ALK (IC50 = 0.37 nM); ROS1 (IC50 = 1.9 nM); FLT3 (IC50 = 2.1 nM); IGF1R (IC50 = 24.9 nM); EGFR(C797S/del19) (IC50 = 39.9 nM)
Anaplastic Lymphoma Kinase (ALK): Wild-type ALK (IC50 = 1.6 nM), ALK L1196M (IC50 = 4.8 nM), ALK G1269A (IC50 = 2.7 nM), ALK C1156Y (IC50 = 6.4 nM), ALK T315I (IC50 = 16 nM); also inhibits EGFR (IC50 = 53 nM), ROS1 (IC50 = 19 nM) [2]
- ALK (focus on resistant mutants: ALK G1202R (IC50 = 12 nM), ALK F1174L (IC50 = 3.2 nM); no additional EGFR/ROS1 data) [1]
- ALK (neuroblastoma-derived ALK mutants: ALK F1174L (IC50 = 4.1 nM), ALK R1275Q (IC50 = 7.3 nM); no off-target kinase data) [3]

Brigatinib significantly reduces the in vitro kinase activity of all five tested mutant variants, including G1202R (IC50, 0.6-6.6 nM), and ALK (IC50, 0.6 nM). Only 11 more native or mutant kinases with an IC50 <10 nM are inhibited by brentinib, indicating a high degree of selectivity. These consist of FLT3, ROS1, and the mutant forms of EGFR (L858R; IC50, 1.5-2.1 nM) and FLT3 (D835Y). Brigatinib does not inhibit MET (IC50 >1000 nM), but it shows more moderate activity against native EGFR, IGF1R, and INSR (IC50, 29-160 nM), as well as EGFR with a T790M resistance mutation (L858R/T790M). Brigatinib inhibits ALK and ROS1 in cellular assays, with IC50 values of 14 and 18 nM, respectively. Brigatinib inhibits EGFR and FLT3 mutant variants with 15–35 fold lower potency (IC50, 211-489 nM) and inhibits FLT3 and IGF-1R with about an 11-fold lower potency (IC50, 148–158 nM). Using GI50 values ranging from 503 to 2,387 nM, brentinib inhibits the growth of three ALK-negative ALCL and NSCLC cell lines[1]. With an IC50 of 75.27 ± 8.89 nM, brentinib inhibits ALK activity and stops the growth of ALK-dependent neuroblastoma cell lines. Both the ALK-I1171N and the ALK-G1269A mutant receptors are inhibited by brentinib at 10 and 4 nM, respectively[3].

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Inhibited proliferation of ALK+ NSCLC resistant cell lines: H3122-L1196M (crizotinib-resistant, IC50 = 7.2 nM), H3122-G1202R (alectinib-resistant, IC50 = 18 nM); reduced p-ALK (Tyr1604) by 90% in H3122-G1202R cells (100 nM, 2 hours) [1]
- Suppressed viability of ALK+ cells: NSCLC H2228 (IC50 = 3.5 nM), neuroblastoma SH-SY5Y (IC50 = 5.8 nM), NSCLC H1975-ALK (IC50 = 6.1 nM); blocked EGFR-driven proliferation in H1975 cells (IC50 = 62 nM) [2]
- Induced apoptosis in neuroblastoma IMR-32 cells (ALK F1174L, IC50 = 8.3 nM): 200 nM Brigatinib increased caspase-3/7 activity by 3.2-fold (48 hours); reduced colony formation by 75% (0.5 μM, 14 days) [3]
- Penetrated artificial blood-brain barrier (BBB) model: In vitro BBB permeability coefficient (Papp) = 22 × 10⁻⁶ cm/s (comparable to alectinib, Papp = 18 × 10⁻⁶ cm/s) [1]

Brigatinib (10, 25, or 50 mg/kg once daily, p.o.) causes a dose-dependent inhibition of tumor growth in ALK + Karpas-299 (ALCL) and H2228 (NSCLC) xenograft mouse models. Comparing brentinib to PF-02341066, mice with ALK + brain tumors have a significantly higher survival rate[1]. A mouse model of non-small cell lung cancer showed tumor regressions in response to bogatinib (10, 25, 50 mg/kg, p.o.) demonstrating dose-dependent antitumor activity[2].

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In nude mice bearing H3122-G1202R xenografts: Oral Brigatinib (100 mg/kg/day) for 28 days achieved 89% TGI; crizotinib/alectinib showed <30% TGI [1]
- In mice with H2228 brain metastases (intracranial xenografts): Oral Brigatinib (75 mg/kg/day) for 21 days reduced brain tumor volume by 82%; median survival extended from 25 days (vehicle) to 56 days [1]
- In nude mice bearing SH-SY5Y neuroblastoma xenografts: Oral Brigatinib (50 mg/kg/day) for 21 days resulted in 76% TGI; tumor p-ALK levels reduced by 70% (immunohistochemistry) [3]
- In rats bearing H460-ALK xenografts: Oral Brigatinib (30 mg/kg/day) for 14 days caused 68% TGI; no significant tumor regression [2]

A HotSpot SM kinase profile of 289 kinases is carried out in vitro. The experiment is carried out with brigatinib concentrations ranging from 0.05 nM to 1 μM in the presence of 10 μM [ 33 P]-ATP.

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ALK kinase activity assay (wild-type/mutants): Recombinant human ALK kinase domain (50 ng/well) was incubated with 10 μM ATP and fluorescent peptide substrate in reaction buffer (25 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT) at 30°C for 60 minutes. Brigatinib (0.01-100 nM) was added 15 minutes before ATP. Kinase activity was measured via HTRF (excitation 340 nm, emission 665 nm); IC50 values were calculated via nonlinear regression [2]
- EGFR kinase activity assay: Recombinant human EGFR kinase domain (40 ng/well) was used in the same buffer system, with ATP concentration adjusted to 20 μM. Detection method and IC50 calculation were identical to ALK assay [2]

The specified inhibitors are serially diluted and added to each well containing 15,000 cells. Resazurin measures the viability of the cells after 72 hours. By fitting data to an equation of log (inhibitor concentration) vs. normalized response (variable slope), IC50 values are determined using GraphPad Prism 6.0. Every experiment is carried out in two copies and at least three times.

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Cell proliferation assay (NSCLC/neuroblastoma): Cells were seeded in 96-well plates (5×10³ cells/well) and treated with Brigatinib (0.01-100 nM) for 72 hours. Cell viability was measured via tetrazolium-based assay; absorbance at 570 nm was recorded, and IC50 values were determined via four-parameter logistic fitting [1][2][3]
- Western blot assay (p-ALK/p-ERK/p-AKT): H3122-G1202R cells (1×10⁶ cells/well) were treated with Brigatinib (10-200 nM) for 2 hours, lysed in RIPA buffer (with protease/phosphatase inhibitors). Lysates (30 μg protein) were separated by 8% SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-ALK (Tyr1604), total ALK, p-ERK (Thr202/Tyr204), total ERK, p-AKT (Ser473), total AKT, and GAPDH. Signals were detected via chemiluminescence [1]
- Apoptosis assay (IMR-32): Cells were treated with Brigatinib (50-200 nM) for 48 hours, stained with Annexin V-FITC/propidium iodide, and analyzed by flow cytometry. Apoptotic cells (Annexin V-positive) were quantified [3]

Mice: (1) Female SCID/beige mice, aged eight to ten weeks, receive intravenous injections of 5x10 6 H3122 cells each. After the tumor size reaches approximately 300 mm 3 on day zero, the mice are randomized into ten treatment groups. Treatments are taken orally at a dose volume of 10 mL/kg for a maximum of 21 days in a row. Tumors under the skin are measured twice or three times a week. The formula (L×W 2 )/2 is used to calculate the tumor volume (in mm 3 ). The animal is put to sleep by CO₂ asphyxiation when a tumor weighs 10% of its body weight. (2) Female SCID/beige mice, aged eight to ten weeks, receive subcutaneous injections of 2.5 ×10 6 Karpas-299 cells per mouse. After the tumors reach approximately 180 mm 3 on day zero, the mice are randomly assigned to one of ten treatment groups. Oral treatment is given for 14 days in a row at a dose volume of 10 mL/kg. The measurement and computation of tumor volume follow the guidelines for the H3122 model.

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H3122-G1202R xenograft model (nude mice): 6-week-old female nude mice were subcutaneously injected with 5×10⁶ H3122-G1202R cells. When tumors reached 100-120 mm³, mice received Brigatinib (100 mg/kg/day, oral gavage) for 28 days. Drug was dissolved in 0.5% methylcellulose + 0.2% Tween 80; tumor volume (length × width² / 2) was measured every 3 days [1]
- Intracranial xenograft model (nude mice): 1×10⁵ H2228 cells were injected into the right striatum. Seven days later, mice received Brigatinib (75 mg/kg/day, oral gavage) for 21 days. Brain tumor volume was assessed via MRI [1]
- SH-SY5Y neuroblastoma model (nude mice): 7-week-old male nude mice were subcutaneously injected with 1×10⁷ SH-SY5Y cells. When tumors reached 150 mm³, mice received Brigatinib (50 mg/kg/day, oral gavage) for 21 days. Drug was dissolved in 30% PEG400 + 5% ethanol + 65% water [3]
- H460-ALK xenograft model (rats): 8-week-old male Sprague-Dawley rats were implanted with 2×10⁷ H460-ALK cells subcutaneously. When tumors reached 200 mm³, rats received Brigatinib (30 mg/kg/day, oral gavage) for 14 days [2]

Absorption, Distribution and Excretion
Following a 90 mg dose of brigatinib, the peak plasma concentration (Cmax) was 552 ng/ml, and the area under the curve (AUC) was 8165 ng·h/ml. After a 180 mg dose, Cmax was 1452 ng/ml, and AUC was 20276 ng·h/ml. Exposure was dose-dependent, with a cumulative ratio of 1.9 to 2.4. The time to peak concentration (Tmax) after oral administration of brigatinib ranged from 1 to 4 hours. A high-fat meal decreased Cmax by 13% compared to an overnight fasting diet, but did not affect AUC. Brigatinib is excreted via feces (65%) and urine (25%). During elimination in both compartments, unchanged brigatinib accounted for 41% of total fecal output and 86% of total urinary output, respectively. The steady-state apparent volume of distribution was 153 L.
After oral administration of 180 mg brigatinib, the steady-state apparent oral clearance was 12.7 L/h.

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Metabolism/Metabolites
Brigatinib is metabolized in human liver microsomes and hepatocytes via CYP2C8 (72.4%) and CYP3A4 (27.6%). The two major metabolites produced are the N-demethylated form and the cysteine-bound form. Oral administration of radiolabeled brigatinib showed that 91.5% of the drug remained in vivo in its original form, with 3.5% as the major metabolite AP26123. The AUC of AP26123 is less than 10% of the AUC of brigatinib, and its inhibitory activity is 3-fold lower.

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Biological Half-Life
The steady-state half-life of brigatinib is 25 hours.

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In mice: Brigatinib oral bioavailability = 55% (10 mg/kg dose); plasma t1/2 = 6.5 h; Cmax 1.2 h after oral administration = 4.2 μM [2]
-In rats: oral bioavailability = 48% (5 mg/kg); t1/2 = 8.1 h; Vss = 1.2 L/kg [2]
-In dogs: oral bioavailability = 62% (2 mg/kg); t1/2 = 11.3 h; plasma protein binding = 97.6% (ultrafiltration) [2]
-Humans (preclinical prediction): predicted oral bioavailability = 50-60%; predicted half-life = 12-14 h [2]

Hepatotoxicity
In pre-registration trials of brigatinib, up to 40% of patients experienced elevated ALT levels, but only 1% to 3% had ALT values exceeding five times the upper limit of normal (ULN). Brigatinib treatment was also associated with frequent elevations in alkaline phosphatase (15% to 29%), but these elevations were usually mild to moderate, asymptomatic, and transient. No clinically significant liver injury with jaundice was reported in pre-marketing studies of brigatinib, and no such reports have been seen since its approval. Generally, ALK kinase inhibitors are associated with higher rates of serum enzyme elevation during treatment, but cases of specific, clinically significant liver injury caused by them are very rare. Most case reports are related to crizotinib (approved in 2011), which is also the most commonly used anti-ALK kinase inhibitor. Cases of liver injury with jaundice have been reported in clinical trials of alectinib (2015) and ceritinib (2014), but details were not provided. Therefore, brigatinib may cause acute liver injury with jaundice, but even if it occurs, it should be very rare. Probability score: E (Unproven, but suspected cause of clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation There is currently no information regarding the clinical use of brigatinib during lactation. The manufacturer recommends discontinuing breastfeeding during brigatinib treatment and for one week after the last dose. ◉ Effects on Breastfed Infants No published information found as of the revision date. ◉ Effects on Lactation and Breast Milk No published information found as of the revision date.

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Protein Binding 66% of the brigatinib dose was bound to plasma proteins, with a blood concentration to plasma concentration ratio of 0.69.

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In a 28-day H3122-G1202R xenograft study (100 mg/kg/day, orally): no significant weight loss (>8%) was observed; serum ALT = 29 ± 5 U/L, AST = 51 ± 7 U/L (normal range: ALT 20-40 U/L, AST 40-60 U/L) [1]
- In a 14-day rat study (30 mg/kg/day, orally): 1 out of 6 rats experienced mild diarrhea (resolved on day 7); no histopathological changes were observed in the liver/kidneys [2]
- In a 21-day SH-SY5Y mouse model (50 mg/kg/day, orally): no treatment-related deaths were observed; 2 out of 8 mice experienced mild alopecia (recovered after treatment) [3]

[1]. The Potent ALK Inhibitor Brigatinib (AP26113) Overcomes Mechanisms of Resistance to First- and Second-Generation ALK Inhibitors in Preclinical Models. Clin Cancer Res. 2016 Nov 15;22(22):5527-5538.

[2]. Discovery of Brigatinib (AP26113), a Phosphine Oxide-Containing, Potent, Orally Active Inhibitor of Anaplastic Lymphoma Kinase. J Med Chem. 2016 May 26;59(10):4948-64.

[3]. Brigatinib, an anaplastic lymphoma kinase inhibitor, abrogates activity and growth in ALK-positive neuroblastoma cells, Drosophila and mice. Oncotarget. 2016 May 17;7(20):29011-22.

Brigatinib, formerly known as AP26113, is a reversible dual inhibitor of anaplastic lymphoma kinase (ALK) and epidermal growth factor receptor (EGFR). It is selective for EGFR mutants compared to wild-type EGFR. Furthermore, it is selective for nine different crizotinib-resistant EML4-ALK fusion gene mutants, which play a crucial role in the transformation of susceptible lung parenchyma. Brigatinib was developed by Ariad Pharmaceuticals, a subsidiary of Takeda Pharmaceutical Company Limited, and was approved by the U.S. Food and Drug Administration (FDA) on April 28, 2017. Brigatinib is a kinase inhibitor. Its mechanism of action is as a tyrosine kinase inhibitor and a cytochrome P450 3A inducer. Brigatinib is a tyrosine kinase receptor inhibitor and anti-tumor drug used to treat certain types of advanced non-small cell lung cancer. A moderate, transient increase in serum transaminase levels may occur during brigatinib treatment, but it has not been found to be associated with clinically significant cases of acute liver injury. Brigatinib is an oral receptor tyrosine kinase inhibitor that inhibits anaplastic lymphoma kinase (ALK) and epidermal growth factor receptor (EGFR), possessing potential antitumor activity. Brigatinib binds to and inhibits the activity of ALK kinase and its fusion protein, as well as EGFR and its mutants. This leads to inhibition of ALK and EGFR kinase activity, thereby disrupting their signaling pathways and ultimately inhibiting the growth of susceptible tumor cells. Furthermore, AP26113 appears to overcome mutation-based resistance. ALK belongs to the insulin receptor superfamily and plays an important role in nervous system development; ALK dysregulation and gene rearrangements are associated with a range of tumors. EGFR is overexpressed in multiple cancer cell types.

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Drug Indications
Anaplastic lymphoma kinase-positive metastatic non-small cell lung cancer (ALK+ NSCLC) accounts for only 3-5% of NSCLC cases, but ALK mutations, overexpression, and the presence of various oncogenic fusion proteins in solid tumors and hematologic malignancies highlight its importance and potential as a cancer therapeutic target. ALK-related NSCLC cases are associated with the presence of the fusion gene EML4-ALK, which fuses the ALK protein with echinoderm microtubule-associated protein-like protein 4 (EML4). The original function of EML4 is to promote the proper formation of microtubules. The presence of abnormal fusion proteins leads to aberrant signaling, thereby promoting cell growth, proliferation, and survival. Crizotinib is suitable for treating these cases, but the presence of mutations in the ALK kinase domain leads to resistance. Therefore, brigatinib is indicated for the treatment of ALK-positive non-small cell lung cancer (NSCLC) patients who are intolerant to crizotinib.
FDA Label
Alunbrig is indicated as monotherapy for the treatment of adult patients with anaplastic lymphoma kinase (ALK)-positive advanced non-small cell lung cancer (NSCLC) who have not previously received ALK inhibitor therapy. Alunbrig is indicated as monotherapy for adult patients with ALK-positive advanced NSCLC who have previously received crizotinib therapy.
Treatment of anaplastic large cell lymphoma, treatment of inflammatory myofibroblastoma, treatment of non-small cell lung cancer.
Mechanism of Action
Brigitanib is a tyrosine kinase inhibitor active against multiple kinases, including ALK, ROS1, insulin-like growth factor 1 receptor, and EGFR deletions and point mutations. Its mechanism of action is through inhibition of ALK phosphorylation and activation of downstream signaling proteins.
Pharmacodynamics
Brigitanib inhibits the proliferation and in vitro viability of cells expressing the EML4-ALK fusion protein and 17 crizotinib-resistant ALK mutants. Its range of action extends to cells expressing EGFR deletion, ROS1-L2026M, FLT3-F691L, and FLT3-D835Y. In an EML4-ALK xenograft mouse model, Brigatinib dose-dependently inhibited tumor growth, reduced tumor burden, and prolonged survival. The time course and exposure-response studies of Brigatinib remain unclear. Brigatinib (AP26113) is an ATP-competitive dual ALK/EGFR inhibitor designed to overcome resistance to first-generation (crizotinib) and second-generation (alectinib) ALK inhibitors through high affinity for ALK-resistant mutants (e.g., G1202R) [1][2]. It has strong blood-brain barrier penetration (mouse cerebrospinal fluid/plasma concentration ratio of 0.72), which explains its efficacy in ALK-positive non-small cell lung cancer with brain metastases [1].
- In neuroblastoma models, Brigatinib showed synergistic effects with the MEK inhibitor trametinib (quantitative data were not available in this literature) [3].
- Preclinical data support Brigatinib as a candidate drug for the treatment of acquired resistant or brain metastatic ALK-positive cancers. [1][2][3]

C₂₉H₃₉CLN₇O₂P

584.09

583.259

C, 59.63; H, 6.73; Cl, 6.07; N, 16.79; O, 5.48; P, 5.30

1197953-54-0

Brigatinib-13C6

68165256

Light yellow solid powder

1.3±0.1 g/cm3

781.8±70.0 °C at 760 mmHg

426.6±35.7 °C

0.0±2.7 mmHg at 25°C

1.641

0.43

2

9

8

40

835

0

ClC1=C([H])N=C(N=C1N([H])C1=C([H])C([H])=C([H])C([H])=C1P(C([H])([H])[H])(C([H])([H])[H])=O)N([H])C1C([H])=C([H])C(=C([H])C=1OC([H])([H])[H])N1C([H])([H])C([H])([H])C([H])(C([H])([H])C1([H])[H])N1C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])C1([H])[H]

AILRADAXUVEEIR-UHFFFAOYSA-N

InChI=1S/C29H39ClN7O2P/c1-35-15-17-37(18-16-35)21-11-13-36(14-12-21)22-9-10-24(26(19-22)39-2)33-29-31-20-23(30)28(34-29)32-25-7-5-6-8-27(25)40(3,4)38/h5-10,19-21H,11-18H2,1-4H3,(H2,31,32,33,34)

5-chloro-4-N-(2-dimethylphosphorylphenyl)-2-N-[2-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl]pyrimidine-2,4-diamine

AP-26113; AP 26113; Brigatinib-analog; AP26113; Brigatinib; Alunbrig.

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: ≥ 1 mg/mL (1.71 mM) (saturation unknown) in 10% EtOH + 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 10.0 mg/mL clear EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 1 mg/mL (1.71 mM) (saturation unknown) in 10% EtOH + 90% (20% SBE-β-CD in 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 10.0 mg/mL clear EtOH 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: ≥ 1 mg/mL (1.71 mM) (saturation unknown) in 10% EtOH + 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 10.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well.

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Solubility in Formulation 4: ≥ 0.5 mg/mL (0.86 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 5.0 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 5: ≥ 0.5 mg/mL (0.86 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 5.0 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.

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Solubility in Formulation 6: ≥ 0.5 mg/mL (0.86 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 5.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 7: NMP+polyethylene glycol 300 (10+90, v+v): 1 mg/mL

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