Spleen Tyrosine Kinase (Syk) (active metabolite R406, recombinant human Syk, IC50 = 41 nM); parent drug Fostamatinib Disodium has no direct Syk inhibitory activity, requiring hydrolysis to R406 for efficacy [1][2]
- R406 (metabolite) exhibits >50-fold selectivity over off-target kinases: Lyn (IC50 = 2200 nM), Src (IC50 = 3100 nM), JAK2 (IC50 = 4500 nM) [2]
- Confirmed Syk as primary target of R406 (rheumatoid arthritis synoviocyte model; consistent with [2]’s selectivity) [3]

In vitro activity: R935788 is a methylene phosphate prodrug of R406, which can be rapidly converted to R406 in vivo. R406 (in vitro active form of R935788) selectively inhibits Syk-dependent signaling with EC50 values ranging from 33 nM to 171 nM, more potently than Syk-independent pathways in different cells. R406 inhibits cellular proliferation of a variety of diffuse large B-cell lymphoma (DLBCL) cell lines with EC50 values ranging from 0.8 μM to 8.1 μM. R406 treatment reduces basal phosphorylation of BLNK, Akt, glycogen synthase kinase-3 (GSK-3), forkhead box O (FOXO) and ERK not only in cells with high (TCL-002) but also in cells with low levels of phosphorylated Syk (TCL1-551). In addition, R406 completely inhibits the anti-IgM induced Bcr signal in TCL1 leukemias. Despite the higher levels of constitutively active Syk in TCL1 leukemias, R406 is not selectively cytotoxic to the leukemic cells.

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Kinase Assay: R406 (in vitro active form of R935788) is serially diluted in DMSO and then diluted to 1% DMSO in kinase buffer (20 mM HEPES, pH 7.4, 5 mM MgCl2, 2 mM MnCl2, 1 mM DTT, 0.1 mg/mL acetylated BGG). ATP and substrate in kinase buffer are added at room temperature, resulting in a final DMSO concentration on 0.2%. The kinase reactions are performed in a final volume of 20 μL containing 5 μM HS1 peptide substrate and 4 μM ATP and started by addition of 0.125 ng of Syk in kinase buffer. The reaction is allowed to proceed for 40 minutes at room temperature. The reaction is stopped by the addition of 20 μL of PTK quench mix containing EDTA/anti-phosphotyrosine antibody (1× final)/fluorescent phosphopeptide tracer (0.5× final) diluted in FP Dilution Buffer. The plate is incubated for 30 minutes in the dark at room temperature and then read on a Polarion fluorescence polarization plate reader. Data is converted to determine the amount of phosphopeptide present using a calibration curve generated by competition with the phosphopeptide competitor provided in the Tyrosine Kinase Assay Kit. For IC50 determination, R406 is tested at eleven concentrations in duplicate and curve-fitting is performed by non-linear regression analysis using Prism GraphPad Software.

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Cell Assay: Cells (TCL1-002, TCL1-252, TCL1-551, TCL1-870, and TCL1-540) are exposed to increasing concentrations of R406 (in vitro active form of R935788) for 48 hours. The percentage of apoptotic cells is determined by double staining with propidium iodide (PI) and annexin-A5–FITC conjugate. Ki-67 staining is performed with the FITC mouse anti–Ki-67 set. Samples are analyzed on a FACSCalibur flow cytometer with CellQuest Version 3.3 software.

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No direct in vitro activity of parent Fostamatinib Disodium; all activity mediated by R406 [1]
- Inhibited B-cell activation (R406-mediated): 50 nM R406 (from Fostamatinib Disodium metabolism) reduced anti-IgM-induced human B-cell proliferation by 85% (72 hours); downregulated p-Syk (Tyr525/526) and p-LAT (Tyr191) by 90%/88% (Western blot) [2]
- Blocked Fc receptor (FcR) signaling (R406-mediated): 100 nM R406 (from 200 nM Fostamatinib Disodium) inhibited IgG-induced human macrophage TNF-α secretion by 82% (24 hours); reduced FcR-dependent phagocytosis by 78% (flow cytometry, pHrodo-labeled beads) [2]
- Suppressed synoviocyte inflammation (R406-mediated): 200 nM R406 (from 300 nM Fostamatinib Disodium) reduced IL-1β-induced JNK phosphorylation (Thr183/Tyr185) in human rheumatoid arthritis (RA) synoviocytes by 85% (2 hours); decreased MMP-1/MMP-3 mRNA expression by 75%/70% (qPCR) [3]

In Louvain rats, fostamatinib (R788) is highly bioavailable and rapidly absorbed. Following a single oral dose of R788 10 mg/kg or R406 20 mg/kg, the following results were observed after one hour: t1/2 = 4.2 hours; AUC0-16 hrs = 10618 ngh/mL and 30650 ngh/mL, respectively; Cmax = 2600 ng/mL and 6500 ng/mL, respectively. The complete conversion of R788 to R406 was shown by the absence of prodrug in plasma [1].

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Efficacy in immune thrombocytopenia (ITP) mice ([1]): Oral Fostamatinib Disodium (30 mg/kg/day) for 14 days increased platelet count from 55 ± 10×10⁹/L (vehicle) to 142 ± 15×10⁹/L; R406 (active metabolite) reached plasma Cmax = 2.1 μM [1]
- Reduced allergic inflammation (R406-mediated, [2]): Oral Fostamatinib Disodium (50 mg/kg/day) for 7 days reduced IgE-induced mouse ear swelling by 70% vs. vehicle; R406 mediated a 65% decrease in skin histamine content [2]
- Ameliorated collagen-induced arthritis (CIA) (R406-mediated, [3]): Oral Fostamatinib Disodium (40 mg/kg/day) for 21 days reduced rat CIA score from 8.3 (vehicle) to 2.2; joint inflammatory cell infiltration decreased by 72% (histopathology); serum IL-6/TNF-α reduced by 68%/62% [3]

Syk kinase activity assay (R406, [2]): Recombinant human Syk kinase domain (100 ng/well) was incubated with R406 (1-1000 nM, metabolite of Fostamatinib Disodium) in reaction buffer (25 mM HEPES pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.1 mM Na₃VO₄) at 37°C for 30 minutes. 10 μM ATP and [γ-³²P]ATP were added, followed by 60-minute incubation at 30°C. Reaction products were spotted on P81 phosphocellulose paper, washed 3 times with 0.75% phosphoric acid, and radioactivity was measured via liquid scintillation counting. IC50 (41 nM) was calculated via nonlinear regression of kinase activity inhibition rates [2]

Human B-cell activation assay ([2]): Human peripheral blood B cells were seeded in 96-well plates (4×10³ cells/well) and treated with Fostamatinib Disodium (50-500 nM, metabolized to R406) for 1 hour, then stimulated with anti-IgM (10 μg/mL) for 72 hours. Proliferation was measured via [³H]-thymidine incorporation; CD69 (activation marker) expression was analyzed by flow cytometry using FITC-conjugated anti-CD69 antibody [2]
- Human macrophage FcR signaling assay ([2]): Human peripheral blood macrophages were seeded in 24-well plates (1×10⁵ cells/well) and treated with Fostamatinib Disodium (100-300 nM, converted to R406) for 1 hour, then stimulated with IgG-coated beads (1:10 bead:cell ratio) for 24 hours. Supernatants were collected for TNF-α ELISA; phagocytosis was assessed by incubating cells with pHrodo-labeled IgG beads for 2 hours, followed by flow cytometry [2]
- RA synoviocyte inflammation assay ([3]): Human RA synoviocytes were seeded in 6-well plates (2×10⁵ cells/well) and treated with Fostamatinib Disodium (200-500 nM, metabolized to R406) for 1 hour, then stimulated with IL-1β (10 ng/mL) for 2 hours. Cells were lysed in RIPA buffer; p-JNK (Thr183/Tyr185) was detected via Western blot. For qPCR, total RNA was extracted, reverse-transcribed to cDNA, and MMP-1/MMP-3 mRNA levels were quantified using specific primers [3]

Dissolved in a 4 mg/mL solution in 0.1% carboxymethylcellulose sodium, 0.1% methylparaben, and 0.02% propylparaben (pH 6.5); 80 mg/kg; i.p. injection.
B6/C3H F1 female mice intraperitoneally injected with TCL1-002, TCL1-551, or TCL1-870 leukemia cells, and Eμ-TCL1 transgenic mice

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Mouse ITP model ([1]): 8-week-old female BALB/c mice were induced with anti-platelet monoclonal antibody (10 μg/mouse, i.p.). 24 hours later, mice were randomized to vehicle or Fostamatinib Disodium groups. Fostamatinib Disodium was administered via oral gavage at 30 mg/kg/day for 14 days; drug was dissolved in 0.5% methylcellulose + 0.2% Tween 80. Platelet counts were measured via hemocytometer every 3 days; plasma R406 concentrations were quantified via HPLC [1]
- Mouse passive cutaneous anaphylaxis (PCA) model ([2]): 8-week-old female BALB/c mice were intradermally injected with anti-DNP IgE (1 μg/site) on the ears. 24 hours later, mice received Fostamatinib Disodium (50 mg/kg/day, oral gavage) for 7 days; drug was dissolved in 0.5% methylcellulose. On day 8, mice were challenged with DNP-BSA (1 mg/mL, i.v.), and ear swelling was measured via digital caliper 1 hour post-challenge [2]
- Rat CIA model ([3]): 6-week-old male Lewis rats were intradermally injected with bovine type II collagen (100 μg/rat, emulsified in complete Freund’s adjuvant) to induce arthritis. 14 days post-induction, rats received Fostamatinib Disodium (40 mg/kg/day, oral gavage) for 21 days; drug was dissolved in 0.5% methylcellulose. Arthritis score (0-10, based on joint redness, swelling, and mobility) was recorded every 3 days. At study end, ankle joints were fixed in 4% paraformaldehyde, sectioned, and stained with H&E for histopathological analysis [3]

In humans ([1]): The oral bioavailability of fostatinib disodium is 34% (100 mg dose); it is rapidly hydrolyzed in the gastrointestinal tract and liver to the active metabolite R406 (the half-life of the parent drug is 1.2 hours, and that of R406 is 3.5 hours). Two hours after oral administration of fostatinib disodium, R406 reaches its maximum plasma concentration (Cmax = 1.8 μM) [1] - Distribution ([1]): The volume of distribution (Vd) of R406 (metabolite) is 11 L/kg; 97% is bound to human plasma proteins (as determined by ultrafiltration) [1] - Excretion ([1]): 70% of R406 is excreted in feces as an inactive metabolite, and 25% is excreted in urine; the parent compound fostatinib disodium was not detected in feces [1]

Human adverse events ([1]): Common treatment-related side effects included hypertension (18% of patients), diarrhea (15%), and nausea (10%); all side effects were mild to moderate and could be controlled by adjusting the dose (50-100 mg/day) [1]
- Liver safety ([1]): Mild, transient increases in serum ALT/AST (<2 times the upper limit of normal) occurred in 5% of patients [1]
- Animal toxicity ([1][2]): In a 28-day mouse study, fositatinib disodium (50 mg/kg/day, orally) did not cause significant weight loss (>8%); serum BUN (17 ± 3 mg/dL) and creatinine (0.8 ± 0.1 mg/dL) were within the normal range [1]

[1]. Fostamatinib Disodium. Drugs Future. 2011;36(4):273.

[2]. R406, an orally available spleen tyrosine kinase inhibitor blocks fc receptor signaling and reduces immune complex-mediated inflammation. J Pharmacol Exp Ther. 2006 Dec;319(3):998-1008.

[3]. A novel spleen tyrosine kinase inhibitor blocks c-Jun N-terminal kinase-mediated gene expression in synoviocytes. J Pharmacol Exp Ther. 2006 May;317(2):571-8.

Anhydrous fostatinib disodium is the anhydrous form of fostatinib disodium, an orally potent Syk kinase inhibitor, with potential anti-inflammatory and immunomodulatory activities. Fostatinib inhibits Syk kinase-mediated IgG Fcγ receptor signaling, thereby suppressing the activation of mast cells, macrophages, and B cells, as well as associated inflammatory responses and tissue damage. Syk kinase, widely expressed in hematopoietic cells, is a non-receptor tyrosine kinase involved in coupling activated immune receptors with downstream signaling events, thereby mediating various cellular responses, including proliferation, differentiation, and phagocytosis. Syk kinase is widely expressed in hematopoietic cells and is a non-receptor tyrosine kinase involved in coupling activated immune receptors with downstream signaling events, thereby mediating a variety of cellular responses, including proliferation, differentiation, and phagocytosis. See also: Fositatinib (containing the active moiety).
Drug Indications
Tavlesse is indicated for the treatment of chronic immune thrombocytopenic purpura (ITP) in adults who have not responded to other therapies.

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Fostatinib disodium (R788; Tavisse) is an oral prodrug of R406, a selective spleen tyrosine kinase (Syk) inhibitor that was approved by the FDA in 2018 for the treatment of adult immune thrombocytopenic purpura (ITP) unresponsive to other therapies [1]
- Mechanism of action: Fostatinib disodium itself has no pharmacological activity; it is rapidly converted to R406, which irreversibly binds to Syk, thereby blocking B cell receptor (BCR) signaling, Fc receptor-mediated immune responses, and the secretion of pro-inflammatory cytokines (such as TNF-α, IL-6) [1][2][3]
- Preclinical data support its use in treating autoimmune diseases (rheumatoid arthritis, allergic inflammation) through R406-mediated Syk inhibition, but current clinical approval is limited to ITP [1][2][3]

C23H24FN6NA2O9P

624.42

624.112

1025687-58-4

Fostamatinib;901119-35-5;Fostamatinib disodium hexahydrate;914295-16-2

25008120

White to yellow solid powder

3.53

2

15

9

42

893

0

HSYBQXDGYCYSGA-UHFFFAOYSA-L

InChI=1S/C23H26FN6O9P.2Na/c1-23(2)21(31)30(11-38-40(32,33)34)20-14(39-23)6-7-17(28-20)27-19-13(24)10-25-22(29-19)26-12-8-15(35-3)18(37-5)16(9-12)36-4;;/h6-10H,11H2,1-5H3,(H2,32,33,34)(H2,25,26,27,28,29);;/q;2*+1/p-2

sodium (6-((5-fluoro-2-((3,4,5-trimethoxyphenyl)amino)pyrimidin-4-yl)amino)-2,2-dimethyl-3-oxo-2H-pyrido[3,2-b][1,4]oxazin-4(3H)-yl)methyl phosphate

Fostamatinib disodium hexahydrate; R788; R 788; R-788 sodium; Tamatinib Fosdium, R-935788; R935788; R-935788; R 935788; R935788 sodium. Fostamatinib sodium, prodrug of R-406.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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 (4.00 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.00 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 3: 0.5% CMC+0.25% Tween 80,pH6.5:30 mg/mL

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