Chk1 (Ki = 0.9 nM); Chk1 (IC50 <1 nM); Chk2 (IC50 = 8 nM)
Prexasertib 2HCl (LY-2606368) is a highly selective inhibitor of checkpoint kinase 1 (CHK1), with an IC50 of ~1.2 nM for recombinant human CHK1 kinase activity (measured by radiometric kinase assay) [2]
; - It exhibits extreme selectivity over other PI3K-like kinases and cell cycle kinases: IC50 > 1000 nM for CHK2, > 1000 nM for ATR, > 1000 nM for ATM, > 1000 nM for mTOR, and > 1000 nM for CDK2 [2][3]
;

Prexasertib (also know LY2606368) is a novel, potent, selective and ATP-competitive inhibitor of the protein kinase CHK1 (checkpoint kinase 1) with IC50 values of less than 1 nM for CHK1 and 8 nM for CHK2, respectively. The multifunctional protein kinase CHK1 is essential for the regulation of the number of active replication forks in cells as well as the response of the cells to damage to DNA. Because CHK1 establishes DNA damage checkpoints in the cell cycle, CHK1 inhibitors are currently being studied as chemopotentiating agents. When taken by itself, prexasertib breaks double-stranded DNA and eliminates the DNA damage checkpoints' defenses. Prexasertib works by inhibiting CHK1, which raises CDC25A activation of CDK2, increasing the number of replication forks while decreasing their stability. TUNEL and pH2AX-positive double-stranded DNA breaks quickly manifest in the S-phase cell population following Prexasertib treatment. Ex vivo tumor models demonstrate comparable responses to ixasertib, including marked inhibition of tumor growth. In summary, Prexasertib is a powerful example of a new class of cancer treatment medications that works by causing a replication catastrophe.

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Antiproliferative activity in solid tumor cell lines: - HCT116 (colon cancer, p53-wt): Prexasertib 2HCl (0.1–100 nM) inhibited proliferation with an IC50 of ~35 nM (72 h MTT assay); 50 nM treatment reduced colony formation by ~50% (48 h clonogenic assay) [2]
; - MCF-7 (breast cancer, ER+): IC50 of ~42 nM (72 h MTT assay); combination with 2 Gy ionizing radiation (IR) decreased IC50 to ~12 nM (radiosensitization) [2]
; - HT-29 (colon cancer, p53-mut): IC50 of ~58 nM (72 h CellTiter-Glo assay); 100 nM treatment increased γH2AX levels by ~4-fold (24 h Western blot) [3]
; - CHK1 pathway inhibition and mechanism: - HCT116 cells (50 nM Prexasertib 2HCl, 4 h): Western blot showed ~90% reduction in p-CHK1 (Ser296) and ~80% reduction in p-CDC25C (Ser216, downstream CHK1 substrate) [2]
; - EdU incorporation assay (HCT116, 50 nM, 24 h): ~3-fold increase in stalled replication forks vs. vehicle [2]
; - Annexin V-FITC/PI staining (SGC7901 gastric cancer, 50 nM + 100 nM BMN673, 48 h): apoptotic rate of ~55% vs. ~15% with single-agent Prexasertib 2HCl [3]
;

Prexasertib (LY2606368), when used both alone and in conjunction with other agents, inhibited the growth of tumors in cancer xenografts. LY2606368 was found to suppress the growth of primary tumors and significantly lower the incidence of metastases and ascites accumulation in an orthotopic SKOV3 ovarian cancer model. Additionally, LY2606368 showed promise in an orthotopic pancreatic cancer model based on SW1990, leading to a 92% reduction in the growth of the primary tumor and the removal of metastases to the intestine, spleen, and lymph node.

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Animal xenograft models (文献[2][3]): - HCT116 subcutaneous xenografts (female nude mice, 6–8 weeks old): Prexasertib 2HCl (10 mg/kg, i.p., once daily for 14 days) reduced tumor volume by ~65% and tumor weight by ~60% vs. vehicle; IHC showed ~80% reduction in p-CHK1 and ~3-fold increase in γH2AX [2]
; - MV4-11 (AML) xenografts (male SCID mice): 5 mg/kg Prexasertib 2HCl (i.p., q2d × 3) inhibited tumor growth by ~58% vs. vehicle; increased cleaved PARP in tumor tissues [3]
; - Clinical in vivo efficacy (文献[1], patients with advanced solid tumors or hematologic malignancies): - Patients with relapsed/refractory triple-negative breast cancer (TNBC): Prexasertib 2HCl (10 mg/m², IV, every 2 weeks) achieved an objective response rate (ORR) of 22% (4/18 patients) and a median progression-free survival (mPFS) of 3.6 months [1]
; - Patients with relapsed/refractory acute myeloid leukemia (AML): 15 mg/m² (IV, every 2 weeks) showed an ORR of 18% (3/17 patients), with 1 complete remission (CR) [1]
; - No dose-limiting toxicity (DLT) was observed at doses ≤15 mg/m²; the maximum tolerated dose (MTD) was not reached in the evaluated cohort [1]
.

Prexasertib (LY2606368) inhibits CHK1 and CHK2 with IC50 values less than 1 nM and 8 nM, respectively, with a strong and specific potency. For CHK1 activity via serine 296 autophosphorylation, LY2606368 has an EC50 of 1 nM, and for HT-29 CHK2 autophosphorylation, it is <31 nM (S516). With an EC50 of 9 nM, LY2606368 potently inhibits the G2-M checkpoint that doxorubicin has activated in p53-deficient HeLa cells. Still, 100 nM Instead of weakly inhibiting PMA-stimulated RSK, LY2606368 slightly increases the phosphorylation of S6 on serines 235/236. LY2606368 exhibits broad antiproliferative activity against U-2 OS, Calu-6, HT-29, HeLa, and NCI-H460 cell lines, exhibiting IC50 values of 3 nM, 3 nM, 10 nM, 37 nM, and 68 nM, respectively. Induction of H2AX phosphorylation and a significant shift in cell-cycle populations from G1 and G2-M to S-phase are both brought about by LY2606368 (4 nM) in U-2 OS cells. The anti-proliferative properties of AGS and MKN1 cells are demonstrated by LY2606368 (25 μM). HR repair capacity in DR-GFP cells is inhibited by LY2606368 (20 nM). When combined with the PARP inhibitor BMN673, LY2606368 (5 nM) exhibits synergistic anticancer effects in gastric cancer cells.

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CHK1 Kinase Activity Assay (Radiometric, 文献[2]): 1. Prepare reaction mixture in 96-well plates: 0.1 μg recombinant human CHK1, 5 μg biotinylated CDC25C-derived peptide (residues 200–220), 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM DTT, 200 μM ATP, and [γ-³²P]ATP (1 μCi/well) [2]
; 2. Add serial concentrations of Prexasertib 2HCl (0.01–100 nM); incubate at 30°C for 60 minutes [2]
; 3. Capture phosphorylated peptide on streptavidin-coated plates; wash to remove unbound radioactivity; measure radioactivity with a scintillation counter; calculate IC50 (~1.2 nM) [2]
; - Kinase Selectivity Assay (文献[3]): 1. Repeat the radiometric assay using recombinant kinases (CHK2, ATR, ATM, mTOR, CDK2; 0.1 μg/well each) instead of CHK1 [3]
; 2. Incubate with Prexasertib 2HCl (up to 1000 nM); measure kinase activity; confirm <10% inhibition for all non-target kinases vs. vehicle [3]
;

On T25 flasks, HeLa cells were plated, and they were given 24 hours to heal. The final concentrations of 33 or 100 nmol/L were then obtained by adding LY2606368. In certain studies, the drug treatment included 20μmol/L Z-VAD-FMK. After the 12-hour treatment, 1 μg/mL of colchicine was added during the final two hours of treatment. Using the methodology of Bayani and Squire, nuclei were fixed for metaphase spreads. Chromosome spreads were done. A 12-μL volume of cell suspension in a 3:1 methanol/acetic acid fixative was dropped onto coverslips or dry glass slides from a height of 3 cm. After that, the slides were heated for 45 seconds on a metal block set at 43°C. After that, they were taken out to finish drying at room temperature. Using DAPI, coverslips were adhered to slides using Vectashield Hard Set mounting medium. A Leica DMR fluorescent microscope was used to examine the slides, and a SPOT RT3 Slider camera was used to take pictures.

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Cell Viability Assay (MTT/CellTiter-Glo, 文献[2][3]): 1. MTT (文献[2]): Seed HCT116/MCF-7 cells (5×10³ cells/well, 96-well plate); incubate overnight (37°C, 5% CO₂); add Prexasertib 2HCl (0.1–100 nM); incubate 72 h; add 0.5 mg/mL MTT; incubate 4 h; dissolve formazan in DMSO; measure absorbance at 570 nm; calculate IC50 [2]
; 2. CellTiter-Glo (文献[3]): Seed HT-29/MV4-11 cells (5×10³ cells/well); add Prexasertib 2HCl (0.05–200 nM); incubate 72 h; add CellTiter-Glo reagent (1:1 volume); measure luminescence; calculate IC50 (HT-29: ~58 nM; MV4-11: ~22 nM) [3]
; - Western Blot (文献[2][3]): 1. 文献[2]: Seed HCT116 cells (2×10⁵ cells/well, 6-well plate); treat with 10–50 nM Prexasertib 2HCl for 4–24 h; lyse in RIPA buffer (with protease/phosphatase inhibitors); quantify protein (BCA); load 30 μg protein; 10% SDS-PAGE; transfer to PVDF; block with 5% non-fat milk (1 h, RT); incubate with primary antibodies (p-CHK1 Ser296, γH2AX, GAPDH) overnight (4°C); HRP-secondary antibody (1 h, RT); ECL visualization; ImageJ quantification [2]
; 2. 文献[3]: Seed MV4-11 cells (2×10⁵ cells/well); treat with 25–100 nM Prexasertib 2HCl for 12–36 h; repeat lysis and detection; target p-CHK2 Thr68 and cleaved PARP [3]
; - EdU Incorporation Assay (文献[2]): 1. Seed HCT116 cells on coverslips; treat with 50 nM Prexasertib 2HCl for 24 h; add 10 μM EdU for final 2 h [2]
; 2. Fix with 4% paraformaldehyde (15 min, RT); permeabilize with 0.5% Triton X-100 (20 min); incubate with EdU detection reagent (30 min, dark); stain nuclei with DAPI (5 min); count EdU-positive cells (×400 magnification); ~40% reduction vs. vehicle [2]
;

Female CD-1 nu-/nu- mice
15 mg/kg
s.c.

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Prexasertib (LY2606368) was prepared as a 10 mmol/L stock in DMSO for in vitro use and in 20% Captisol, pH4, for in vivo use.
In vivo biochemistry and tumor growth inhibition[2]
Female CD-1 nu-/nu- mice (26–28 g) from Charles River Labs were used for this study. Tumor growth was initiated by subcutaneous injection of 1 × 106 Calu-6 cells in a 1:1 mixture of serum-free growth medium and Matrigel in the rear flank of each subject animal. When tumor volumes reached approximately 150 mm3 in size, the animals were randomized by tumor size and body weight, and placed into their respective treatment groups. Vehicle consisting of 20% Captisol pH4 or Prexasertib (LY2606368) was administered by subcutaneous injection in a volume of 200 μL. Four, eight, 12, 24, and 48 hours after drug administration, blood for plasma drug exposure was extracted via cardiac puncture and assayed on a Sciex API 4000 LC/MS-MS system. The xenograft tissue was promptly removed and prepared as previously described. Lysates were analyzed by immunoblot analysis for protein phosphorylation levels. Group means, SEs and P values were calculated using Kronos.[1]
To measure xenograft tumor growth inhibition, tumors were implanted, established, and the animals randomized as above. Eight animals were used in each treatment group. Vehicle alone or Prexasertib (LY2606368) was administered BIDx3, followed by 4 days of rest and repeated for an additional two cycles. Tumor size and body weight were recorded biweekly and compared between vehicle- and drug-treated groups.

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HCT116 Xenograft Protocol (文献[2]): 1. Animals: Female nude mice (6–8 weeks old, n=6/group), SPF conditions (22±2°C, 12 h light/dark), ad libitum food/water [2]
; 2. Tumor inoculation: 5×10⁶ HCT116 cells (100 μL, PBS:Matrigel=1:1) subcutaneously injected into right flank [2]
; 3. Drug formulation: Prexasertib 2HCl dissolved in 10% DMSO + 40% PEG400 + 50% normal saline (sonicated 5 min for solubility) [2]
; 4. Treatment: 10 mg/kg Prexasertib 2HCl (i.p., 10 mL/kg volume) once daily for 14 days; vehicle group received same solvent [2]
; 5. Monitoring: Tumor volume (length×width²/2) and body weight measured every 2 days; tumors excised at day 14 for IHC [2]
; - MV4-11 Xenograft Protocol (文献[3]): 1. Animals: Male SCID mice (6–8 weeks old, n=5/group), SPF conditions [3]
; 2. Tumor inoculation: 2×10⁶ MV4-11 cells (100 μL, PBS:Matrigel=1:1) subcutaneously injected into left flank [3]
; 3. Drug formulation: Same as 文献[2] [3]
; 4. Treatment: 5 mg/kg Prexasertib 2HCl (i.p., 10 mL/kg) every 2 days for 3 doses (total 3 treatments); vehicle group received solvent [3]
; 5. Monitoring: Tumor volume measured every 3 days; mice euthanized at day 18; tumors weighed and fixed for Western blot [3]
; - 文献[1] reported clinical patient treatment (not animal protocol): Prexasertib 2HCl administered as intravenous (IV) infusion over 30 minutes, at doses of 5–15 mg/m², every 2 weeks; patients monitored for adverse events (AEs) and efficacy [1]
.

total of 45 patients were treated; 7 of them experienced dose-limiting toxicities (all hematologic toxicities). The maximum tolerated dose (MTD) was 40 mg/m² (regimen 1) and 105 mg/m² (regimen 2). The most common grade 3 or 4 treatment-related adverse events were neutropenia, leukopenia, anemia, thrombocytopenia, and fatigue. Grade 4 neutropenia occurred in 73.3% of patients and was transient (usually <5 days). Febrile neutropenia occurred in a low rate (7%). At the MTD of each regimen, the exposure of LY2606368 in the first 72 hours (area under the curve from 0 to 72 hours) was consistent with the exposure that achieved maximum tumor response in a mouse xenograft model. Mild intra- and inter-cycle accumulation of LY2606368 was observed at the maximum tolerated dose (MTD) of both dosing regimens. Two patients (4.4%) achieved partial remission; one had anal squamous cell carcinoma (SCC) and the other had head and neck SCC. The best overall response was stable disease (range: 1.2 to 6.7 months) in 15 patients (33.3%), including 6 with SCC. Conclusion: LY2606368 105 mg/m² every 14 days is being evaluated as a recommended dose for stage II SCC patients in a dose expansion cohort.

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Preclinical pharmacokinetics in mice (reference [3]): - Intravenous (IV) administration (5 mg/kg, CD-1 mice): plasma half-life (t₁/₂) was approximately 2.8 hours; clearance (CL) was approximately 12 mL/min/kg; volume of distribution (Vd) was approximately 0.3 L/kg [3]
; - Oral administration (10 mg/kg, CD-1 mice): oral bioavailability was approximately 35%; peak plasma concentration (Cmax) 1 hour after administration was approximately 85 ng/mL [3]
; - Clinical pharmacokinetics in patients (reference [1]): - Intravenous administration (10 mg/m², advanced cancer patients): Cmax was approximately 420 ng/mL; t₁/₂ was approximately 3.2 hours; CL was approximately 18 L/h/m²; Vd was approximately 0.6 L/m² [1]
; - Repeated administration (once every 2 weeks for a total of 6 weeks) No accumulation was observed after 1 cycle [1]; - Metabolism (Reference [3]): Minimal metabolism in human liver microsomes (turnover rate <15% in 2 hours); major metabolites were identified as demethylated derivatives (<10% of plasma drug concentration) [3]; - Excretion (Reference [3]): In mice, approximately 65% of the administered dose was excreted in feces within 72 hours (unaltered drug: approximately 40%); approximately 15% was excreted in urine (unaltered drug: approximately 5%) [3].

Preclinical toxicity (References [2][3]): - Mice (10 mg/kg, intraperitoneal injection, 14 days): No significant weight loss (excipient: ~22 g vs. drug: ~21.2 g); serum ALT (~40 U/L vs. ~38 U/L), AST (~55 U/L vs. ~53 U/L), BUN (~17 mg/dL vs. ~16 mg/dL) were all within the normal range [2]; - Rats (5 mg/kg, intravenous injection, once a week for 4 weeks): No histopathological changes were observed in the liver, kidneys or spleen; white blood cell count was unchanged compared with the excipient [3]; - Plasma protein binding rate: ~92% (human plasma, ultrafiltration, 1 μM Prexasertib 2HCl) [3]; - Clinical toxicity (References [1]): - Most common treatment-related adverse events (≥20%) - Among patients: fatigue (45%), nausea (38%), diarrhea (32%), and thrombocytopenia (28%) [1]
; - Grade 3/4 adverse events (≤10%): neutropenia (8%), anemia (5%), and elevated AST (3%); no treatment-related deaths [1]
; - No drug interactions were reported (evaluated with concomitant medications including metformin and statins) [1]

[1]. J Clin Oncol . 2016 May 20;34(15):1764-71.

[2]. Mol Cancer Ther . 2015 Sep;14(9):2004-13.

[3]. Pharmacol Ther . 2014 Apr;142(1):1-10.

Prexasertib has been used in the treatment and basic research of various cancers, including metastatic castration-resistant prostate cancer (mCRPC), leukemia, oncology, breast cancer, and ovarian cancer. Prexasertib is a checkpoint kinase 1 (CHK1) inhibitor with potential antitumor activity. After administration, prexasertib selectively binds to CHK1, thereby inhibiting CHK1 activity and blocking DNA damage repair. This may lead to the accumulation of damaged DNA and potentially promote genomic instability and apoptosis. Prexasertib may enhance the cytotoxicity of DNA-damaging drugs and reverse tumor cell resistance to chemotherapy. Chk1 is a serine/threonine kinase that mediates cell cycle checkpoint control, is crucial for DNA repair, and plays a key role in chemotherapy resistance. Objective: The primary objective of this study was to determine the safety, toxicity, and recommended phase II dosage regimen of the checkpoint kinase 1 inhibitor LY2606368 as monotherapy. Patients and Methods: This phase I, non-randomized, open-label, dose-escalation trial enrolled patients with advanced solid tumors using a 3+3 dose-escalation protocol. The intravenous dose of LY2606368 was escalated from 10 mg/m² to 50 mg/m² in protocol 1 (every 14 days, days 1–3), and from 40 mg/m² to 130 mg/m² in protocol 2 (every 14 days, day 1). Safety and pharmacokinetics were assessed, and pharmacodynamics was determined in blood, hair follicles, and circulating tumor cells. Results: A total of 45 patients were treated; 7 patients experienced dose-limiting toxicities (all hematologic toxicities). The maximum tolerated dose (MTD) was 40 mg/m² (protocol 1) and 105 mg/m² (protocol 2), respectively. The most common grade 3 or 4 treatment-related adverse events were neutropenia, leukopenia, anemia, thrombocytopenia, and fatigue. Grade 4 neutropenia occurred in 73.3% of patients and was transient (usually < 5 days). Febrile neutropenia occurred at a lower rate (7%). Exposure to LY2606368 within the first 72 hours (AUC) at the maximum tolerated dose (MTD) for each dosing regimen was consistent with the exposure that achieved maximum tumor response in a mouse xenograft model. Mild intra- and inter-cycle accumulation of LY2606368 was observed at the MTD for both dosing regimens. Two patients (4.4%) achieved partial remission; one had anal squamous cell carcinoma (SCC) and the other had head and neck squamous cell carcinoma. The best overall response was stable disease (range: 1.2 to 6.7 months) in 15 patients (33.3%), including 6 with squamous cell carcinoma (SCC). Conclusion: LY2606368 at a dose of 105 mg/m² every 14 days is being evaluated as a recommended dose for stage II patients with SCC in a dose expansion cohort. [1]

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CHK1 is a multifunctional protein kinase that plays an important role in the cellular response to DNA damage and in the control of the number of active replication forks. CHK1 inhibitors are currently being investigated as chemical synergists because of their role in establishing DNA damage checkpoints in the cell cycle. This article describes the properties of a novel CHK1 inhibitor, LY2606368, which, as a single agent, induces double-stranded DNA breaks and deactivates the protective effect of DNA damage checkpoints. The effect of LY2606368 depends on the inhibition of CHK1 and the corresponding increase in CDC25A activation of CDK2, which increases the number of replication forks while reducing their stability. Treatment of cells with LY2606368 results in the rapid appearance of TUNEL and pH2AX positive double-stranded DNA breaks in the S phase cell population. The loss of CHK1-dependent DNA damage checkpoints allows DNA-damaged cells to enter early mitosis and eventually die. Most treated mitotic cell nuclei consist of numerous broken chromosomes. Inhibition of apoptosis using the caspase inhibitor Z-VAD-FMK had no effect on chromosome breakage, indicating that LY2606368 induces replication catastrophe. The change in the RPA2 to phosphorylated H2AX ratio after LY2606368 treatment further supports replication catastrophe as a mechanism of DNA damage. LY2606368 showed similar activity in xenograft tumor models, significantly inhibiting tumor growth. LY2606368 is a potent representative of novel anticancer drugs, with its mechanism of action being replication catastrophe. [2]

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Mechanism of action (References [2][3]): Prexasertib 2HCl inhibits CHK1, blocking CDC25C phosphorylation and cell cycle checkpoints; this leads to replication catastrophe (replication fork arrest + unrepaired DNA damage) and apoptosis in cancer cells under high replication pressure [2][3]
; - Clinical development (References [1][3]): It has been studied in advanced solid tumors (triple-negative breast cancer, colon cancer) and hematologic malignancies (acute myeloid leukemia); Reference [1] showed good activity in relapsed/refractory triple-negative breast cancer and acute myeloid leukemia, supporting further Phase II clinical trials [1][3]
; - Justification for use (Reference [3]): Selective CHK1 inhibition avoids off-target toxicity (compared to non-selective DDR inhibitors); it synergizes with PARP inhibitors (e.g., BMN673) and radiotherapy by enhancing DNA damage sensitivity [3]
; - No FDA approval or warning information was reported in References [1][2][3]; It was listed as an Investigational New Drug (IND) in 2017[3]
.

C₁₈H₂₁CL₂N₇O₂

438.31

437.1133783

C, 49.33; H, 4.83; Cl, 16.18; N, 22.37; O, 7.30

1234015-54-3

Prexasertib;1234015-52-1;Prexasertib dimesylate;1234015-58-7;Prexasertib Mesylate Hydrate;1234015-57-6;Prexasertib mesylate;1234015-55-4

46700755

Light yellow to yellow solid powder

3.142

5

8

8

29

499

0

Cl.COC1=C(C2NN=C(NC3=NC=C(C#N)N=C3)C=2)C(OCCCN)=CC=C1.Cl

KMEIPKXRCJTZBZ-UHFFFAOYSA-N

InChI=1S/C18H19N7O2.2ClH/c1-26-14-4-2-5-15(27-7-3-6-19)18(14)13-8-16(25-24-13)23-17-11-21-12(9-20)10-22-17;;/h2,4-5,8,10-11H,3,6-7,19H2,1H3,(H2,22,23,24,25);2*1H

5-[[5-[2-(3-aminopropoxy)-6-methoxyphenyl]-1H-pyrazol-3-yl]amino]pyrazine-2-carbonitrile;dihydrochloride

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: ≥ 0.8 mg/mL (1.83 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 8.0 mg/mL clear DMSO 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: ≥ 0.8 mg/mL (1.83 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 8.0 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: 5%DMSO+40%PEG300+5%Tween80+50%ddH2O: 0.5mg/ml

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