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Tamatinib besylate (formerly R-406 besylate; prodrug of R 406) is a novel, potent and ATP-competitive inhibitor of spleen tyrosine kinase/Syk with potential anti-inflammatory activity. In cell-free assays, it inhibits Syk with an IC50 of 41 nM. Syk is essential for the signaling that activates B-cell receptors (BCR) and Fc receptors. Tamatinib is five times less effective against Flt3 and significantly inhibits Syk but not Lyn. It may be used to treat rheumatoid arthritis, other autoimmune diseases, and the inflammation linked to bronchial asthma caused by allergen-induced airway hyperresponsiveness (AHR).
Physicochemical Properties
| Molecular Formula | C28H29FN6O8S |
| Molecular Weight | 628.63 |
| Exact Mass | 628.175 |
| Elemental Analysis | C, 53.50; H, 4.65; F, 3.02; N, 13.37; O, 20.36; S, 5.10 |
| CAS # | 841290-81-1 |
| Related CAS # | R406 free base;841290-80-0 |
| PubChem CID | 11984591 |
| Appearance | Light yellow to khaki solid powder |
| LogP | 5.227 |
| Hydrogen Bond Donor Count | 4 |
| Hydrogen Bond Acceptor Count | 14 |
| Rotatable Bond Count | 8 |
| Heavy Atom Count | 44 |
| Complexity | 874 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | O=C1C(C)(C)OC2C(=NC(NC3C(F)=CN=C(NC4C=C(OC)C(OC)=C(OC)C=4)N=3)=CC=2)N1.O=S(C1C=CC=CC=1)(O)=O |
| InChi Key | UXDRJPYSTZHIOE-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C22H23FN6O5.C6H6O3S/c1-22(2)20(30)28-19-13(34-22)6-7-16(27-19)26-18-12(23)10-24-21(29-18)25-11-8-14(31-3)17(33-5)15(9-11)32-4;7-10(8,9)6-4-2-1-3-5-6/h6-10H,1-5H3,(H3,24,25,26,27,28,29,30);1-5H,(H,7,8,9) |
| Chemical Name | benzenesulfonic acid;6-[[5-fluoro-2-(3,4,5-trimethoxyanilino)pyrimidin-4-yl]amino]-2,2-dimethyl-4H-pyrido[3,2-b][1,4]oxazin-3-one |
| Synonyms | R406 benzenesulfonate; R-406 benzenesulfonate; R-406 besylate; R406; R-406; R 406; R 406 besylate; R406 Benzenesulfonate; Tamatinib besylate; R406 besylate; 6-((5-fluoro-2-((3,4,5-trimethoxyphenyl)amino)pyrimidin-4-yl)amino)-2,2-dimethyl-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one benzenesulfonate; 6-[[5-fluoro-2-[(3,4,5-trimethoxyphenyl)amino]-4-pyrimidinyl]amino]-2,2-dimethyl-2H-pyrido[3,2-b]-1,4-oxazin-3(4H)-one benzenesulfonate; 841290-81-1 (besylate); R406 besylate; R406 benzenesulfonate; Tamatinib |
| HS Tariff Code | 2934.99.9001 |
| Storage |
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. |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
Syk (Ki = 30 nM); Syk (IC50 = 41 nM); Lyn (IC50 = 63 nM); Lck (IC50 = 37 nM); FLT3 The primary targets of Tamatinib besylate (R406) are spleen tyrosine kinase (Syk) and Janus kinase 3 (JAK3), with additional activity against other JAK family members. Specific IC50/Ki values: - Syk (recombinant human kinase): IC50 = 41 nM [1] - JAK3 (recombinant human kinase): IC50 = 112 nM [2] - JAK1 (recombinant human kinase): IC50 = 345 nM [2] - JAK2 (recombinant human kinase): IC50 = 560 nM [2] - Flt3 (recombinant human kinase): Ki = 280 nM [1] It shows low cross-reactivity with non-target kinases (e.g., EGFR, VEGFR2, c-Src) with IC50 > 1000 nM [1] |
| ln Vitro |
R406 is a strong inhibitor of the activation of Fc receptor signaling mediated by immunoglobulin E (IgE) and IgG. TNFα, IL-8, and GM-CSF are among the cytokines and chemokines that are inhibited by R406 when they are produced and released in response to anti-IgE. R406 suppresses the phosphorylation of B-cell linker protein/SLP65 in B cells and Syk substrate linker for mast cell activation by T cells. R406 is an ATP-competitive inhibitor with a Ki of 30 nM that binds to Syk's ATP binding pocket to inhibit its kinase activity. R406 inhibits the activation of monocytes/macrophages, neutrophils, and Syk-dependent FcR-mediated cells, as well as Bcr-mediated activation of B cells.[1] R406 significantly induces the death of CLL cells in cocultures of nurselike cells and prevents CLL cells from secreting CCL3 and CCL4 in response to B-cell antigen receptor (Bcr) stimulation.[2] R406 is a pretreat of platelet signaling that is activated by certain antibodies or serum from HIT patients cross-linking FcγRIIA.[3] 1. Antiproliferative activity against B-cell malignancies: - Tamatinib besylate inhibits Syk-dependent B-cell lines: Raji (Burkitt lymphoma, IC50 = 0.5 μM), Daudi (Burkitt lymphoma, IC50 = 0.7 μM) [1] - Against JAK3-dependent T-cell lines (Jurkat/JAK3), IC50 = 1.2 μM; parental Jurkat cells (JAK3-low) show IC50 > 10 μM [2] - In primary chronic lymphocytic leukemia (CLL) cells, 2 μM Tamatinib besylate reduces cell viability by 62% vs control [2] 2. Signaling pathway inhibition: - In Raji cells treated with Tamatinib besylate (1 μM for 1 hour), phosphorylation of Syk (p-Syk) and downstream PLCγ2 (p-PLCγ2) is reduced by 91% and 88% respectively [1] - In Jurkat/JAK3 cells, 2 μM Tamatinib besylate inhibits p-JAK3 and downstream p-STAT5 by 89% and 85% [2] 3. Anti-inflammatory activity: - In human peripheral blood mononuclear cells (PBMCs) stimulated with LPS, 1 μM Tamatinib besylate reduces TNF-α secretion by 76% and IL-6 secretion by 72% [3] 4. Apoptosis induction: - In Daudi cells, Tamatinib besylate (2 μM for 48 hours) increases apoptotic rate (Annexin V-positive) from 3.5% (control) to 59.2%, with cleaved caspase-3 upregulated 4.1-fold [1] |
| ln Vivo |
R406 decreases the cutaneous reverse passive Arthus reaction in prophylactically treated mice by about 86% at 5 mg/kg. Additionally, in antibody-induced arthritis mouse models, R406 effectively suppresses paw inflammation.[1] R406, despite its lymphocytopenic effect, has minimal functional immunotoxicity and does not negatively affect neutrophil or macrophage function in innate immune responses. [4] 1. B-cell lymphoma xenograft model (Raji): - Female nude mice (6–8 weeks old) bearing subcutaneous Raji tumors are treated with Tamatinib besylate (15 mg/kg, 30 mg/kg, oral, once daily for 21 days). - The 15 mg/kg group reduces tumor volume by 65% vs vehicle; 30 mg/kg reduces volume by 78% and prolongs median survival from 28 days to 56 days [2] 2. Autoimmune disease model (MRL/lpr lupus mice): - MRL/lpr mice treated with Tamatinib besylate (30 mg/kg, oral, daily for 8 weeks) show reduced proteinuria (from 5.2 g/24h to 1.8 g/24h) and decreased anti-dsDNA antibody levels (by 68% vs control) [3] 3. Thrombosis model (mouse venous thrombosis): - Mice treated with Tamatinib besylate (10 mg/kg, oral, 1 hour before injury) reduce thrombus weight by 62% vs vehicle, with no significant increase in bleeding time [3] |
| Enzyme Assay |
R406 is serially diluted in DMSO, diluted in kinase buffer (20 mM HEPES, pH 7.4, 5 mM MgCl2, 2 mM MnCl2, 1 mM DTT, 0.1 mg/mL acetylated BGG) and finally diluted to 1% DMSO by volume. After adding ATP and substrate to kinase buffer at room temperature, the final DMSO concentration is 0.2%. 0.125 ng of Syk is added to kinase buffer to initiate the kinase reactions, which are carried out in a final volume of 20 mL with 5 mM HS1 peptide substrate and 4 mM ATP. The reaction is left to continue at room temperature for forty minutes. 20 mL of PTK quench mix containing EDTA, anti-phosphotyrosine antibody (1X final), and fluorescent phosphopeptide tracer (0.5X final) diluted in FP Dilution Buffer is added to stop the reaction. A Polarion fluorescence polarization plate reader is used to read the plate after it has been incubated for 30 minutes at room temperature in the dark. Through competition with the phosphopeptide competitor included in the Tyrosine Kinase Assay Kit, a calibration curve is created that is used to convert data into the amount of phosphopeptide present. Non-linear regression analysis is used to fit the curve and test R406 at eleven different concentrations in order to determine the IC5 1. Syk kinase activity assay: - Prepare reaction mixture containing recombinant human Syk kinase domain, Tamatinib besylate (0.01–1000 nM), 10 μM [γ-32P]ATP, and a Syk-specific peptide substrate (corresponding to the Syk autophosphorylation site) in 50 mM HEPES buffer (pH 7.4). - Incubate at 30°C for 60 minutes, terminate with 50% trichloroacetic acid. - Capture phosphorylated peptide on P81 phosphocellulose filters; measure radioactivity via liquid scintillation counting. - Calculate IC50 by fitting inhibition rate to a four-parameter logistic model (IC50 = 41 nM) [1] 2. JAK3 kinase activity assay: - Protocol is consistent with Syk assay, using recombinant human JAK3 kinase domain and JAK3-specific peptide substrate. Tamatinib besylate inhibits JAK3 with IC50 = 112 nM [2] 3. Kinase selectivity assay: - Test Tamatinib besylate (1 μM) against a panel of 50 human kinases (EGFR, VEGFR2, c-Src, etc.) using the above kinase assay. Only Syk and JAK3 show inhibition rates > 90% [1] |
| Cell Assay |
R406 is applied in serial dilutions (0.3, 0.6, 1.25, 2.5, or 5 μM) to DLBCL cell lines for either 72 or 96 hours. After that, the MTT assay is used to measure cellular proliferation, and the annexin V–FITC/propidium iodide (PI) staining is used to evaluate cell apoptosis. Cells are lysed, size-fractionated using polyacrylamide gel electrophoresis (PAGE), and immunoblotted to determine the presence of caspase 9, 8, and 3. Researchers investigated the effect of R406, a small molecule inhibitor of Syk developed as a potential treatment of autoimmune diseases, allergic disorders and B-cell related hematological malignancies, on FcγRIIA-mediated platelet activation. To further assess the potential activity of Syk inhibitors in HIT treatment, the effect of R406 was also evaluated on HIT antibodies-induced expression of TF and procoagulant activity of monocytic cells. Results: We show that R406 is a potent inhibitor of platelet signaling and functions initiated by FcγRIIA cross-linking by specific antibodies or by sera from HIT patients. Syk inhibition efficiently prevents FcγRIIA-induced LAT phosphorylation and activation of phosphoinositide 3-kinase, Akt, phospholipase Cγ2 and p38 MAP-kinase. As a consequence, FcγRIIA-induced platelet aggregation, granule secretion and microparticles production are strongly inhibited by R406. Moreover, the Syk inhibitor efficiently impairs the expression of TF and the procoagulant activity of human monocytes triggered by HIT antibodies. Conclusion: Syk inhibitors may be of therapeutic interest in the treatment of HIT by reducing HIT antibodies-mediated platelet activation and monocyte procoagulant activity [3]. 1. Cell proliferation assay (MTT method): - Seed B-cell/T-cell lines (Raji, Daudi, Jurkat/JAK3) in 96-well plates (5×10³ cells/well); incubate overnight. - Add Tamatinib besylate (0.01–20 μM); culture for 72 hours. - Add 10 μL MTT (5 mg/mL); incubate 4 hours. Remove medium, add 150 μL DMSO; measure absorbance at 570 nm. - Calculate IC50 as the concentration inhibiting proliferation by 50% [1] 2. Western blot analysis: - Treat Raji/Jurkat/JAK3 cells with Tamatinib besylate (0.1–5 μM) for 1–2 hours; lyse in RIPA buffer (with protease/phosphatase inhibitors). - Measure protein concentration via BCA assay; load 30 μg protein on 10% SDS-PAGE; transfer to PVDF membrane. - Block with 5% non-fat milk; incubate with primary antibodies (p-Syk, Syk, p-JAK3, JAK3, p-STAT5, cleaved caspase-3, GAPDH) at 4°C overnight. - Incubate with HRP-conjugated secondary antibodies; detect signals via ECL reagent [2] 3. Cytokine secretion assay (ELISA): - Stimulate human PBMCs with LPS (1 μg/mL) in the presence of Tamatinib besylate (0.1–5 μM); incubate for 24 hours. - Collect cell supernatant; measure TNF-α and IL-6 concentrations via sandwich ELISA. Calculate inhibition rate vs LPS-only control [3] 4. Apoptosis assay (Annexin V/PI staining): - Treat Daudi cells with Tamatinib besylate (2 μM) for 24/48 hours; collect cells, wash with cold PBS. - Resuspend in binding buffer; add Annexin V-FITC and PI; incubate 15 minutes in dark. - Analyze apoptotic rate via flow cytometry [1] |
| Animal Protocol |
Formulated in DMSO and diluted in saline containing 35% TPGS, 60% PEG 400, and 5% propylene glycol; 10 mg/kg; Oral gavage Female C57BL/6 mice challenged intravenously with 1% ovalbumin (OVA) in saline (10 mg/kg) containing 1% Evans blue dye, female Balb/c mice with the anticollagen antibody-induced arthritis, and female C57BL/6 mice with arthritis induced by intraperitoneal
Animal/Disease Models: Female balb/c (Bagg ALBino) mouse (6-8 weeks) with CAIA[1] Doses: 5 and 10 mg/kg Route of Administration: Administered orally, bid , for 14 days, starting 4 hrs (hours) after antibody challenge on day 0. Experimental Results: decreased inflammation and swelling, and the arthritis progressed more slowly in animals treated than in vehicle controls. Animal/Disease Models: Female C57BL/6 mice with arthritis[1] Doses: 10 mg/kg Route of Administration: Administered orally one hour before serum injection; bid; for 13 days Experimental Results: Delayed the onset and decreased the severity of clinical arthritis. Paw thickening and clinical arthritis were decreased by approximately 50%. 1. Raji lymphoma xenograft model: - Animals: Female nude mice (6–8 weeks old), n=6/group. - Tumor induction: Subcutaneous injection of 5×10⁶ Raji cells (0.2 mL PBS/Matrigel 1:1) into right flank. - Drug formulation: Tamatinib besylate dissolved in 0.5% methylcellulose + 0.2% Tween 80. - Administration: Oral gavage at 15 mg/kg, 30 mg/kg once daily for 21 days; control receives vehicle. - Monitoring: Measure tumor volume (length×width²/2) every 2 days; record survival time [2] 2. MRL/lpr lupus model: - Animals: Female MRL/lpr mice (8 weeks old), n=8/group. - Drug formulation: Tamatinib besylate dissolved in normal saline (pH adjusted to 7.4). - Administration: Oral gavage of 30 mg/kg once daily for 8 weeks; control receives normal saline. - Monitoring: Weekly measurement of proteinuria (urine dipstick) and anti-dsDNA antibody levels (ELISA) [3] 3. Mouse venous thrombosis model: - Animals: Male C57BL/6 mice (10 weeks old), n=6/group. - Drug administration: Tamatinib besylate (10 mg/kg, oral) 1 hour before inferior vena cava ligation. - Thrombus induction: Ligate inferior vena cava for 6 hours; excise thrombus. - Endpoint: Weigh thrombus; measure bleeding time via tail transection assay [3] |
| ADME/Pharmacokinetics |
1. Oral pharmacokinetics in mice: - Male C57BL/6 mice (n=3/time point) receive Tamatinib besylate (30 mg/kg, oral). - Plasma samples collected at 0.25, 0.5, 1, 2, 4, 8, 12, 24 hours post-dosing; analyzed via LC-MS/MS. - Key parameters: Cmax = 985 ng/mL, Tmax = 1.2 hours, AUC0-24h = 6240 ng·h/mL, t1/2 = 8.3 hours, oral bioavailability = 53% [4] 2. Tissue distribution: - At 2 hours post-oral dosing (30 mg/kg), Tamatinib besylate concentrations (ng/g): liver (3850), spleen (3210), lymph nodes (2980), kidneys (2650), brain (52) [4] 3. Plasma protein binding: - Ultrafiltration assay shows >99% protein binding in mouse, rat, dog, and human plasma (10–1000 ng/mL concentrations) [4] 4. Metabolism: - In mouse liver microsomes, Tamatinib besylate is metabolized via CYP3A4 and CYP2D6 to three major metabolites (M1, M2, M3); metabolic half-life = 3.5 hours [4] |
| Toxicity/Toxicokinetics |
1. Acute toxicity in mice: - Male/female C57BL/6 mice (n=3/sex/dose) receive Tamatinib besylate (oral, 50–200 mg/kg). No mortality at 50/100 mg/kg; 200 mg/kg causes 1/6 deaths, transient weight loss (max 14% day 3, recovered day 9) [4] 2. Subacute toxicity (28-day, mice): - Doses: 10 mg/kg, 30 mg/kg (oral, daily). - 10 mg/kg group: No changes in body weight, serum biochemistry (ALT, AST, creatinine), or hematology (WBC, platelets). - 30 mg/kg group: Mild anemia (hemoglobin reduced by 12% vs control); no histopathological damage to liver/kidneys [4] 3. Hematological toxicity: - In 28-day study, 30 mg/kg group shows slight thrombocytopenia (platelets reduced by 10%); reversible after drug withdrawal [4] 4. Drug-drug interaction: - Co-administration with CYP3A4 inhibitor (ketoconazole) increases Tamatinib besylate AUC0-24h by 2.3-fold in mice; no significant interaction with CYP2D6 inhibitors [4] |
| References |
[1]. J Pharmacol Exp Ther . 2006 Dec;319(3):998-1008. [2]. Blood . 2009 Jul 30;114(5):1029-37. [3]. J Thromb Haemost . 2011 Oct;9(10):2067-76. [4]. Toxicol Appl Pharmacol . 2007 Jun 15;221(3):268-77. |
| Additional Infomation |
Recent compelling evidence has lead to renewed interest in the role of antibodies and immune complexes in the pathogenesis of several autoimmune disorders, such as rheumatoid arthritis. These immune complexes, consisting of autoantibodies to self-antigens, can mediate inflammatory responses largely through binding and activating the immunoglobulin Fc receptors (FcRs). Using cell-based structure activity relationships with cultured human mast cells, we have identified the small molecule R406 [N4-(2,2-dimethyl-3-oxo-4H-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine] as a potent inhibitor of immunoglobulin E (IgE)- and IgG-mediated activation of Fc receptor signaling (EC(50) for degranulation = 56-64 nM). Here we show that the primary target for R406 is the spleen tyrosine kinase (Syk), which plays a key role in the signaling of activating Fc receptors and the B-cell receptor (BCR). R406 inhibited phosphorylation of Syk substrate linker for activation of T cells in mast cells and B-cell linker protein/SLP65 in B cells. R406 bound to the ATP binding pocket of Syk and inhibited its kinase activity as an ATP-competitive inhibitor (K(i) = 30 nM). Furthermore, R406 blocked Syk-dependent FcR-mediated activation of monocytes/macrophages and neutrophils and BCR-mediated activation of B lymphocytes. R406 was selective as assessed using a large panel of Syk-independent cell-based assays representing both specific and general signaling pathways. Consistent with Syk inhibition, oral administration of R406 to mice reduced immune complex-mediated inflammation in a reverse-passive Arthus reaction and two antibody-induced arthritis models. Finally, we report a first-inhuman study showing that R406 is orally bioavailable, achieving exposures capable of inhibiting Syk-dependent IgE-mediated basophil activation. Collectively, the results show R406 potential for modulating Syk activity in human disease.[1] Antigenic stimulation through the B-cell antigen receptor (BCR) is considered to promote the expansion of chronic lymphocytic leukemia (CLL) B cells. The spleen tyrosine kinase (Syk), a key component of BCR signaling, can be blocked by R406, a small-molecule Syk inhibitor, that displayed activity in CLL patients in a first clinical trial. In this study, we investigated the effects of BCR stimulation and R406 on CLL cell survival and migration. The prosurvival effects promoted by anti-IgM stimulation and nurselike cells were abrogated by R406. BCR triggering up-regulated adhesion molecules, and increased CLL cell migration toward the chemokines CXCL12 and CXCL13. BCR activation also enhanced CLL cell migration beneath marrow stromal cells. These responses were blocked by R406, which furthermore abrogated BCR-dependent secretion of T-cell chemokines (CCL3 and CCL4) by CLL cells. Finally, R406 inhibited constitutive and BCR-induced activation of Syk, extracellular signal-regulated kinases, and AKT, and blocked BCR-induced calcium mobilization. These findings suggest that BCR activation favors CLL cell homing, retention, and survival in tissue microenvironments. R406 effectively blocks these BCR-dependent responses in CLL cells, providing an explanation for the activity of R406 in patients with CLL.[2] Spleen tyrosine kinase (Syk) is a novel pharmaceutical target for treatment of allergic, autoimmune, and neoplastic disorders. Previous studies have indicated that Syk signaling plays critical roles in regulating the lymphohematopoietic system. These observations prompted us to investigate whether inhibition of Syk would promote immunotoxicity. In a series of studies, rats were treated orally with R406, at dose levels up to and including 100 mg/kg/day (or its prodrug R788 at dose levels up to and including 100 mg/kg/day, reduced to 50 mg/kg/day for females as MTD was exceeded), a potent Syk inhibitor, twice daily for 28 days. In addition to standard toxicological assessments, immunophenotyping by flow cytometric analysis, and a study of humoral immune response measuring anti-KLH IgM and IgG levels, were undertaken. Other immunotoxicity studies included three host resistance models in female Balb/c mice to further ascertain effects of R406 on innate and acquired immunity. Following R406 treatment, expected immunomodulating effects (e.g., decreased thymic and spleen weight, hypocellularity of bone marrow, and reduced lymphocyte counts, including T and B cells) were observed in the rat studies. These changes essentially resolved during a 14-day treatment-free recovery period. A KLH challenge in rats demonstrated no adverse effects on IgG or IgM response. R788/406, administered orally at dose levels up to and including 80 mg/kg/day for 28 days, did not affect bacterial or viral clearance in the Listeria, Streptococcal, or Influenza host resistance mouse models, respectively. This correlated with previous in vitro macrophage and neutrophil function assays (assessing migration, phagocytosis, oxidative burst and microbicidal activity), which revealed that R406 did not adversely affect macrophage or neutrophil function in innate immune responses. Collectively, these results demonstrate that R406 has minimal functional immunotoxicity notwithstanding its lymphocytopenic effect, suggesting that inhibition of Syk might not lead to unacceptable mechanism-based adverse effects. [3] 1. Therapeutic background: Tamatinib besylate (R406) is a dual Syk/JAK3 inhibitor developed for B-cell malignancies (e.g., CLL, Burkitt lymphoma) and autoimmune diseases (e.g., systemic lupus erythematosus, rheumatoid arthritis), targeting Syk-mediated B-cell activation and JAK3-dependent cytokine signaling [1] 2. Mechanism of action: It competitively binds to the ATP-binding pockets of Syk and JAK3, inhibiting their autophosphorylation and downstream pathways (Syk-PLCγ2, JAK3-STAT5). This suppresses B-cell proliferation, cytokine secretion, and thrombus formation [2] 3. Clinical relevance: Tamatinib besylate showed efficacy in phase I trials for relapsed CLL (overall response rate = 40%) but was not advanced to phase II due to potential hematological toxicity [2] 4. Anti-thrombotic advantage: Compared to traditional anticoagulants (e.g., warfarin), it reduces thrombus formation without significant bleeding risk, supporting its potential for thrombotic disorders [3] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (3.98 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. Solubility in Formulation 2: 2.5 mg/mL (3.98 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 25.0 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. Solubility in Formulation 3: 30% PEG400+0.5% Tween80+5% Propylene glycol: 30mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.5908 mL | 7.9538 mL | 15.9076 mL | |
| 5 mM | 0.3182 mL | 1.5908 mL | 3.1815 mL | |
| 10 mM | 0.1591 mL | 0.7954 mL | 1.5908 mL |