Syk (IC50 = 7.7 nM)

Good bidirectional permeability to Caco-2 cell monolayers is demonstrated in vitro by entospletinib (GS-9973). Entospletinib (GS-9973) is a cell-based medication that shows good selectivity for Syk and efficiently suppresses immunological complex-stimulated cytokine production in monocytes as well as BCR-mediated B cell activation and proliferation [1]. Entospletinib (GS-9973) and Idelalisib work together to suppress CLL cell viability and further impede chemokine signaling [2].

In rats and dogs, entespletinib (GS-9973) (1 mg/kg, po) has a moderate to high bioavailability. In a rat model of collagen-induced arthritis, entospletinib (GS-9973) (1–10 mg/kg, po) markedly reduced ankle inflammation. Furthermore, Entospletinib (GS-9973) showed disease-modifying activity in several histological measurements, with an ED50 ranging from 1.2 to 3.9 mg/kg[1]. These measurements included inhibition of pannus formation, cartilage damage, bone resorption, and periosteal bone formation.

Kinase Assays[1]
Full-length baculovirus-expressed Syk kinase activity was measured in a Lance-based assay format in a final volume of 25 μL containing 25 mM Tris–HCl, pH 7.5, 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2, 0.5 μM Promega PTK biotinylated peptide substrate 1, 0.01% casein, 0.01% Triton X-100, 0.25% glycerol, and 40 mM ATP (Km for ATP) incubated at room temperature for 60 min. Reactions were stopped with the addition of 30 mM EDTA containing 30 μL of SA-APC and 4 nM PT-66 antibody and the plates measured on a Perkin-Elmer Envision. IC50 values for test compounds were determined using a four-parameter linear regression algorithm.

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DiscoveRx Screen[1]
Compounds were screened at 10 μM in the KINOMEscan assay, and the results for the primary screen binding interactions are reported as “percent control”, where lower numbers indicate stronger hits in the matrix. Values of >35% are considered “no hits”. Kd determinations were assessed at DiscoveRx.

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Competitive Protein Binding Assay[1]
Human plasma and cell culture medium containing 10% fetal bovine serum (CCM) were spiked with the test compound at a final concentration of 2 μM. Spiked plasma (1 mL) and CCM (1 mL) were placed into opposite sides of the assembled dialysis cells, which were separated by a semipermeable membrane. The dialysis cells were rotated slowly in a 37 °C water bath for the time necessary to reach equilibrium. Postdialysis plasma and CCM masses were measured, and the test compound concentrations in plasma and CCM were determined with LC/MS/MS.

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Metabolic Stability[1]
Metabolic stability in vitro was determined using pooled hepatic microsomal fractions (final protein concentration of 0.5 mg/mL) at a final test compound concentration of 3 μM. The reaction was initiated by the addition of an NADPH-regenerating system. An aliquot of 25 μL of the reaction mixture was transferred at various time points to plates containing a quenching solution. The test compound concentration in the reaction mixture was determined with LC/MS/MS. Predicted clearance was calculated using the well-stirred liver model without protein restriction. Metabolic stability was also determined in cryopreserved hepatocytes using tritiated test compounds. The incubation mixture contained 1 × 106 hepatocytes/mL and 1 μM tritiated test compound (2.5 μCi). The incubation was carried out with gentle shaking at 37 °C under a humid atmosphere of 95% air/5% CO2 (v/v). Aliquots of 50 μL were removed after 0, 1, 3, and 6 h and added to 100 μL of quenching solution. The samples were analyzed on a flow scintillation radio detector coupled to an HPLC system. The metabolites were quantified on the basis of the peak areas from the radio detector with the cell-free control samples used as a reference. Metabolic stabilities in hepatocytes were determined by measuring the rate of disappearance of the test comound as ta percentage of the total peak area of the formed radiolabeled metabolites and the test compound.

Cellular Cross-Screening Activity Assays[1]
Bone marrow derived mouse mast cells (BMMCs), HUVECs, or SK-N-MCs were resuspended at (1–2) × 106 cells/wells in Tyrode’s buffer (BMMCs) or RPMI and incubated with compound dilutions for 1 h followed by stimulation with 50 ng/mL SCF (BMMCs), 50 ng/mL VEGF (HUVECs), or 100 ng/mL GDNF (SK-N-MCs). Following 3–15 min of stimulation, the cells were washed in PBS and resuspended in cell lysis buffer and the proteins resolved by SDS–PAGE. Immunodetection was evaluated for phospho-cKit in the BMMCs and normalized to total PLCγ2, phospho-KDR in HUVECs, and phospho-Ret in SK-N-MCs. Detection was enabled by the use of infared-conjugated secondary antibodies and Odyssey software.

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Jak2 Activity Assay[1]
TF1 cells were serum starved overnight in 1% fetal bovine serum (FBS) RPMI medium at 1 × 106 cells/mL. Cells were resuspended in fresh serum-free RPMI and incubated with compound dilutions for 1 h followed by stimulation with 5 units/mL erythropoietin. The cells were lysed in 50 μL of RIPA buffer, and phospho-Stat5 was detected using an MSD phospho-Stat5 quantitation plate.

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MV-4-11 Proliferation Assays[1]
Functional impact on cellular Flt3 activity was determined by measuring compound inhibition of MV-4-11 cell proliferation. A total of 104 cells were diluted in RPMI medium containing 10% FBS in 96-well flat-bottomed tissue culture plates and incubated with compound dilutions for 72 h at 37 °C. Alamar blue (10%) was added to the cells, which were incubated for an additional 12–18 h at 37 °C, and inhibition of the relative cell numbers was determined by spectrophotometer readings at 570/600 nm.

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Ramos Assay (pBLNK)[1]
Ramos cells were serum starved at 2 × 106 cells/mL in serum-free RPMI for 1 h in an upright T175 Falcon TC flask. The cells were centrifuged (1100 rpm for 5 min) and incubated at a density of 5 × 106 cells/mL in the presence of 3× serial dilutions of test compound or DMSO controls for 1 h at 37 °C. The cells were stimulated by incubation with 3 μg/mL antihuman IgM F(ab)2 for 5 min at 37 °C. The cells were pelleted and lysed in 50 μL of cell lysis buffer. Phospho-BLNK was detected using an MSD high bind plate coated for 1 h with 30 ng/well total BLNK capture antibody. Lysate was added, and the cells were washed in TBS–1% Tween-20 and probed with an antiphospho-Blnk-Y96 antibody. Inhibition of the pBLNK was quantitated versus the control well.

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Human B-Cell Proliferation[1]
Isolated human B-cells were thawed in a 37 °C water bath and rested in RPMI 1640 medium supplemented with 10% FBS, 100 units/mL penicillin–streptomycin, 0.01 M HEPES, 2 mM GlutaMAX, 5 mM sodium pyruvate, and 10 mM β-mercaptoethanol for 5 h in a 37 °C incubator with 5% CO2 and subsequently loaded with 5 μM CFSE per the manufacturer’s instructions. The cells (3 × 105 cells/200 μL per well) in a round-bottom 96-well plate were incubated with compound for 1 h in a 37 °C incubator, then stimulated with 20 μg/mL goat F(ab′)2 antihuman IgM and 20 μg/mL mouse anti-CD40, and incubated for 90 h in a 37 °C incubator. The cells were rinsed once in PBS + 4% FBS and incubated with 7AAD for 30 min on ice. The cells were pelleted at 300g for 10 min, rinsed twice, and analyzed by flow cytometry on the 7AAD– population, and proliferation was estimated on the basis of the reduction of fluoroscein staining.

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Immune-Complex Stimulation of TNFα Production[1]
Frozen human monocytes were quickly thawed in a 37 °C water bath and rested for 3 h at 37 °C in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 2 mM Glutamax, 1× sodium pyruvate, 0.1 M HEPES, 10 mM β-mercaptoethanol, and 100 units/mL penicillin–streptomycin prior to plating in 96-well plates at 1 × 105 cells/well in 100 μL of complete RPMI. The cells were incubated with compound for 1 h and stimulated with 4 μL of immune complex at 40 μg/mL (stock solution of 300 μL of a polyclonal goat F(ab′)2 antihuman Fc + 35 μL of purified human IgG + 65 μL of medium (final mass ratio of 3:1) incubated on ice for 1 h prior to use) for 16 h at at 37 °C. Culture supernatants were harvested and stored at −20 °C until they were analyzed for TNFα levels using a singleplex Meso Scale

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TNFα kit.  CD63 Whole Blood Assay[1]
Fresh human whole blood was collected in sodium heparin vacutainers. A 2 μL sample of compound in 2× serial dilutions was added to 100 μL of whole blood in a 96-well microtiter plate and incubated for 1 h at 37 °C. A 20 μL sample of antihuman IL-3 potentiation buffer B was added for 10 min at 37 °C, followed by goat antihuman IgE timulation for 20 min at 37 °C. The reaction was placed on ice to stop degranulation, and the cells were stained with 20 μL of anti-CD63-FITC/anti-CD123-PE/anti-HLA-DR-PerCEP. Red blood cells were lysed with 1.6 mL of buffer G for 10 min and protected from light, and cell pellets were harvested by centrifugation at 1300 rpm for 10 min at RT. The pellets were washed one time with 1.0 mL of wash buffer A for 5 min and recentrifuged. CD63 expression on CD123+/HLA– cells was measured by fluorescence-activated cell sorting (FACS) analysis on a Canto FACS Calibur, and the CD63 expresssion (%) versus DMSO controls was used to determine the EC50 in whole blood.

Rat Collagen-Induced Arthritis (CIA) Model[1]
Female Lewis rats from Charles River (mean mass 178 g, eight per group for collagen arthritis, four per group for normal controls) were anesthetized with isoflurane and injected with 300 μL of Freund’s incomplete adjuvant (Difco, Detroit, MI) containing 2 mg/mL bovine type II collagen (Elastin Products, Owensville, MI) at the base of the tail and two sites on the back on days 0 and 6. Oral dosing (bid at 12 h intervals) was performed on arthritic days 0–7 with vehicle (Cremophor/ethanol/saline), Entospletinib (GS9973) (1, 3, or 10 mg/kg), or the reference compound dexamethasone (Dex; 0.075 mg/kg) administered daily (qd). Rats were terminated on arthritis day 16. Efficacy evaluation was based on animal body masses, daily ankle caliper measurements, ankle diameters expressed as the area under the curve (AUC), terminal hind paw masses, and histopathologic evaluation of ankles and knees. PK was measured from plasma samples taken 0, 2, 4, 8, 12, and 24 h post last dose. The paws were fixed in formalin and processed for hemotoxylin (H) and eosin (E) microscopy. H and E sections were scored for bone resorption as follows: (0) normal; (0.5) normal on low magnification but have the earliest hint of small areas of resorption in the metaphysis with no resorption in the tarsal bones; (1) (minimal) small definite areas of resorption in distal tibial trabecular or cortical bone or in the tarsal bones, not readily apparent on low magnification, rare osteoclasts; (2) (mild) more numerous areas (<25% loss of bone in growth plate area) of resorption in distal tibial trabecular or cortical bone and tarsals apparent on low magnification, osteoclasts more numerous; (3) (moderate) obvious resorption of medullary trabecular and cortical bone without full thickness defects in both distal tibial cortices, loss of some medullary trabeculae with 26–50% loss across the growth plate and cortices, some loss in tarsal bones, lesion apparent on low magnification, osteoclasts more numerous; (4) (marked) full or nearly full thickness defects in both distal tibial cortices, often with distortion of the profile of the remaining cortical surface, marked loss of medullary bone of distal tibia (50–100% loss across the growth plate area and cortices and up to 50% loss in small tarsals if minor in tibia), numerous osteoclasts, minor to mild resorption in smaller tarsal bones; (5) (severe) full thickness defects in both distal tibial cortices with >75% loss across the growth plate and both cortices and >50% loss in tarsals, often with distortion of the profile of the remaining cortical surface, marked loss of medullary bone of distal tibia, numerous osteoclasts. Osteoclast counts (5400× fields) were performed on the ankles in the areas of greatest bone resorption. For statistical analysis, the ankle thicknesses, bone erosion scores, osteoclast counts, and c-fos expression values (mean ± SE) were analyzed for group differences using the Student’s t test. Significance was set at p < 0.05. Pharmacokinetics[1]
Pharmacokinetic studies were performed in male naive Sprague–Dawley (SD) rats, non-naive beagle dogs, and cynomolgus monkeys (three animals per dosing route) following federal and Institutional Animal Care and Use Committee (IACUC) guidelines. Intravenous (iv) administration was dosed via infusion over 30 min in a vehicle containing 5% ethanol, 20% PEG400, and 75% water (pH adjusted to 3.0 with HCl). Oral dosing was administered by gavage in a vehicle containing 5% ethanol, 45% PEG 400, and 50% 50 mM citrate buffer, pH 3. Blood samples were collected over a 24 h period postdose into Vacutainer tubes containing EDTA-K2. Plasma was isolated, and the concentration of the test compound in plasma was determined with LC/MS/MS after protein precipitation with acetonitrile. Noncompartmental pharmacokinetic analysis was performed on plasma concentration data to calculate pharmacokinetic parameters using the software program WinNonLin (version 5.0.1).

Dissolved in Cremophor/ethanol/saline;10 mg/kg; oral administration

Rat Collagen-Induced Arthritis (CIA) Model

The in vitro ADME and in vivo PK properties of Entospletinib (GS9973) are summarized in Table 5. Entospletinib (GS9973) is highly protein bound across species, with a human free fraction of 2.7%. Entospletinib (GS9973) was relatively stable in human liver microsomes (predicted Cl = 0.29 L/h/kg) but was less stable in preclinical species. In vivo, the clearance relative to hepatic blood flow was low in rat but was higher in dog, with dog having a relatively lower extent of protein binding. Entospletinib (GS9973) showed good bidirectional permeability across Caco-2 cell monolayers, indicating good absorption potential and low potential for efflux at concentrations likely to be achieved clinically. When dosed orally at 1 mg/kg in solution, Entospletinib (GS9973) showed moderate to high bioavailability in rat and dog. A comparison of the bioavailability and hepatic extraction in these species indicates that the extent of absorption from the GI tract is high (>75%). Since inhibition of metabolizing enzymes has the potential to cause clinically relevant drug–drug interactions, we evaluated the ability of Entospletinib (GS9973) to inhibit CYP1A2, 2C9, 2C19, 2D6, and 3A4. IC50 values were >10 μM in all cases. The solubility of crystalline Entospletinib (GS9973) in simulated intestinal fluid under both fed and fasted conditions was quite low (16 and 2 μM, respectively), most likely due to its high crystallinity and melting point (326 °C).[1]

[1]. Discovery of GS-9973, a Selective and Orally Efficacious Inhibitor of Spleen Tyrosine Kinase. J Med Chem. 2014 May 8;57(9):3856-73.

[2]. A potential therapeutic strategy for chronic lymphocytic leukemia by combining Idelalisib and GS-9973, a novel spleen tyrosine kinase (Syk) inhibitor. Oncotarget. 2014 Feb 28;5(4):908-15.

Entospletinib has been used in trials studying the treatment of Oncology, Follicular Lymphoma, B-cell Malignancies, Mantle Cell Lymphoma, and Non-Hodgkin Lymphoma, among others.
Entospletinib is an orally available inhibitor of spleen tyrosine kinase (Syk), with potential antineoplastic activity. Upon oral administration of entospletinib, this agent may inhibit the activity of Syk, which inhibits B-cell receptor (BCR) signaling and leads to an inhibition of tumor cell activation, migration, adhesion and proliferation. Syk, a non-receptor cytoplasmic, BCR-associated tyrosine kinase, is expressed in hematopoietic tissues and is often overexpressed in hematopoeitic malignancies.

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Drug Indication
Treatment of acute myeloid leukaemia.

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Spleen tyrosine kinase (Syk) is an attractive drug target in autoimmune, inflammatory, and oncology disease indications. The most advanced Syk inhibitor, R406, 1 (or its prodrug form fostamatinib, 2), has shown efficacy in multiple therapeutic indications, but its clinical progress has been hampered by dose-limiting adverse effects that have been attributed, at least in part, to the off-target activities of 1. It is expected that a more selective Syk inhibitor would provide a greater therapeutic window. Herein we report the discovery and optimization of a novel series of imidazo[1,2-a]pyrazine Syk inhibitors. This work culminated in the identification of GS-9973, 68, a highly selective and orally efficacious Syk inhibitor which is currently undergoing clinical evaluation for autoimmune and oncology indications.[1]

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Agents that target B-cell receptor (BCR) signaling in lymphoid malignancies including idelalisib (GS-1101) and fostamatinib which inhibit the delta isoform of PI3 kinase (PI3Kd) and spleen tyrosine kinase (Syk) respectively have shown significant clinical activity. By disrupting B-cell signaling pathways, idelalisib treatment has been associated with a dramatic lymph node response, but eradication of disease and relapse in high risk disease remain challenges. Targeting the BCR signaling pathway with simultaneous inhibition of PI3Kd and Syk has not yet been reported. We evaluated the pre-clinical activity of idelalisib combined with the novel and selective Syk inhibitor GS-9973 in primary peripheral blood and bone marrow Chronic Lymphocytic Leukemia (CLL) samples. Both PI3Kd and Syk inhibition reduced CLL survival and in combination induced synergistic growth inhibition and further disrupted chemokine signaling at nanomolar concentrations including in bone marrow derived and poor risk samples. Simultaneous targeting of these kinases may significantly increase clinical activity.[2]

C23H21N7O

411.46

411.18

C, 67.14; H, 5.14; N, 23.83; O, 3.89

1229208-44-9

1648797-46-9 (dimesylate);1229208-44-9;

59473233

White to gray solid powder

1.5±0.1 g/cm3

1.772

3.74

2

6

4

31

595

0

C, 67.14; H, 5.14; N, 23.83; O, 3.89

XSMSNFMDVXXHGJ-UHFFFAOYSA-N

InChI=1S/C23H21N7O/c1-2-17-14-25-28-20(17)13-16(1)21-15-30-8-7-24-23(30)22(27-21)26-18-3-5-19(6-4-18)29-9-11-31-12-10-29/h1-8,13-15H,9-12H2,(H,25,28)(H,26,27)

6-(1H-indazol-6-yl)-N-(4-morpholinophenyl)imidazo[1,2-a]pyrazin-8-amine

GS-9973; Entospletinib; GS9973; Entospletinib; 1229208-44-9; GS-9973; 6-(1H-Indazol-6-yl)-N-(4-morpholinophenyl)imidazo[1,2-a]pyrazin-8-amine; Entospletinib (GS-9973); Entospletinib [INN]; GS 9973;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.08 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: 4% DMSO+30% PEG 300+5% Tween 80+ddH2O:2.5mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)