Accepted Manuscript Title: A novel inhaled Syk inhibitor blocks mast cell degranulation and early asthmatic response Author: Isabel Ramis Raquel Otal Cristina Carre˜no Anna Dom`enech Peter Eichhorn Adelina Orellana M´onica Maldonado Jorge De Alba Neus Prats Joan-Carles Fern´andez Bernat Vidal Montserrat Miralpeix PII: DOI: Reference:
S1043-6618(15)00109-7 http://dx.doi.org/doi:10.1016/j.phrs.2015.05.011 YPHRS 2838
To appear in:
Pharmacological Research
Received date: Revised date: Accepted date:
23-3-2015 14-5-2015 26-5-2015
Please cite this article as: Ramis I, Otal R, Carre˜no C, Dom`enech A, Eichhorn P, Orellana A, Maldonado M, De Alba J, Prats N, Fern´andez J-C, Vidal B, Miralpeix M, A novel inhaled Syk inhibitor blocks mast cell degranulation and early asthmatic response, Pharmacological Research (2015), http://dx.doi.org/10.1016/j.phrs.2015.05.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A novel inhaled Syk inhibitor blocks mast cell degranulation and early asthmatic response Isabel Ramisa, Raquel Otala, Cristina Carreñoa, Anna Domènecha, Peter Eichhorna, Adelina
ip t
Orellanaa, Mónica Maldonadoa, Jorge De Albaa, Neus Pratsa, Joan-Carles Fernándezb, Bernat
cr
Vidala, Montserrat Miralpeixa Almirall R&D Center, Sant Feliu de Llobregat, Barcelona, Spain
b
Almirall-Barcelona Science Park Unit, Barcelona Science Park, Barcelona, Spain.
an
us
a
M
Corresponding author: Isabel Ramis, Almirall R&D Centre, Laureà Miró 408-410, 08980 Sant Feliu de Llobregat, Barcelona, Spain. Tel.:+34 933 128 860; fax: +34 932 912 827
d
e-mail: [email protected]
te
CC: [email protected], AD: [email protected],
Ac ce p
PE: [email protected], AD: [email protected] M Maldonado: [email protected], JDA: [email protected], NP: [email protected], JCF: [email protected], BV: [email protected], M. Miralpeix: [email protected] Keywords: Syk, inhalation, asthma, mast cells, early asthmatic response, Brown Norway rat Nonstandard abbreviations: ADME: absorption, distribution, metabolism, elimination; ANOVA: analysis of variance; BCR: B cell receptor; BN: Brown Norway; BSA: bovine serum albumin; EAR: early asthmatic response; ELISA: enzyme-linked immunosorbent assay; FBS: fetal bovine serum; IC50: half-maximal inhibitory concentration; i.p.: 1
Page 1 of 34
intraperitoneal; i.t.: intratracheal; i.v.: intravenous; LAR: late asthmatic response; PK: pharmacokinetic; OVA: ovalbumin; PBS: Dulbecco’s phosphate buffered saline; Syk: spleen
ip t
tyrosine kinase.
cr
LAS189386 is a new inhaled Syk inhibitor that blocks airway mast cell degranulation and
Ac ce p
te
d
M
an
us
EAR in rats
2
Page 2 of 34
ABSTRACT Spleen tyrosine kinase (Syk) is essential for signal transduction of immunoreceptors. Inhibition of Syk abrogates mast cell degranulation and B cell responses. We hypothesized
ip t
that Syk inhibition in the lung by inhaled route could block airway mast cells degranulation and the early asthmatic response without the need of systemic exposure. We discovered
cr
LAS189386, a novel Syk inhibitor with suitable properties for inhaled administration. The
us
aim of this study was to characterize the in vitro and in vivo profile of LAS189386.
The compound was profiled in Syk enzymatic assay, against a panel of selected kinases and in
an
Syk-dependent cellular assays in mast cells and B cells. Pharmacokinetics and in vivo efficacy was assessed by intratracheal route. Airway resistance and mast cell degranulation after OVA
M
challenge was evaluated in an ovalbumin-sensitized Brown Norway rat model.
d
LAS189386 potently inhibits Syk enzymatic activity (IC50 7.2 nM), Syk phosphorylation
te
(IC50 41 nM), LAD2 cells degranulation (IC50 56 nM), and B cell activation (IC50 22 nM). LAS189386 inhibits early asthmatic response and airway mast cell degranulation without
Ac ce p
affecting systemic mast cells.
The present results support the hypothesis that topical inhibition of Syk in the lung, without systemic exposure, is sufficient to inhibit EAR in rats. Syk inhibition by inhaled route constitutes a promising therapeutic option for asthma.
Keywords: Syk, inhalation, asthma, mast cells, Early Asthmatic Response, Brown Norway rat
3
Page 3 of 34
1 Introduction Spleen tyrosine kinase (Syk) is a cytosolic, non-receptor protein tyrosine kinase expressed predominantly in hematopoietic cells that signals in a variety of immune receptors including
ip t
FcRI and Fc receptors, B cell receptor (BCR), integrin and lectin receptors 1, 2. Syk
couples activated immune receptors in mast cells, B cells, macrophages and neutrophils to
cr
downstream cellular responses such as degranulation, antibody production, phagocytosis, cell
us
adhesion, cytokine production, proliferation and differentiation 2. Therefore, Syk constitutes a therapeutic target for the treatment of immune-mediated disorders such as asthma, allergy,
an
autoimmune diseases and hematological malignancies.
M
A number of ATP-competitive Syk inhibitors have been described 3 and it has been reported that pharmacological inhibition of Syk activity modulates mast cell degranulation 4 and
d
leukocyte immune function 5 and suppresses inflammation in vivo 6, 7. Interestingly, since
te
Syk is located upstream in the cell signaling pathway of multiple immune receptors, therapies with Syk inhibitors may be potentially more efficacious than drugs that inhibit a single
Ac ce p
downstream event.
Few Syk inhibitors are in clinical development. Fostamatinib (also known as R788, Rigel), the soluble pro-drug of R406 6, has been evaluated in clinical trials for rheumatoid arthritis 8, 9 and currently is in development for immune thrombocytopenia and IgA nephropathy. The dual Syk/Janus Kinase inhibitor cerdulatinib (PRT062070, Portola) [10] is in phase I for leukemia and lymphoma. The more selective Syk inhibitor PRT062607 (Portola) 11 has been evaluated in phase I. Recently, entospletinib (GS-9973, Gilead) [12] started phase II trials for refractory hematologic conditions.
4
Page 4 of 34
For respiratory diseases, the Syk inhibitor R343 (Rigel) 13 has been evaluated by inhaled route in clinical trials for asthma 14. As an alternative approach, a Syk small interfering RNA (Excellair
TM
, Zabecor) has been evaluated in phase II for asthma. Our research efforts
ip t
for the discovery of Syk inhibitors led to the discovery of a series of potent indazole derivatives with a pharmacokinetic profile suitable for inhaled administration. LAS189386
cr
was selected for in vivo characterization.
us
We hypothesize that topical Syk inhibition in the lung by inhaled route could block airway mast cells degranulation and the antigen-induced early asthmatic response (EAR). The aim of
an
this study has been to describe the in vitro and in vivo profile by intratracheal (i.t.) route of LAS189386. For comparison purposes the Syk inhibitor R343 was also tested in the in vitro
M
assays. Our data show that LAS189386 is a potent and selective Syk inhibitor with a suitable profile for inhaled administration that reduces the EAR in an ovalbumin (OVA)-challenged
d
BN rat and completely blocks airway mast cell degranulation. Our data support the Syk
Ac ce p
te
inhibition by inhaled route for the treatment of asthma.
2 Methods
2.1 Synthesis of Syk inhibitors LAS189386,
1-{2-[(1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl]pyridin-4-yl}-N-pyrazin-2-yl-
1H-indazol-3-amine, was synthesized by the department of Medicinal Chemistry of Almirall with a 99% purity determined by liquid chromatography–mass spectrometry, following the procedure described in the patent application EP2489663. Low resolution mass spectrometry (m/z): 385 (M+1)+ and 1H nuclear magnetic resonance (400 MHz, CDCl3) delta ppm: 1.84 - 2.00 (m, 2 H), 3.15 (s, 2 H), 3.32 (d, J=9.57 Hz, 1 H), 3.70 (dd, J=9.38, 1.95 Hz, 1 H), 3.88 (s, 1 H), 4.85
5
Page 5 of 34
(bs, 1 H), 6.74 (d, J=1.56 Hz, 1 H), 7.03 (dd, J=5.67, 1.76 Hz, 1 H), 7.24 - 7.31 (m, 2 H), 7.50 (bs, 1 H), 7.54 (ddd, J=8.45, 7.18, 0.98 Hz, 1 H), 7.73 (d, J=8.21 Hz, 1 H), 7.88 (d, J=8.60 Hz, 1 H), 8.16 8.29 (m, 3 H), 9.57 (s, 1 H ) confirmed the structural identity.
N4-[(2,2-difluoro-4H-benzo[1,4]oxazin-3-one)-6-yl]-5-fluoro-N2-[3-(methylamino
carbonylmethyleneoxy)phenyl]-2,4-pyrimidinediamine,
was
also
ip t
R343,
synthesized
by
the
cr
department of Medicinal Chemistry of Almirall following the procedure described in
an
us
WO2003063794.
2.2 Materials and drug preparation
M
Imject® Alum Adjuvant was purchased from Thermo Fisher Scientific (Rockford, IL), sterile saline (0.9% NaCl) from B. Braun Medical Ltd (Sheffield, UK), Dulbecco’s phosphate
d
buffered saline (PBS), OVA and methyl cellulose from Sigma-Aldrich (Ayrshire, UK) and
te
Tween®80 (Polysorbat) from Merck (Hohenbrunn, Germany). Methysergide maleate was
Ac ce p
purchased from Tocris Bioscience (Bristol, UK), montelukast (sodium salt) from Cayman Chemical Company (Ann Arbor, MI). Ketamine chlorhydrate (Imalgene®) and xylazine hydrochloride (Rompun®) were obtained from Merial (Lyon, France) and KVP Pharma und Veterinär Produkte GmbH (Kiel, Germany), respectively. For oral administration, the suspension of montelukast (volume 2 ml/kg) was prepared in 0.5% methyl cellulose, 0.1 % Tween®80 in water. For i.t. administration, LAS189386 was prepared in 0.2 % Tween®80 in PBS and R343 was prepared in 0.2 % Tween®80, 0.5% methyl cellulose in PBS. Both Syk inhibitors were administered (volume 0.8 ml/kg) with a Liquid MicroSprayer® (model IA-1B, rat size) with the help of a small animal laryngoscope (model LS-1), both from Penn-Century Inc. (Wyndmoor, PA).
6
Page 6 of 34
2.3 Cell lines
ip t
LAD2 cell line is a human mast cell line established at the National Institutes of Health (Bethesda, Maryland, US) 15. LAD2 cells were provided by Dr. Arnold Kirshenbaum
cr
through a biological materials license agreement. RBL-2H3 cell were obtained from the
us
ATCC.
an
2.4 Animals
M
Care and use of animals were undertaken in compliance with the European Directive 2010/63/EU for the use of laboratory animals and was approved by the Almirall Ethics
d
Committee.
te
Male BN rats (200-250 g) were purchased from Charles River (Lyon, France) and housed at
Ac ce p
20–24 ºC, relative humidity 40-65 % with 15 air changes/h under a 12-h light/dark cycle for at least five days before use. Animals were allowed free access to standard laboratory food (Harlan Teklad 2014) and water.
2.5 Syk enzymatic assay
In order to identify Syk inhibitors that directly interact with the ATP binding pocket in the Syk catalytic domain a biochemical Syk activity assay was used. The effect of LAS189386 on Syk activity was assessed by measuring the extent of substrate phosphorylation by recombinant full-length Syk (Millipore, Dundee, UK) using a radiometric filtration assay.
7
Page 7 of 34
Briefly, the assay was run in 50 mM Tris-HCl (pH 7.5), 0.1 mM EGTA, 4 mM Mg(CH3COO)2, 0.1 mM Na3VO4, 0.1 % (v/v) β-mercaptoethanol, 0.133 mg/ml bovine serum albumin (BSA) in the presence of 100 µg/mL poly-GT, 1 nM Syk and different
ip t
concentrations of Syk inhibitors (ranging 50 µM-10 pM) in 5% dimethylsulfoxide. The reaction was initiated by the addition of 0.36 µCi [γ-33P] ATP (10 mCi/mL, PerkinElmer,
cr
Boston, MA). After 40 min incubation at room temperature, 15 µL of the reaction was
transferred to a filter plate (Millipore, Billerica, MA) previously pre-wetted with a solution of
us
75 mM phosphoric acid. The filter plate was washed three times with 200 µl phosphoric acid. Finally, 30 µL Optiphase™ Supermix (PerkinElmer) were added to each test well and
an
incubated for at least 1 h before radioactivity counting in a Wallac MicroBeta®
d
M
(PerkinElmer).
te
2.6 Biochemical kinase screening panel
The selectivity of LAS189386 and R343 was assessed at 1 µM in a KinaseProfiler panel
Ac ce p
(Millipore) comprising 42 kinases. Assays were performed at the corresponding Km of ATP for each enzyme. Kinases were selected according to common cross-reactivity described for other Syk inhibitors (mainly tyrosine kinases such as Flt3, Lyn, Lck), kinases being most homologous to Syk (ZAP70, Pyk2, Fak) as well as a number of kinases representative of the kinome. In addition, IC50 values for kinases involved in FcRI receptor cross-linking pathway (Fyn, Lyn, Btk) and ZAP70 were also calculated in Millipore.
2.7 LAD2 cells degranulation assay
8
Page 8 of 34
LAD2 cells were sensitized in their normal growth medium, Complete StemPro-34 SFM, (Prepotech, Rocky Hill, NJ) by adding 100 ng/ml of biotin-labeled human IgE (US Biological, Swampscott, MA) overnight (37 ºC, 5 % CO2) in a humidified atmosphere (90 %).
ip t
Sensitized LAD2 cells were washed, transferred to the assay plate (10,000 cells/well), preincubated with the test compounds for 30 min and then activated with 125 ng/mL of
cr
streptavidin (Thermo Scientific Pierce, Rockford, IL) for 30 min. After activation, supernatants were removed and transferred to another well for β-hexosaminidase
us
determination. Cells were lysed in 2 freeze-thaw cycles and then incubated for 90 min at 37ºC
an
with 1 mM -hexosaminidase substrate (p-nitrophenyl-N-acetyl-D-glucosamide (SigmaAldrich), in citric buffer, pH 4.5). The reaction was stopped with 0.4 M glycine (Merck)
d
M
solution (pH 10) and absorbance was read at 405 nm.
te
2.8 Syk phosphorylation (Tyr525/526) in LAD2 cells assay LAD2 cells prepared as described above were pre-incubated with the test compounds for 30
Ac ce p
min and then activated with 125 ng/mL of streptavidin for 1 min at room temperature. Cells were lysed and phosphorylated Syk (Tyr525/526) was analyzed in supernatants by ELISA (#7970 Cell Signaling Technology, Izasa; Barcelona, Spain).
2.9 Degranulation in RBL-2H3 cells assay RBL-2H3 cells were seeded in a 96-well plate (0.1x106 cells per well) and sensitized with 100 ng/mL mouse monoclonal anti-DNP antibody (clone SPE-7) (Sigma-Aldrich) for 90 min (37ºC, 5 % CO2). Cells were washed twice with Tyrodes buffer and pre-incubated with the test compounds for 30 min at 37 ºC. Cells were then stimulated with 100 ng/mL 9
Page 9 of 34
dinitrophenyl-BSA (Biosearch Technologies; Novato, CA) for 60 min at 37 ºC.
ip t
β-hexosaminidase determination was performed as described above.
2.10 Human B cell activation assay
cr
B cells were isolated from blood of healthy volunteers by using RossetteSep® Human B Cell
us
Enrichment Cocktail (StemCell Technologies; Grenoble, France). Isolated cells were adjusted to a density of 0.2x106 cells/mL in PBS with 2% fetal bovine serum (FBS) and transferred to a
an
5-ml polystyrene round-bottom tube (20,000 cells per tube). Cells were pre-incubated with the test compounds for 30 min (37 ºC, 5 % CO2) and then activated with 10 µg/mL of anti-human
M
IgM (Jackson ImmunoResearch; Suffolk, UK) for 2 h. After activation, cells were stained with FITC-conjugated mouse anti-human CD19 and PE-conjugated mouse anti-human CD69
d
(BD Biosciences; Erembodegem, Belgium) in the dark for 15 min at room temperature and
te
fixed with BD FACSTM Lysis solution for 15 min. Cells were washed and suspended with PBS, 1 % FBS and 0.1% sodium azide. Flow cytometry analysis was performed to quantify
Ac ce p
CD69 expression as a B cell activation marker.
2.11 in vitro ADME profiling
Plasma protein binding of LAS189386 was determined by equilibrium dialysis (Rapid Equilibrium Dialysis kit; Thermo Scientific Pierce, San José, CA). Rat plasma was spiked at 1 M and dialyzed against phosphate buffer (pH 7.4) for 4 h at 37ºC. The concentrations of LAS189386 in both dialysis chambers were quantified by LC-MS/MS. Plasma protein binding was used to estimate the unbound plasma levels in efficacy study.
10
Page 10 of 34
The oxidative metabolism was assessed by incubating LAS189386 (5 M) with rat liver microsomes (protein concentration 1 mg/ml) in the presence of NADPH. The reaction was quenched after 30 min and the samples were analyzed by LC-MS/MS to determine substrate
ip t
disappearance and metabolite formation.
cr
2.12 Pharmacokinetic study and samples analysis
Male Wistar rats were obtained from Charles River Laboratories (Santa Perpètua de Mogoda,
us
Spain). For i.t. dosing (three animals per time point), overnight-fasted rats weighing approximately 250 g were anesthetized with 4% isofluorane and placed in a supine position.
an
Two hundred µL of the suspension of the test compounds were administered directly into the trachea using a Liquid MicroSprayer®. At pre-defined time points (0.01, 0.1, 1, 6, 24 h),
M
blood samples were collected and centrifuged to obtain blood plasma. Lungs were harvested after perfusion with saline through the right ventricle of the heart. Plasma samples were
te
d
precipitated with acetonitrile/trifluoroacetic acid, centrifuged and the supernatant analyzed by LC-MS/MS. Lungs were homogenized with methanol (4:1; w/v)) using an UltraTurrax (IKA-
Ac ce p
Werke, Staufen, Germany). The homogenates were then sonicated, centrifuged and the supernatants analyzed by LC-MS/MS. Chromatographic analyses were accomplished on a reversed-phase column (C18) followed by mass spectrometric detection in MRM mode.
2.13 OVA-induced early asthmatic response in BN rats and tissue mast cell degranulation BN rats were sensitized on days 0, 14 and 21 with OVA (100 µg i.p.) and Imject® Alum (20 mg i.p.). From day 28 to 32, LAS189386 was administered at 0.1, 0.3 or 1 mg/kg by i.t. route 1 h before challenge. Rats were anaesthetized (99 mg/kg ketamine chlorhydrate and 8 mg/kg xylazine hydrochloride by i.p. route) and instrumented as previously described [16]. Animals
11
Page 11 of 34
were challenged either with OVA (2 mg i.v.) or with control vehicle (sterile saline) and the airways resistance (cm H2O/mL/s) was measured over a 10-min period, using a FinePointe™ RC System (Buxco Research Systems, Wantage, UK). The changes in airways resistance
ip t
were assessed using the difference between the baseline resistance (average value of 1-min period) and the peak resistance (maximum value) in the 10-min period after saline or OVA
cr
administration. After EAR measurement the rats were sacrificed by severing the abdominal aorta. Subsequently, the trachea, left lung, mesentery and two inter-scapular skin samples
us
were carefully removed, fixed in 10 % formalin (pH 7.4), paraffin-embedded, and 3-µm cross-sections were stained with 0.1 % toluidine blue to evaluate the integrity of mast cells.
an
Mast cell degranulation was assessed blindly by two independent investigators and expressed
M
as percentage of degranulated mast cells both locally (trachea and left main bronchus) and systemically (mesentery and skin). Briefly, for each animal, total and degranulated mast cells
d
were counted in the tracheal dorsal wall (four cross sections) and bronchial wall (one cross
te
section). In the mesentery and skin, mast cell degranulation was determined by counting 200
Ac ce p
mast cells in randomly selected fields.
2.14 Statistical analysis
In the in vivo experiments comparisons between groups was done by one way analysis of variance (ANOVA) followed by Dunnett's post hoc test using GraphPad Prism software (GraphPad Software Inc; San Diego, California, US). Differences were considered significant when p
S1043-6618(15)00109-7 http://dx.doi.org/doi:10.1016/j.phrs.2015.05.011 YPHRS 2838
To appear in:
Pharmacological Research
Received date: Revised date: Accepted date:
23-3-2015 14-5-2015 26-5-2015
Please cite this article as: Ramis I, Otal R, Carre˜no C, Dom`enech A, Eichhorn P, Orellana A, Maldonado M, De Alba J, Prats N, Fern´andez J-C, Vidal B, Miralpeix M, A novel inhaled Syk inhibitor blocks mast cell degranulation and early asthmatic response, Pharmacological Research (2015), http://dx.doi.org/10.1016/j.phrs.2015.05.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A novel inhaled Syk inhibitor blocks mast cell degranulation and early asthmatic response Isabel Ramisa, Raquel Otala, Cristina Carreñoa, Anna Domènecha, Peter Eichhorna, Adelina
ip t
Orellanaa, Mónica Maldonadoa, Jorge De Albaa, Neus Pratsa, Joan-Carles Fernándezb, Bernat
cr
Vidala, Montserrat Miralpeixa Almirall R&D Center, Sant Feliu de Llobregat, Barcelona, Spain
b
Almirall-Barcelona Science Park Unit, Barcelona Science Park, Barcelona, Spain.
an
us
a
M
Corresponding author: Isabel Ramis, Almirall R&D Centre, Laureà Miró 408-410, 08980 Sant Feliu de Llobregat, Barcelona, Spain. Tel.:+34 933 128 860; fax: +34 932 912 827
d
e-mail: [email protected]
te
CC: [email protected], AD: [email protected],
Ac ce p
PE: [email protected], AD: [email protected] M Maldonado: [email protected], JDA: [email protected], NP: [email protected], JCF: [email protected], BV: [email protected], M. Miralpeix: [email protected] Keywords: Syk, inhalation, asthma, mast cells, early asthmatic response, Brown Norway rat Nonstandard abbreviations: ADME: absorption, distribution, metabolism, elimination; ANOVA: analysis of variance; BCR: B cell receptor; BN: Brown Norway; BSA: bovine serum albumin; EAR: early asthmatic response; ELISA: enzyme-linked immunosorbent assay; FBS: fetal bovine serum; IC50: half-maximal inhibitory concentration; i.p.: 1
Page 1 of 34
intraperitoneal; i.t.: intratracheal; i.v.: intravenous; LAR: late asthmatic response; PK: pharmacokinetic; OVA: ovalbumin; PBS: Dulbecco’s phosphate buffered saline; Syk: spleen
ip t
tyrosine kinase.
cr
LAS189386 is a new inhaled Syk inhibitor that blocks airway mast cell degranulation and
Ac ce p
te
d
M
an
us
EAR in rats
2
Page 2 of 34
ABSTRACT Spleen tyrosine kinase (Syk) is essential for signal transduction of immunoreceptors. Inhibition of Syk abrogates mast cell degranulation and B cell responses. We hypothesized
ip t
that Syk inhibition in the lung by inhaled route could block airway mast cells degranulation and the early asthmatic response without the need of systemic exposure. We discovered
cr
LAS189386, a novel Syk inhibitor with suitable properties for inhaled administration. The
us
aim of this study was to characterize the in vitro and in vivo profile of LAS189386.
The compound was profiled in Syk enzymatic assay, against a panel of selected kinases and in
an
Syk-dependent cellular assays in mast cells and B cells. Pharmacokinetics and in vivo efficacy was assessed by intratracheal route. Airway resistance and mast cell degranulation after OVA
M
challenge was evaluated in an ovalbumin-sensitized Brown Norway rat model.
d
LAS189386 potently inhibits Syk enzymatic activity (IC50 7.2 nM), Syk phosphorylation
te
(IC50 41 nM), LAD2 cells degranulation (IC50 56 nM), and B cell activation (IC50 22 nM). LAS189386 inhibits early asthmatic response and airway mast cell degranulation without
Ac ce p
affecting systemic mast cells.
The present results support the hypothesis that topical inhibition of Syk in the lung, without systemic exposure, is sufficient to inhibit EAR in rats. Syk inhibition by inhaled route constitutes a promising therapeutic option for asthma.
Keywords: Syk, inhalation, asthma, mast cells, Early Asthmatic Response, Brown Norway rat
3
Page 3 of 34
1 Introduction Spleen tyrosine kinase (Syk) is a cytosolic, non-receptor protein tyrosine kinase expressed predominantly in hematopoietic cells that signals in a variety of immune receptors including
ip t
FcRI and Fc receptors, B cell receptor (BCR), integrin and lectin receptors 1, 2. Syk
couples activated immune receptors in mast cells, B cells, macrophages and neutrophils to
cr
downstream cellular responses such as degranulation, antibody production, phagocytosis, cell
us
adhesion, cytokine production, proliferation and differentiation 2. Therefore, Syk constitutes a therapeutic target for the treatment of immune-mediated disorders such as asthma, allergy,
an
autoimmune diseases and hematological malignancies.
M
A number of ATP-competitive Syk inhibitors have been described 3 and it has been reported that pharmacological inhibition of Syk activity modulates mast cell degranulation 4 and
d
leukocyte immune function 5 and suppresses inflammation in vivo 6, 7. Interestingly, since
te
Syk is located upstream in the cell signaling pathway of multiple immune receptors, therapies with Syk inhibitors may be potentially more efficacious than drugs that inhibit a single
Ac ce p
downstream event.
Few Syk inhibitors are in clinical development. Fostamatinib (also known as R788, Rigel), the soluble pro-drug of R406 6, has been evaluated in clinical trials for rheumatoid arthritis 8, 9 and currently is in development for immune thrombocytopenia and IgA nephropathy. The dual Syk/Janus Kinase inhibitor cerdulatinib (PRT062070, Portola) [10] is in phase I for leukemia and lymphoma. The more selective Syk inhibitor PRT062607 (Portola) 11 has been evaluated in phase I. Recently, entospletinib (GS-9973, Gilead) [12] started phase II trials for refractory hematologic conditions.
4
Page 4 of 34
For respiratory diseases, the Syk inhibitor R343 (Rigel) 13 has been evaluated by inhaled route in clinical trials for asthma 14. As an alternative approach, a Syk small interfering RNA (Excellair
TM
, Zabecor) has been evaluated in phase II for asthma. Our research efforts
ip t
for the discovery of Syk inhibitors led to the discovery of a series of potent indazole derivatives with a pharmacokinetic profile suitable for inhaled administration. LAS189386
cr
was selected for in vivo characterization.
us
We hypothesize that topical Syk inhibition in the lung by inhaled route could block airway mast cells degranulation and the antigen-induced early asthmatic response (EAR). The aim of
an
this study has been to describe the in vitro and in vivo profile by intratracheal (i.t.) route of LAS189386. For comparison purposes the Syk inhibitor R343 was also tested in the in vitro
M
assays. Our data show that LAS189386 is a potent and selective Syk inhibitor with a suitable profile for inhaled administration that reduces the EAR in an ovalbumin (OVA)-challenged
d
BN rat and completely blocks airway mast cell degranulation. Our data support the Syk
Ac ce p
te
inhibition by inhaled route for the treatment of asthma.
2 Methods
2.1 Synthesis of Syk inhibitors LAS189386,
1-{2-[(1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl]pyridin-4-yl}-N-pyrazin-2-yl-
1H-indazol-3-amine, was synthesized by the department of Medicinal Chemistry of Almirall with a 99% purity determined by liquid chromatography–mass spectrometry, following the procedure described in the patent application EP2489663. Low resolution mass spectrometry (m/z): 385 (M+1)+ and 1H nuclear magnetic resonance (400 MHz, CDCl3) delta ppm: 1.84 - 2.00 (m, 2 H), 3.15 (s, 2 H), 3.32 (d, J=9.57 Hz, 1 H), 3.70 (dd, J=9.38, 1.95 Hz, 1 H), 3.88 (s, 1 H), 4.85
5
Page 5 of 34
(bs, 1 H), 6.74 (d, J=1.56 Hz, 1 H), 7.03 (dd, J=5.67, 1.76 Hz, 1 H), 7.24 - 7.31 (m, 2 H), 7.50 (bs, 1 H), 7.54 (ddd, J=8.45, 7.18, 0.98 Hz, 1 H), 7.73 (d, J=8.21 Hz, 1 H), 7.88 (d, J=8.60 Hz, 1 H), 8.16 8.29 (m, 3 H), 9.57 (s, 1 H ) confirmed the structural identity.
N4-[(2,2-difluoro-4H-benzo[1,4]oxazin-3-one)-6-yl]-5-fluoro-N2-[3-(methylamino
carbonylmethyleneoxy)phenyl]-2,4-pyrimidinediamine,
was
also
ip t
R343,
synthesized
by
the
cr
department of Medicinal Chemistry of Almirall following the procedure described in
an
us
WO2003063794.
2.2 Materials and drug preparation
M
Imject® Alum Adjuvant was purchased from Thermo Fisher Scientific (Rockford, IL), sterile saline (0.9% NaCl) from B. Braun Medical Ltd (Sheffield, UK), Dulbecco’s phosphate
d
buffered saline (PBS), OVA and methyl cellulose from Sigma-Aldrich (Ayrshire, UK) and
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Tween®80 (Polysorbat) from Merck (Hohenbrunn, Germany). Methysergide maleate was
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purchased from Tocris Bioscience (Bristol, UK), montelukast (sodium salt) from Cayman Chemical Company (Ann Arbor, MI). Ketamine chlorhydrate (Imalgene®) and xylazine hydrochloride (Rompun®) were obtained from Merial (Lyon, France) and KVP Pharma und Veterinär Produkte GmbH (Kiel, Germany), respectively. For oral administration, the suspension of montelukast (volume 2 ml/kg) was prepared in 0.5% methyl cellulose, 0.1 % Tween®80 in water. For i.t. administration, LAS189386 was prepared in 0.2 % Tween®80 in PBS and R343 was prepared in 0.2 % Tween®80, 0.5% methyl cellulose in PBS. Both Syk inhibitors were administered (volume 0.8 ml/kg) with a Liquid MicroSprayer® (model IA-1B, rat size) with the help of a small animal laryngoscope (model LS-1), both from Penn-Century Inc. (Wyndmoor, PA).
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2.3 Cell lines
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LAD2 cell line is a human mast cell line established at the National Institutes of Health (Bethesda, Maryland, US) 15. LAD2 cells were provided by Dr. Arnold Kirshenbaum
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through a biological materials license agreement. RBL-2H3 cell were obtained from the
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ATCC.
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2.4 Animals
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Care and use of animals were undertaken in compliance with the European Directive 2010/63/EU for the use of laboratory animals and was approved by the Almirall Ethics
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Committee.
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Male BN rats (200-250 g) were purchased from Charles River (Lyon, France) and housed at
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20–24 ºC, relative humidity 40-65 % with 15 air changes/h under a 12-h light/dark cycle for at least five days before use. Animals were allowed free access to standard laboratory food (Harlan Teklad 2014) and water.
2.5 Syk enzymatic assay
In order to identify Syk inhibitors that directly interact with the ATP binding pocket in the Syk catalytic domain a biochemical Syk activity assay was used. The effect of LAS189386 on Syk activity was assessed by measuring the extent of substrate phosphorylation by recombinant full-length Syk (Millipore, Dundee, UK) using a radiometric filtration assay.
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Briefly, the assay was run in 50 mM Tris-HCl (pH 7.5), 0.1 mM EGTA, 4 mM Mg(CH3COO)2, 0.1 mM Na3VO4, 0.1 % (v/v) β-mercaptoethanol, 0.133 mg/ml bovine serum albumin (BSA) in the presence of 100 µg/mL poly-GT, 1 nM Syk and different
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concentrations of Syk inhibitors (ranging 50 µM-10 pM) in 5% dimethylsulfoxide. The reaction was initiated by the addition of 0.36 µCi [γ-33P] ATP (10 mCi/mL, PerkinElmer,
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Boston, MA). After 40 min incubation at room temperature, 15 µL of the reaction was
transferred to a filter plate (Millipore, Billerica, MA) previously pre-wetted with a solution of
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75 mM phosphoric acid. The filter plate was washed three times with 200 µl phosphoric acid. Finally, 30 µL Optiphase™ Supermix (PerkinElmer) were added to each test well and
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incubated for at least 1 h before radioactivity counting in a Wallac MicroBeta®
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(PerkinElmer).
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2.6 Biochemical kinase screening panel
The selectivity of LAS189386 and R343 was assessed at 1 µM in a KinaseProfiler panel
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(Millipore) comprising 42 kinases. Assays were performed at the corresponding Km of ATP for each enzyme. Kinases were selected according to common cross-reactivity described for other Syk inhibitors (mainly tyrosine kinases such as Flt3, Lyn, Lck), kinases being most homologous to Syk (ZAP70, Pyk2, Fak) as well as a number of kinases representative of the kinome. In addition, IC50 values for kinases involved in FcRI receptor cross-linking pathway (Fyn, Lyn, Btk) and ZAP70 were also calculated in Millipore.
2.7 LAD2 cells degranulation assay
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LAD2 cells were sensitized in their normal growth medium, Complete StemPro-34 SFM, (Prepotech, Rocky Hill, NJ) by adding 100 ng/ml of biotin-labeled human IgE (US Biological, Swampscott, MA) overnight (37 ºC, 5 % CO2) in a humidified atmosphere (90 %).
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Sensitized LAD2 cells were washed, transferred to the assay plate (10,000 cells/well), preincubated with the test compounds for 30 min and then activated with 125 ng/mL of
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streptavidin (Thermo Scientific Pierce, Rockford, IL) for 30 min. After activation, supernatants were removed and transferred to another well for β-hexosaminidase
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determination. Cells were lysed in 2 freeze-thaw cycles and then incubated for 90 min at 37ºC
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with 1 mM -hexosaminidase substrate (p-nitrophenyl-N-acetyl-D-glucosamide (SigmaAldrich), in citric buffer, pH 4.5). The reaction was stopped with 0.4 M glycine (Merck)
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solution (pH 10) and absorbance was read at 405 nm.
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2.8 Syk phosphorylation (Tyr525/526) in LAD2 cells assay LAD2 cells prepared as described above were pre-incubated with the test compounds for 30
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min and then activated with 125 ng/mL of streptavidin for 1 min at room temperature. Cells were lysed and phosphorylated Syk (Tyr525/526) was analyzed in supernatants by ELISA (#7970 Cell Signaling Technology, Izasa; Barcelona, Spain).
2.9 Degranulation in RBL-2H3 cells assay RBL-2H3 cells were seeded in a 96-well plate (0.1x106 cells per well) and sensitized with 100 ng/mL mouse monoclonal anti-DNP antibody (clone SPE-7) (Sigma-Aldrich) for 90 min (37ºC, 5 % CO2). Cells were washed twice with Tyrodes buffer and pre-incubated with the test compounds for 30 min at 37 ºC. Cells were then stimulated with 100 ng/mL 9
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dinitrophenyl-BSA (Biosearch Technologies; Novato, CA) for 60 min at 37 ºC.
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β-hexosaminidase determination was performed as described above.
2.10 Human B cell activation assay
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B cells were isolated from blood of healthy volunteers by using RossetteSep® Human B Cell
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Enrichment Cocktail (StemCell Technologies; Grenoble, France). Isolated cells were adjusted to a density of 0.2x106 cells/mL in PBS with 2% fetal bovine serum (FBS) and transferred to a
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5-ml polystyrene round-bottom tube (20,000 cells per tube). Cells were pre-incubated with the test compounds for 30 min (37 ºC, 5 % CO2) and then activated with 10 µg/mL of anti-human
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IgM (Jackson ImmunoResearch; Suffolk, UK) for 2 h. After activation, cells were stained with FITC-conjugated mouse anti-human CD19 and PE-conjugated mouse anti-human CD69
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(BD Biosciences; Erembodegem, Belgium) in the dark for 15 min at room temperature and
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fixed with BD FACSTM Lysis solution for 15 min. Cells were washed and suspended with PBS, 1 % FBS and 0.1% sodium azide. Flow cytometry analysis was performed to quantify
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CD69 expression as a B cell activation marker.
2.11 in vitro ADME profiling
Plasma protein binding of LAS189386 was determined by equilibrium dialysis (Rapid Equilibrium Dialysis kit; Thermo Scientific Pierce, San José, CA). Rat plasma was spiked at 1 M and dialyzed against phosphate buffer (pH 7.4) for 4 h at 37ºC. The concentrations of LAS189386 in both dialysis chambers were quantified by LC-MS/MS. Plasma protein binding was used to estimate the unbound plasma levels in efficacy study.
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The oxidative metabolism was assessed by incubating LAS189386 (5 M) with rat liver microsomes (protein concentration 1 mg/ml) in the presence of NADPH. The reaction was quenched after 30 min and the samples were analyzed by LC-MS/MS to determine substrate
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disappearance and metabolite formation.
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2.12 Pharmacokinetic study and samples analysis
Male Wistar rats were obtained from Charles River Laboratories (Santa Perpètua de Mogoda,
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Spain). For i.t. dosing (three animals per time point), overnight-fasted rats weighing approximately 250 g were anesthetized with 4% isofluorane and placed in a supine position.
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Two hundred µL of the suspension of the test compounds were administered directly into the trachea using a Liquid MicroSprayer®. At pre-defined time points (0.01, 0.1, 1, 6, 24 h),
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blood samples were collected and centrifuged to obtain blood plasma. Lungs were harvested after perfusion with saline through the right ventricle of the heart. Plasma samples were
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d
precipitated with acetonitrile/trifluoroacetic acid, centrifuged and the supernatant analyzed by LC-MS/MS. Lungs were homogenized with methanol (4:1; w/v)) using an UltraTurrax (IKA-
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Werke, Staufen, Germany). The homogenates were then sonicated, centrifuged and the supernatants analyzed by LC-MS/MS. Chromatographic analyses were accomplished on a reversed-phase column (C18) followed by mass spectrometric detection in MRM mode.
2.13 OVA-induced early asthmatic response in BN rats and tissue mast cell degranulation BN rats were sensitized on days 0, 14 and 21 with OVA (100 µg i.p.) and Imject® Alum (20 mg i.p.). From day 28 to 32, LAS189386 was administered at 0.1, 0.3 or 1 mg/kg by i.t. route 1 h before challenge. Rats were anaesthetized (99 mg/kg ketamine chlorhydrate and 8 mg/kg xylazine hydrochloride by i.p. route) and instrumented as previously described [16]. Animals
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were challenged either with OVA (2 mg i.v.) or with control vehicle (sterile saline) and the airways resistance (cm H2O/mL/s) was measured over a 10-min period, using a FinePointe™ RC System (Buxco Research Systems, Wantage, UK). The changes in airways resistance
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were assessed using the difference between the baseline resistance (average value of 1-min period) and the peak resistance (maximum value) in the 10-min period after saline or OVA
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administration. After EAR measurement the rats were sacrificed by severing the abdominal aorta. Subsequently, the trachea, left lung, mesentery and two inter-scapular skin samples
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were carefully removed, fixed in 10 % formalin (pH 7.4), paraffin-embedded, and 3-µm cross-sections were stained with 0.1 % toluidine blue to evaluate the integrity of mast cells.
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Mast cell degranulation was assessed blindly by two independent investigators and expressed
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as percentage of degranulated mast cells both locally (trachea and left main bronchus) and systemically (mesentery and skin). Briefly, for each animal, total and degranulated mast cells
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were counted in the tracheal dorsal wall (four cross sections) and bronchial wall (one cross
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section). In the mesentery and skin, mast cell degranulation was determined by counting 200
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mast cells in randomly selected fields.
2.14 Statistical analysis
In the in vivo experiments comparisons between groups was done by one way analysis of variance (ANOVA) followed by Dunnett's post hoc test using GraphPad Prism software (GraphPad Software Inc; San Diego, California, US). Differences were considered significant when p
