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

In vitro activity: R788 is a prodrug of the spleen tyrosine kinase (Syk) inhibitor R406. R788 is a competitive inhibitor for ATP binding with a Ki of 30 nM. R788 dose-dependently inhibits anti-IgE-mediated CHMC degranulation with an EC50 of 56 nM. R788 also inhibits the anti-IgE-induced production and release of LTC4 and cytokines and chemokines, including TNFα, IL-8, and GM-CSF. Inhibition of Syk by R788 results in inhibition of all phosphorylation events downstream of Syk signaling. Next to FcϵRI signaling in CHMC, R788 most potently inhibits the signaling of IL-4 and IL-2 receptors. R788 specifically inhibits FcγR signaling in human mast cells, macrophages, and neutrophils. R788 can inhibit local inflammatory injury mediated by immune complexes. R788 induces apoptosis of the majority of examined DLBCL cell lines. In R788-sensitive DLBCL cell lines, R788 specifically inhibits both tonic- and ligand-induced BCR signaling (autophosphorylation of SYK525/526 and SYK-dependent phosphorylation of the B-cell linker protein [BLNK]).

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Kinase Assay: The fluorescence polarization reactions are performed. For Ki determination, duplicate 200-μL reactions are set up at eight different ATP concentrations from 200 μM (2-fold serial dilutions) in the presence of either DMSO or R788 at 125, 62.5, 31.25, 15.5, or 7.8 nM. At different time points, 20 μL of each reaction is removed and quenched to stop the reaction. For each concentration of R788, the rate of reaction at each concentration of ATP is determined and plotted against the ATP concentration to determine the apparent Km and Vmax (maximal rate). Finally the apparent Km (or apparent Ki/Vmax) is plotted against the inhibitor concentration to determine the Ki.

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Cell Assay: Cultured human mast cells (CHMC) are derived from cord blood CD34+ progenitor cells and grown, primed, and stimulated and shown in supplemental data. Before stimulation, cells are incubated with R788 or DMSO for 30 minutes. Cells are then stimulated with either 0.25 to 2 mg/mL anti-IgE or anti-IgG or 2 μM ionomycin. For tryptase measurement, ∼1500 cells per well are stimulated for 30 min in modified Tyrodes buffer. For LTC4 and cytokine production, 100,000 cells per well are stimulated for 1 or 7 hours, respectively. Tryptase activity is measured by luminescence readout of a peptide substrate, and LTC4 and cytokines are measured using Luminex multiplex technology.

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Inhibited B-cell activation via R406: 100 nM Fostamatinib (metabolized to R406) reduced anti-IgM-induced human B-cell proliferation by 85% (72 hours); R406 (50 nM) downregulated p-Syk (Tyr525/526) by 90% in B cells (Western blot) [1][2]
- Blocked Fc receptor (FcR)-mediated immune responses (R406 activity): Fostamatinib (200 nM, converted to R406) inhibited IgG-induced human macrophage TNF-α secretion by 82% (24 hours); R406 (100 nM) reduced FcR-dependent phagocytosis by 78% (flow cytometry) [2]
- Suppressed synoviocyte inflammation (R406 activity): Fostamatinib (300 nM, metabolized to R406) reduced IL-1β-induced JNK phosphorylation in rheumatoid arthritis (RA) synoviocytes by 85% (2 hours); R406 (200 nM) decreased MMP-1 mRNA by 75% (qPCR) [3]

In Louvain rats, fostamatinib (R788) is highly bioavailable and rapidly absorbed. AUC0–16 hours were 10618 ngh/mL and 30650 ngh/mL, respectively; Cmax=2600 ng/mL and 6500 ng/mL (1 hour observation); t1/2=4.2 hours were recorded in R406 following a single oral dosage of R788 10 mg/kg or 20 mg/kg. The absence of prodrug in plasma suggests that R788 has fully transformed into R406 [1].

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Efficacy in immune thrombocytopenia (ITP) mice ([1]): Oral Fostamatinib (30 mg/kg/day) for 14 days increased platelet count from 55 ± 10×10⁹/L (vehicle) to 142 ± 15×10⁹/L; R406 (active metabolite) detected in peripheral blood (Cmax = 2.1 μM) [1]
- Reduced allergic inflammation (R406 activity): Fostamatinib (50 mg/kg/day, oral) for 7 days (metabolized to R406) reduced IgE-induced mouse ear swelling by 70% vs. vehicle; R406 mediated histamine reduction by 65% [2]
- Ameliorated arthritis (R406 activity): Fostamatinib (40 mg/kg/day, oral) for 21 days (converted to R406) reduced rat collagen-induced arthritis (CIA) score from 8.3 (vehicle) to 2.2; R406 inhibited joint inflammatory infiltration by 72% [3]

Syk kinase activity assay (R406, [1][2]): Recombinant human Syk kinase domain (100 ng/well) was incubated with R406 (1-1000 nM, metabolite of Fostamatinib) 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 with 0.75% phosphoric acid, and radioactivity was measured via liquid scintillation counting. IC50 (41 nM) was calculated via nonlinear regression [2]

Human B-cell activation assay ([1][2]): B cells were seeded in 96-well plates (4×10³ cells/well) and treated with Fostamatinib (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; R406 (50 nM) reduced CD69 expression by 82% (flow cytometry) [2]
- Macrophage FcR signaling assay ([2]): Human macrophages were seeded in 24-well plates (1×10⁵ cells/well) and treated with Fostamatinib (100-300 nM, converted to R406) for 1 hour, then stimulated with IgG-coated beads for 24 hours. TNF-α secretion was measured via ELISA; R406 (100 nM) inhibited phagocytosis by 78% [2]
- RA synoviocyte assay ([3]): Synoviocytes were seeded in 6-well plates (2×10⁵ cells/well) and treated with Fostamatinib (200-500 nM, metabolized to R406) for 1 hour, then stimulated with IL-1β (10 ng/mL) for 2 hours. p-JNK was detected via Western blot; R406 (200 nM) decreased MMP-1 mRNA by 75% (qPCR) [3]

Dissolved in 35% TPGS, 60% PEG 400, 5% propylene glycol;1 mg/kg or 5 mg/kg; oral administration
Balb/c mice with arthritis

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Mouse ITP model ([1]): 8-week-old female BALB/c mice were induced with anti-platelet antibody. Mice received Fostamatinib (30 mg/kg/day, oral gavage) for 14 days; drug was dissolved in 0.5% methylcellulose + 0.2% Tween 80. Platelet counts were measured via hemocytometer every 3 days; R406 plasma concentrations were quantified via HPLC [1]
- Mouse PCA model ([2]): Mice were intradermally injected with anti-DNP IgE (1 μg/site). 24 hours later, mice received Fostamatinib (50 mg/kg/day, oral) for 7 days (dissolved in 0.5% methylcellulose). On day 8, mice were challenged with DNP-BSA; ear swelling was measured via caliper [2]
- Rat CIA model ([3]): Arthritis was induced with bovine type II collagen. 14 days post-induction, rats received Fostamatinib (40 mg/kg/day, oral) for 21 days (dissolved in 0.5% methylcellulose). Arthritis scores were recorded every 3 days; joint histopathology was analyzed at study end [3]

Absorption, Distribution and Excretion
Fositatinib is a methylene phosphate prodrug of its active metabolite R406. It is primarily hydrolyzed by intestinal alkaline phosphatase. Only a very small amount of fositatinib enters systemic circulation. The absolute bioavailability of R406 is 55%, with peak plasma concentrations reached in approximately 1.5 hours. Concomitant administration with a high-calorie, high-fat meal can increase exposure by 23% and peak plasma concentration by 15%. This may prolong the time to peak plasma concentration to approximately 3 hours. R406 exposure is known to be dose-dependent. A dose of 200 mg twice daily is recommended. A dose of 100-160 mg twice daily results in a 2-3 fold increase in R406 accumulation. Approximately 80% of R406 is excreted in feces, primarily as O-glucuronide conjugates and O-demethylated metabolites produced by intestinal bacteria. The remaining 20% is excreted in urine as N-glucuronide conjugates.
The apparent oral volume of distribution of R406 is approximately 400 L.
The apparent oral clearance of R406 is approximately 300 mL/min.

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Metabolism/Metabolites
Fostatinib is metabolized in the intestine by alkaline phosphatase to the active metabolite R406. R406 is further oxidized by CYP3A4 and glucuronidated by UGT1A9. Plasma metabolites include O-glucuronide conjugate, N-glucuronide conjugate, O-demethylated metabolite, and sulfate conjugate. 3,5-benzenediol metabolite is formed in feces after the O-demethylated metabolite is metabolized by intestinal bacteria.

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Biological Half-Life
The elimination half-life of R406 is approximately 15 hours.

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In humans ([1]): The oral bioavailability of fostatinib is 34% (100 mg dose); it is rapidly metabolized to active R406 (the half-life of fostatinib is 1.2 hours, and the half-life of R406 is 3.5 hours). Two hours after oral administration of fostatinib, the peak plasma concentration (Cmax) of R406 is 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 (ultrafiltration) [1]
- Excretion ([1]): 70% of R406 is excreted in feces as metabolites, and 25% is excreted in urine; maternal fostatinib was not detected in excrement [1]

Hepatotoxicity
In premarket controlled trials, 9% of subjects treated with fositatinib experienced serum transaminase levels exceeding 3 times the upper limit of normal, compared to none in the placebo group. 5% of treated subjects experienced ALT levels exceeding 5 times the upper limit of normal. These elevations were usually transient but led some patients to discontinue treatment prematurely; however, in most cases, they resolved spontaneously without dose adjustment. No clinically significant liver injury related to fositatinib was observed in premarket studies. Since fositatinib's approval and widespread use, despite its limited application, no published reports of hepatotoxicity have been received. Probability Score: E (Suspected but not confirmed as a specific cause of clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation Currently, there is no information regarding the clinical use of fositatinib during lactation. Because the active metabolite of fostatinib (R406) binds to plasma proteins at a rate as high as 98.3%, its concentration in breast milk may be low. However, the half-life of this active metabolite is 15 hours, which may allow it to accumulate in the infant. The manufacturer recommends discontinuing breastfeeding during fostatinib treatment and for at least one month after the last dose.
◉ Effects on breastfed infants
As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding R406 binds to plasma proteins at a rate of 98.3%.

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Common adverse reactions in humans ([1]): hypertension (18% of patients), diarrhea (15%), nausea (10%); these can be controlled by adjusting the dose [1]
- Liver safety ([1]): mild, transient increases in ALT/AST (<2 times the normal value) occurred in 5% of patients; no serious hepatotoxicity [1]
- In animal studies ([1][2]): fositatinib (50 mg/kg/day, 28 days) did not cause significant weight loss (>8%); serum urea nitrogen (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.

Pharmacodynamics
The active metabolite of fostatinib, R406, inhibits Fcγ receptor-mediated signal transduction, which is involved in the immune cell-mediated antibody destruction of platelets in chronic ITP. This can lead to elevated platelet counts in this population. R406 inhibits T-cell and B-cell activation, respectively, via the T-cell receptor (TCR) and B-cell receptor (BCR). It also inhibits Fcε receptor signaling, which may help treat allergy symptoms by preventing mast cell degranulation. The inhibition of the Fc receptor signaling system by R406 also inhibits dendritic cell maturation and antigen presentation, which may contribute to the efficacy of fostatinib. The inhibition of Fc receptor, TCR, and BCR signaling leads to a reduction in the production of inflammatory mediators and cytokines such as tumor necrosis factor-α, leukotriene C4, interleukin-8, and granulocyte-macrophage colony-stimulating factor. Fusattatinib can cause hypertension through off-target effects. Fusattatinib (R788; Tavalisse) is an oral prodrug of R406 (an active Syk inhibitor) and has been approved for the treatment of immune thrombocytopenic purpura (ITP) in adults[1]. Mechanism of action: Fusattatinib is rapidly hydrolyzed to R406, which irreversibly inhibits Syk, thereby blocking B cell activation, FcR-mediated immune responses and inflammatory signaling pathways (e.g., JNK/MMP)[1][2][3]. Preclinical data support the use of R406 to treat autoimmune diseases (arthritis, allergic inflammation) by inhibiting Syk; current clinical applications are mainly focused on ITP[1][2][3]. FDA approval information: Approved by the FDA in 2018 for the treatment of ITP; approval for other indications is not mentioned in specific literature[1].

C23H26FN6O9P

580.46

580.148

901119-35-5

Fostamatinib Disodium;1025687-58-4;Fostamatinib disodium hexahydrate;914295-16-2

11671467

White to off-white solid powder

1.5±0.1 g/cm3

814.2±75.0 °C at 760 mmHg

446.2±37.1 °C

0.0±3.1 mmHg at 25°C

1.629

2.12

4

15

10

40

904

0

GKDRMWXFWHEQQT-UHFFFAOYSA-N

InChI=1S/C23H26FN6O9P/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)

[6-[[5-fluoro-2-(3,4,5-trimethoxyanilino)pyrimidin-4-yl]amino]-2,2-dimethyl-3-oxopyrido[3,2-b][1,4]oxazin-4-yl]methyl dihydrogen phosphate

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: ≥ 2.08 mg/mL (3.58 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 20.8 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: 2.08 mg/mL (3.58 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (3.58 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly..

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

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Solubility in Formulation 5: 10 mg/mL (17.23 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; Need ultrasonic and warming and heat to 40°C.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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