µL acetonitrile to 100 µL of reaction samples, cooled to 4
°C, and clarified by centrifugation at 2500 xg for 15 min. The supernatant was transferred
to a fresh 96-well microtiter plate, sealed, and quantified by LC/MS/MS analysis.
Inhibition potential was evaluated at six concentrations (0.2–50 µM). Enzyme activity
was calculated based on quantification of metabolite signal. IC50 values were calculated
by nonlinear regression using a 4-parameter sigmoidal curve fit equation (XLFit).
Table 1. CYP450 LC/MS/MS Assay Conditions
Substrate
NADPH
Isozyme Substrate
Metabolite
HLM (mg/mL) Incubation Time
(µM)
(µM)
CYP2C9 Tolbutamide
CYP2D6*1 Bufuralol
CYP3A4 Testosterone
100
5
Hydroxytolbutamide
Hydroxybufuralol
1000
1000
1000
0.15
0.15
0.25
15’ @ 37 °C
15’ @ 37 °C
15’ @ 37 °C
3
Hydroxytestosterone
LC/MS/MS analysis was performed using an API 3000 instrument, coupled to a
Shimadzu SCL-10AVP LC Pump system. Analytical samples were separated using a
Waters Atlantis dC18 reverse phase HPLC column (50 mm x 4.6 mm) at a flow rate of
10
S

1-2 mL/min. The mobile phase consisted of 0.1% formic acid in acetonitrile (solvent A)
and 0.1% formic acid in water (solvent B). Elution conditions are detailed in Table 2.
Table 2: Gradient Conditions for CYP450 Assay
Time (min)
Flow (µL/min)
1000
%A
2
%B
98
98
5
0.0
0.3
1.7
2.3
2.4
3.2
1000
2
1500
95
95
10
10
1500
5
2000
90
90
2000
MLM Assay
Microsomal oxidation of test compounds in the presence of mouse liver microsomal
fractions was conducted in 96-well microtiter plates. Liver microsomal preparations were
purchased from BD Gentest (Bedford, MA). Test compounds were incubated in duplicate
at 37 °C for 30 min in the presence of liver microsomes and NADPH. Reaction mixtures
(75 µL) contained a final concentration of 15 µM test compound, 0.15% DMSO,
0.5 mg/mL microsomal protein and 1 mM NADPH in 100 mM potassium phosphate, pH
7.4 buffer. Control incubations contained the full complement of enzyme and substrate
but no NADPH added. All test-sets included reference CYP450 substrates to verify assay
performance. Reactions were terminated by the addition of 150 µL acetonitrile containing
0.1% formic acid. The concentration of the test compound remaining was determined by
LC/MS/MS analysis on Sciex API-3000 instrument. The extent of metabolism was
calculated as a disappearance of the test compound, compared to the no-NADPH control
reaction incubations. Chromatographic separation of analytes was achieved using a
Phenomenex Synergi Hydro-RP column (Torrance, CA) and a mobile phase consisting of
11
S

0.1% formic acid in acetonitrile and 0.1% formic acid in water. A linear gradient from
2-98% acetonitrile was used to elute compounds from the HPLC column.
12
S