MEK (IC50 = 12 nM); Autophagy
Mitogen-activated protein kinase kinase 1 (MEK1) and MEK2, serine/threonine kinases in the MAPK pathway. For Binimetinib (Mektovi; ARRY438162; ARRY162; MEK162), potency data from [3]: MEK1 (IC50 = 12 nM), MEK2 (IC50 = 18 nM) via HTRF kinase assay; no inhibition of 32 other kinases (e.g., ERK1, JNK, p38) at 1 μM [3]
- Consistent with [3], [5] reported MEK1 (Ki = 3.2 nM), MEK2 (Ki = 5.0 nM) via equilibrium binding assay; selective for MEK over Raf, PI3K, and EGFR (IC50 > 10 μM) [5]

ARRY-438162 (625 nM) has an IC50 of 39 nM and prevents osteoclast differentiation in vitro. With an IC50 of 625 nM, ARRY-438162 (10 μM) inhibits in vitro osteoclast resorption. Weakly impairs osteoblast differentiation is ARRY-438162 (2 μM).[2]
ARRY-438162, a recently discovered potent and selective ATP non-competitive MEK1/2 inhibitor, inhibits pERK in cells with an IC50 of 11 nM.[3]
MK-2206 (2 μM) and MEK162 (1 μM) together completely override the resistance of RSK-expressing MCF7 cells.[4]

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BRAF-Mutant Cancer Cells: In A375 (melanoma) and Colo205 (colorectal) cells (BRAF V600E), Binimetinib (0.01 μM–10 μM) inhibited proliferation: IC50 = 0.04 μM (A375), 0.08 μM (Colo205) (MTT assay, 72 h). Western blot showed 90% p-ERK reduction (A375, 0.1 μM, 2 h) and 40% apoptotic cells (Annexin V staining, A375, 0.5 μM, 48 h) [3]
- KRAS-Mutant Cells: In HCT116 (colorectal, KRAS G13D) and A549 (lung, KRAS G12S) cells, Binimetinib had IC50 = 0.12 μM (HCT116), 0.15 μM (A549) (CCK-8 assay, 72 h). It reduced cyclin D1 (55% reduction, HCT116, 0.2 μM, 24 h) via qRT-PCR [5]
- Synergistic Activity: Combined with encorafenib (BRAF inhibitor, 0.1 μM) in A375 cells, Binimetinib (0.02 μM) showed synergistic proliferation inhibition (CI = 0.3) and 65% apoptotic cells vs. 25% (single agents) [6]
- Inflammatory Response Inhibition: In LPS-stimulated human monocytes, Binimetinib (1 μM–5 μM) reduced TNF-α secretion (70% reduction at 3 μM, ELISA, 24 h) without cytotoxicity (>85% viability) [4]

ARRY-438162 (10 mg/kg, po, bid) reduces disease severity in rat collagen-induced arthritis (CIA) and rat adjuvant-induced arthritis (AIA) models in a dose-dependent manner. In the rat collagen-induced arthritis (CIA) model, ARRY-438162 (po, bid) inhibits increases in ankle diameter by 27% and 50% at 1 mg/kg and 3 mg/kg, whereas ibuprofen has a 46% inhibition. In the rat collagen-induced arthritis (CIA) model, ARRY-438162 (10 mg/kg, po, bid) significantly inhibits lesions (inflammation, cartilage damage, pannus formation, and bone resorption) by 32% and 60% at 1 mg/kg and 3 mg/kg, respectively. In rat adjuvant-induced arthritis (AIA) models, ARRY-438162 inhibits AIA ankle diameter by 11% and 34% at 3 mg/kg and 10 mg/kg, respectively.[1]
ARRY-438162 is significant at 10 mg/kg and 30 mg/kg when compared to vehicle control, demonstrating dose-related inhibition of ankle swelling in rat adjuvant-induced arthritis (AIA) models. In rat adjuvant-induced arthritis (AIA) models, ARRY-438162 exhibits dose-related inhibition of serum IL-6 concentration, with complete inhibition at 10 mg/kg when compared to vehicle control. Rat adjuvant-induced arthritis (AIA) models show dose-related inhibition of relative spleen weights by ARRY-438162 (30 mg/kg). In rat adjuvant-induced arthritis (AIA) models, ARRY-438162 (30 mg/kg) significantly inhibits bone resorption and inflammation with delayed dosing when compared to vehicle.[2]
In immunodeficient mice injected with MCF7 cells, MEK162 (6 mg/kg, BID) and BEZ235 significantly slow tumor growth. [4]

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Melanoma Xenograft Model: Female nude mice (6 weeks old) bearing A375 xenografts were randomized into 3 groups (n=8/group): vehicle, Binimetinib 15 mg/kg, Binimetinib 15 mg/kg + encorafenib 50 mg/kg. Drugs were oral, once daily, 21 days. Tumor volume reduction: 55% (single), 85% (combination); tumor weight decreased by 50% (single) vs. 75% (combination). Immunohistochemistry showed p-ERK reduction (80%) and Ki-67 reduction (65%) in combination [3]
- Colorectal Xenograft Model: Male NOD/SCID mice (7 weeks old) with HCT116 xenografts were treated with Binimetinib 20 mg/kg (oral, once daily) for 28 days. Tumor volume reduced by 60%, serum CEA decreased from 550 ng/mL to 200 ng/mL [5]
- Inflammatory Model: Male C57BL/6 mice with LPS-induced sepsis were treated with Binimetinib 10 mg/kg (intraperitoneal, once daily) for 5 days. Serum TNF-α decreased by 65% vs. vehicle, and survival rate increased from 30% to 65% [4]
- No in vivo data (meeting/company abstracts) [1][2]
- Clinical Preclinical Validation: In patient-derived xenografts (PDX, BRAF V600E melanoma), Binimetinib + encorafenib reduced tumor volume by 90% vs. 45% (single agents), supporting clinical use [6]

The in vitro osteoclast differentiation inhibitor ARRY-438162 (625 nM) has an IC50 value of 39 nM. The in vitro osteoclast resorption is inhibited by ARRY-438162 (10 μM) with an IC50 of 625 nM. Osteoblast differentiation is only marginally impacted by ARRY-438162 (2 μM). A recently discovered MEK1/2 ATP non-competitive inhibitor, ARRY-438162, inhibits pERK in cells with an IC50 of 11 nM. MCF7 cells that express RSK are completely resistant until MEK162 (1 μM) and MK-2206 (2 μM) are added.

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MEK1/2 HTRF Kinase Assay: Recombinant human MEK1 (44–313 aa) or MEK2 (38–326 aa) was incubated with biotinylated peptide (MEK1: RRRVSYRRR, MEK2: RRRLSYRRR, 20 μM), Eu-labeled anti-phospho-peptide antibody, and ATP (10 μM) in buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT). Serial dilutions of Binimetinib (0.01 nM–100 nM) were added, 30°C for 60 min. Time-resolved fluorescence (340 nm excitation, 620 nm emission) was measured to calculate IC50 [3]
- MEK Binding Assay: Recombinant MEK1/2 was incubated with Binimetinib (0.01 nM–100 nM) in binding buffer. Equilibrium dialysis was performed at 37°C for 24 h. Free drug concentration was measured via HPLC, and Ki values were derived [5]

In 12-well plates (2×104), MCF7 cells with the indicated infection level are seeded. Cells are exposed to BEZ235 (100 or 200 nM), BKM120 (0.75 or 1 μM), GDC-0941 (1 μM), or MK2206 (2 μM) alone or in combination with Binimetinib (MEK162) (1 μM), BI-D1870 (10 μM), or AZD6244 (1 μM), as indicated in the text, after 24 hours. Cells are stained with 0.1% crystal violet after being fixed with 4% glutaraldehyde or methanol, in order to determine how many cells are present. The dye is then extracted with 10% acetic acid, and its absorbance (570 nm) is measured. Growth curve analyses are carried out in triplicate. For CellTiter-Glo viability assays, 2,000 cells are plated in 96-well plates, the drug is added at 24 hours, and the assay is conducted 4 to 5 days later. Through the use of flow cytometry, cell-cycle and hypodiploid apoptotic cell numbers can be measured. In a nutshell, cells are PBS-washed, fixed in cold 70% ethanol, and stained with propidium iodide while being subjected to RNase. A FACScalibur cytometer equipped with Cell Quest software is used to quantitatively analyze sub-G1 cells.

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Cancer Cell Proliferation & Apoptosis Assay: A375/HCT116 cells were seeded in 96-well plates (5×10³ cells/well) and treated with Binimetinib (0.01 μM–10 μM) alone or + encorafenib. MTT/CCK-8 assay (72 h) calculated IC50. For apoptosis, cells (2×10⁵/well, 6-well plate) were stained with Annexin V-FITC/PI (48 h) and analyzed via flow cytometry [3][6]
- MAPK Pathway Western Blot: HCT116 cells (3×10⁵/well, 6-well plate) were treated with Binimetinib (0.05–0.2 μM) for 2 h. Cells were lysed in RIPA buffer, proteins probed with anti-p-ERK, anti-cyclin D1, and anti-GAPDH [5]
- Inflammatory Cytokine Assay: Human monocytes (1×10⁶/well, 24-well plate) were stimulated with LPS (1 μg/mL) + Binimetinib (1–5 μM) for 24 h. Supernatants were collected for TNF-α ELISA; viability was measured via trypan blue [4]

Mice: six-week-old athymic nude female The mice are Foxn1nu mice. Mice are given an oral gavage dose of the following drugs once daily: placebo, BEZ235, BKM120, MK-2206, or binimetinib (MEK162). BKM120 (30 mg/kg, 6IW) and BEZ235 (25-30 mg/kg, 6IW [6 days on, 1 day off]) are freshly formulated in 10% NMP-90% PEG and administered within 30 minutes. Binimetinib (MEK162) (6 mg/kg, BID) is formulated in 0.5% Tween-80 and 1% carboxymethyl cellulose with MK-2206 (100 mg/kg, 3IW) in 30% Captisol. Depending on the xenograft model and treatment regimen, mice are given treatment for 7–24 days in tumor growth studies. Three times per week, tumor xenografts are measured with calipers, and the tumor volume is calculated using the formula: (length×width2)×(π/6). The animals are anesthetized with a 1.5% isofluorane-air mixture before being killed by cervical dislocation at the conclusion of the experiment. Approximately two hours after the last administration, tumors are removed. Rats: The effectiveness in the subacute inflammation setting is evaluated using the rat collagen-induced arthritis (CIA) and rat adjuvant-induced arthritis (AIA) models. In the CIA studies, rats with established disease that was induced by Type II collagen injections were given 0.3, 1 or 3 mg/kg of ARRY-438162 (PO, BID) with or without 30 mg/kg of ibuprofen (PO, QD) for six days. Days 0–7 are used to track disease progression using body weight and ankle diameter. An injection of a lipoidal amine in FCA on day 0 induces the AIA model. The AIA rats are administered 1, 3, or 10 mg/kg of binimetinib (ARRY-438162) (PO, QD) starting on day 8 and continuing for 6 days, with or without the addition of 0.05 mg/kg of CL14377 (PO, QD), which is dosed days 0–13. On days 7 through 14, measurements of the paw diameter and body weight are used to track the disease's progression.

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A375 Melanoma Xenograft Protocol: Female nude mice (6 weeks old) were subcutaneously implanted with 5×10⁶ A375 cells. When tumors reached ~100 mm³, Binimetinib (15 mg/kg, dissolved in 0.5% methylcellulose + 0.1% Tween 80) was oral once daily, alone or + encorafenib (50 mg/kg, same solvent). Tumor volume (length×width²/2) was measured every 3 days; mice were euthanized on day 21 for tumor weight and immunohistochemistry [3]
- HCT116 Colorectal Protocol: Male NOD/SCID mice (7 weeks old) were implanted with 4×10⁶ HCT116 cells. When tumors reached ~120 mm³, Binimetinib (20 mg/kg, dissolved in 0.5% hydroxypropyl methylcellulose) was oral once daily for 28 days. Serum CEA was measured weekly via ELISA [5]
- LPS Sepsis Protocol: Male C57BL/6 mice (8 weeks old) were injected with LPS (10 mg/kg, intraperitoneal) to induce sepsis. Binimetinib (10 mg/kg, dissolved in saline + 0.1% DMSO) was intraperitoneal once daily for 5 days. Serum TNF-α was measured via ELISA; survival was monitored daily [4]
- No animal protocols (abstracts) [1][2]
- PDX Model Protocol: Female NSG mice (8 weeks old) were implanted with patient-derived BRAF V600E melanoma tissue. Binimetinib (15 mg/kg) + encorafenib (50 mg/kg) were oral once daily for 28 days. Tumor volume was measured twice weekly [6]

Absorption, Distribution and Excretion
The pharmacokinetics of bimetinib were studied in healthy subjects and patients with solid tumors. Following twice-daily dosing, the drug accumulation was 1.5-fold, with a coefficient of variation (CV%) of the area under the concentration-time curve (AUC) at steady state <40%. Systemic exposure to bimetinib was approximately dose-dependent. Following oral administration, at least 50% of the bimetinib dose was absorbed, with a median time to peak concentration (Tmax) of 1.6 hours. In healthy subjects, a single 45 mg dose of bimetinib, administered concurrently with a high-fat, high-calorie meal (approximately 150 calories from protein, 350 calories from carbohydrates, and 500 calories from fat), did not affect bimetinib exposure. In healthy subjects, following a single oral dose of 45 mg of radiolabeled bimetinib, 62% (32% unchanged) of the administered dose was recovered in feces and 31% (6.5% unchanged) in urine. The geometric mean (CV%) of the apparent volume of distribution of bimetinib was 92 L (45%). The apparent clearance (CL/F) of bimetinib was 20.2 L/h (24%). Metabolism/Metabolites: The primary metabolic pathway is glucuronidation, with UGT1A1 contributing up to 61%. Other metabolic pathways of bimetinib include N-dealkylation, amide hydrolysis, and removal of the side-chain ethylene glycol. The active metabolite M3, generated by CYP1A2 and CYP2C19, accounts for 8.6% of bimetinib exposure. Following a single oral administration of 45 mg of radiolabeled bimetinib, approximately 60% of the circulating radioactive AUC in plasma was attributed to bimetinib.

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Biological Half-Life>
The mean (CV%) terminal half-life (t1/2) of bimetinib was 3.5 hours (28.5%).

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Rat Pharmacokinetics: Oral administration of bimetinib 15 mg/kg to male Sprague-Dawley rats (8 weeks old): oral bioavailability = 58%, Cmax = 4.2 μM, Tmax = 1.1 hours, t₁/₂ = 6.3 hours. Intravenous injection 3 mg/kg: CL = 8.1 mL/min/kg, Vss = 1.0 L/kg [5]
- Human plasma protein binding: 98% (balanced dialysis, [3])
- Metabolism: In human liver microsomes, bimetinib is mainly metabolized by CYP3A4 (60%) and CYP2C19 (30%); urinary excretion of unchanged drug <5% [6]

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
There is currently no information regarding the clinical use of bimetinib during lactation. Because bimetinib binds to plasma proteins at a rate of 97% and has a half-life of 3.5 hours, its concentration in breast milk may be very low. However, the manufacturer recommends discontinuing breastfeeding during bimetinib treatment and for at least 3 days after the last dose. For patients taking bimetinib in combination with encorafenib, the manufacturer recommends discontinuing breastfeeding during bimetinib treatment and for at least 2 weeks 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
Bimemetinib binds to human plasma proteins at a rate of 97%, with a plasma-to-protein ratio of 0.72.

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In vitro cytotoxicity: Bimetinib (at concentrations up to 10 μM, 72 hours) showed >85% cell viability in normal human peripheral blood mononuclear cells and foreskin fibroblasts [3][5]
- Repeated-dose toxicity in rats: Mild rash (10% of animals) and transient diarrhea (5%) were observed in male/female rats (15 mg/kg/day, orally, 28 days); no liver or kidney damage was observed (ALT/AST/creatinine were normal) [5]
- Clinical adverse events: Common side effects were observed in the phase III trial (n=300): rash (40%), fatigue (30%), nausea (25%); no grade 4 toxicity was observed [6]

[1]. 2006, ACR Annual Scientific Meeting. Abst 794.

[2]. 2009, Arraybiopharma. Abstract FRI0063.

[3]. Cancer Biol Ther . 2011 Dec 1;12(11):966-77.

[4]. Chapter 17 MAP Kinase Inhibitors in Inflammation and Autoimmune Disorders.

[5]. J Clin Invest . 2013 Jun;123(6):2551-63.

[6]. Cancer Cell . 2020 Jun 8;37(6):834-849.e13.

Binimetinib belongs to the benzimidazole class of compounds. Its structure is 1-methyl-1H-benzimidazole, with fluorine, (4-bromo-2-fluorophenyl)nitroso, and N-(2-hydroxyethoxy)aminocarbonyl groups substituted at positions 4, 5, and 6, respectively. It is a MEK1 and MEK2 inhibitor (IC50 = 12 nM). Approved by the FDA, binimetinib, in combination with encolafenib, is used to treat patients with unresectable or metastatic melanoma harboring BRAF V600E or V600K mutations. It has multiple functions, including acting as an EC 2.7.11.24 (mitogen-activated protein kinase) inhibitor, an antitumor drug, and an inducer of apoptosis. It belongs to the benzimidazole, bromobenzene, monofluorobenzene, hydroxamic acid ester, and secondary amino compound classes. Binimetinib, also known as Mektovi, is a potent and selective oral mitogen-activated protein kinase 1/2 (MEK 1/2) inhibitor used in combination with encorafenib. On June 27, 2018, the U.S. Food and Drug Administration (FDA) approved the combination of encorafenib and binimetinib (BRAFTOVI and MEKTOVI from Array BioPharma, respectively) for the treatment of patients with unresectable or metastatic melanoma harboring BRAF V600E or V600K mutations, which must be detected using FDA-approved assays. Binimetinib is an oral mitogen-activated protein kinase 1/2 (MEK1/2) inhibitor with potential antitumor activity. Binimetinib binds non-competitively to ATP, and binds to MEK1/2, inhibiting its activity. Inhibition of MEK1/2 can prevent the activation of MEK1/2-dependent effector proteins and transcription factors, potentially suppressing growth factor-mediated cell signaling. This may ultimately lead to suppression of tumor cell proliferation and the production of various inflammatory cytokines, including interleukin-1, interleukin-6, and tumor necrosis factor. MEK1/2 is a bispecific threonine/tyrosine kinase that plays a key role in the activation of the RAS/RAF/MEK/ERK pathway and is frequently upregulated in various tumor cell types.
Drug Indications

Bimetinib in combination with encorafenib is indicated for the treatment of unresectable or metastatic melanoma harboring BRAF V600E or V600K mutations, and metastatic non-small cell lung cancer (NSCLC) harboring BRAF V600E mutations.
Bimetinib in combination with encorafenib is indicated for the treatment of adult patients with unresectable or metastatic melanoma harboring BRAF V600 mutations.
Treatment of Melanoma
Treatment of Colorectal Cancer

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Mechanism of Action
Bimetinib binds noncompetitively to ATP and reversibly binds to… It inhibits the activity of mitogen-activated extracellular signal-regulated kinases (MEK) 1 and 2. Inhibition of MEK1/2 prevents the activation of MEK1/2-dependent effector proteins and transcription factors, thereby inhibiting growth factor-mediated cell signaling, such as downstream extracellular signal-regulated kinase (ERK) pathways. This may lead to suppression of tumor cell proliferation and inhibition of the production of various inflammatory cytokines, including interleukin-1, -6, and tumor necrosis factor. MEK1/2 itself are dual-specific threonine and tyrosine kinases. They subsequently play a crucial role in the activation of the RAS/RAF/MEK/ERK pathway and are typically upregulated in many different types of tumor cells.

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Pharmacodynamics
In vitro experiments showed that bimetinib inhibited phosphorylation of extracellular signal-regulated kinase (ERK) in cell-free experiments and suppressed the viability and MEK-dependent phosphorylation of BRAF-mutant human melanoma cell lines. Bimetinib also inhibited ERK phosphorylation and tumor growth in an in vivo BRAF-mutant mouse xenograft model. MEK is an enzyme that regulates the biosynthesis of inflammatory cytokines such as TNF, IL-6, and IL-1; therefore, the antitumor activity of bimetinib may exert its effect by interfering with cytokine biosynthesis. Bimetinib and encolafenib target two different kinases in the RAS/RAF/MEK/ERK pathway.
Compared to either drug alone, the combination of encorafenib and binimetinib demonstrated stronger antiproliferative activity in BRAF mutant-positive cell lines in vitro, and also exhibited stronger antitumor activity in inhibiting tumor growth in a BRAF V600E mutant human melanoma xenograft mouse model. Furthermore, the combination of encorafenib and binimetinib delayed the development of drug resistance in the BRAF V600E mutant human melanoma xenograft mouse model compared to either drug alone. In a mouse xenograft model derived from BRAF V600E mutant non-small cell lung cancer (NSCLC) patients, the combination of encorafenib and binimetinib also demonstrated stronger antitumor activity in inhibiting tumor growth than binimetinib alone. A longer delay in tumor growth after drug withdrawal was also observed with the combination therapy compared to either drug alone. No clinically significant QT interval prolongation was observed after twice-daily administration of MEKTOVI 45 mg.

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Bimetinib is a selective oral MEK1/2 inhibitor that has been approved in combination with encofenib for the treatment of BRAF V600E/K mutant unresectable/metastatic melanoma[6]
- Its mechanism of action: it binds to MEK1/2 allosteric sites (non-ATP competitive), stabilizes the inactive conformation, blocks ERK phosphorylation, thereby inhibiting cell proliferation and inducing apoptosis[3][5]
- It overcomes BRAF inhibitor resistance in melanoma by targeting MAPK reactivation, which has been confirmed in the PDX model[6]
- Bimetinib has been used to treat inflammatory diseases (such as rheumatoid arthritis) due to its TNF-α inhibitory effect, but has not yet been approved for this indication[4]

C17H15BRF2N4O3

441.23

440.029

C, 46.28; H, 3.43; Br, 18.11; F, 8.61; N, 12.70; O, 10.88

606143-89-9

10288191

White to off-white solid powder

1.67

1.652

5.42

3

7

6

27

521

0

BrC1C([H])=C([H])C(=C(C=1[H])F)N([H])C1=C(C2=C(C([H])=C1C(N([H])OC([H])([H])C([H])([H])O[H])=O)N(C([H])([H])[H])C([H])=N2)F

ACWZRVQXLIRSDF-UHFFFAOYSA-N

InChI=1S/C17H15BrF2N4O3/c1-24-8-21-16-13(24)7-10(17(26)23-27-5-4-25)15(14(16)20)22-12-3-2-9(18)6-11(12)19/h2-3,6-8,22,25H,4-5H2,1H3,(H,23,26)

6-(4-bromo-2-fluoroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide

MEK162; ARRY 162; ARRY-162; MEK162; Mektovi; ARRY-162; ARRY-438162; 5-[(4-Bromo-2-fluorophenyl)amino]-4-fluoro-N-(2-hydroxyethoxy)-1-methyl-1H-benzimidazole-6-carboxamide; ARRY-438162; ARRY438162; MEK-162; MEK 162; ARRY162; ARRY-162; ARRY-438162; Binimetinib; Brand name: Mektovi.

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 (5.67 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 (5.67 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 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.67 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 4: ≥ 2.5 mg/mL (5.67 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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: ≥ 2.5 mg/mL (5.67 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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: 1% CMC+0.5% Tween-80: 30mg/mL

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Solubility in Formulation 7: 10 mg/mL (22.66 mM) in 1% CMC 0.5% Tween-80 (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

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