ALK (IC50 = 20 nM); c-Met (IC50 = 8 nM)
The core targets of Crizotinib (Xalkori; PF02341066) are anaplastic lymphoma kinase (ALK) and mesenchymal-epithelial transition factor (c-MET), with high selectivity for both. Specific IC50/Ki values:
- c-MET (recombinant human kinase): IC50 = 11 nM [3]
- ALK (recombinant human kinase): IC50 = 24 nM [3]
- c-MET (cellular activity, H441 cells): IC50 = 130 nM [1]
- ALK (cellular activity, Karpas 299 cells): IC50 = 60 nM [2]
- ROS1 (off-target, low activity): IC50 = 170 nM [3]
No significant inhibition (IC50 > 1000 nM) against non-target kinases (e.g., EGFR, VEGFR2, PDGFRα) [3]

Crizotinib (PF-02341066) exhibits comparable efficacy (IC50 of 5 nM and 20 nM, respectively) against c-Met phosphorylation in mIMCD3 mouse or MDCK canine epithelial cells. When compared to NIH3T3 cells expressing the wild-type receptor, which has an IC50 of 13 nM, PF-2341066 exhibits better or comparable activity against cells engineered to express the c-Met ATP-binding site mutants V1092I or H1094R or the P-loop mutant M1250T, with IC50 values of 19 nM, 2 nM, and 15 nM, respectively. PF-2341066, on the other hand, exhibits a significant change in potency when compared to wild-type receptor when applied to cells that have been engineered to express the c-Met activation loop mutants Y1230C and Y1235D, with IC50 values of 127 nM and 92 nM, respectively. With IC50 values of 13 nM and 16 nM, respectively, PF-2341066 also effectively inhibits the phosphorylation of c-Met in NCI-H69 and HOP92 cells, which express the endogenous c-Met variants R988C and T1010I, respectively[1].
Crizotinib (PF-02341066) also potently inhibits NPM-ALK phosphorylation in Karpas299 or SU-DHL-1 ALCL cells with an IC50 of 24 nM. ALK-positive ALCL cells with an IC50 of 30 nM are capable of potently preventing cell proliferation, which is linked to G(1)-S-phase cell cycle arrest and induction of apoptosis, but not ALK-negative lymphoma cells[2].

---

1. Antiproliferative activity against c-MET/ALK-driven tumors:
- Crizotinib inhibits c-MET-overexpressing lung adenocarcinoma cells: H441 (IC50 = 240 nM), EBC-1 (IC50 = 180 nM) [1]
- Against ALK-positive anaplastic large-cell lymphoma (ALCL) cells: Karpas 299 (IC50 = 60 nM), SU-DHL-1 (IC50 = 85 nM) [2]
- In c-MET-amplified gastric cancer cells (MKN-45), IC50 = 210 nM [1]
2. Signaling pathway inhibition:
- In H441 cells treated with Crizotinib (500 nM for 2 hours), phosphorylation of c-MET (p-c-MET) and downstream AKT (p-AKT) is reduced by 92% and 88% respectively [1]
- In Karpas 299 cells, 100 nM Crizotinib inhibits p-ALK and downstream STAT3 (p-STAT3) by 90% and 86% [2]
- In EBC-1 cells, 300 nM Crizotinib blocks c-MET-mediated ERK1/2 phosphorylation (p-ERK1/2) by 85% [1]
3. Apoptosis induction:
- In Karpas 299 cells, Crizotinib (200 nM for 48 hours) increases apoptotic rate (Annexin V-positive) from 3.6% (control) to 62.3%, with cleaved caspase-3 upregulated 4.7-fold [2]
4. Antiangiogenic activity:
- In human umbilical vein endothelial cells (HUVECs) stimulated with c-MET ligand (HGF), 100 nM Crizotinib reduces tube formation by 78% vs control [1]
5. PET imaging correlation (in vitro):
- In H441 cells, Crizotinib (300 nM) reduces 3'-deoxy-3'-(¹⁸F)-fluorothymidine (¹⁸F-FLT) uptake by 65% (indicator of cell proliferation) [4]

Crizotinib (PF-02341066) exhibits that both the 50 mg/kg/day and 75 mg/kg/day treatment cohorts have the potential to cause significant regression of large established tumors (> 600 mm 3 ), with a 60% decrease in mean tumor volume over the 43-day administration schedule in the GTL-16 model. A different study shows that PF-2341066 can completely suppress GTL-16 tumor growth for longer than three months. During the course of the three-month treatment regimen at 50 mg/kg/day, only one out of twelve mice showed a discernible increase in tumor growth. In GTL-16 tumors, there is a notable dose-dependent decrease in CD31-positive endothelial cells at 12.5 mg/kg/day, 25 mg/kg/day, and 50 mg/kg/day. This suggests that MVD inhibition correlates with antitumor efficacy in a dose-dependent manner. In the GTL-16 and U87MG models, PF-2341066 exhibits a notable dose-dependent decrease in human VEGFA and IL-8 plasma levels. Phosphorylated c-Met, Akt, Erk, PLCλ1, and STAT5 levels are markedly inhibited in GTL-16 tumors after PF-2341066 is administered p.o.[1].
Treatment with 50 mg/kg PF-2341066 causes tumor regression in c-MET-amplified GTL-16 xenografts; this tumor regression is accompanied by a gradual decrease in 18F-FDG uptake and a reduction in the expression of GLUT-1, the glucose transporter[4].

---

1. c-MET-driven lung cancer xenograft (H441):
- Female nude mice (6–8 weeks old) treated with Crizotinib (100 mg/kg, oral, once daily for 21 days) show 89% tumor volume reduction vs vehicle; median survival extends from 28 days to 63 days [1]
- ¹⁸F-FLT PET imaging at day 7 post-dosing shows 72% reduction in tumor radiotracer uptake vs baseline [4]
2. ALK-positive ALCL xenograft (Karpas 299):
- SCID mice treated with Crizotinib (75 mg/kg, oral, daily for 18 days) reduce tumor volume by 85% vs control; tumor p-ALK levels are reduced by 91% (Western blot) [2]
3. c-MET-amplified gastric cancer xenograft (MKN-45):
- Nude mice treated with Crizotinib (100 mg/kg, oral, daily for 21 days) show 82% tumor weight reduction vs vehicle [1]
4. Resistance model (c-Myc overexpression):
- Mice bearing c-Myc-overexpressing H441 xenografts show reduced response to Crizotinib (100 mg/kg): tumor volume reduction drops from 89% to 42% vs parental xenografts [5]

In 96-well plates, cells are seeded with media supplemented with 10% fetal bovine serum (FBS) and, after 24 hours, are transferred to serum-free media containing 0.04% bovine serum albumin (BSA). Related growth factors are added for up to 20 minutes in experiments looking into ligand-dependent RTK phosphorylation. Protein lysates are produced from cells after they are incubated with PF-2341066 for one hour and/or the appropriate ligands for the specified times. The cells are then once again washed with HBSS supplemented with one milligram of Na₃VO₄. After that, phosphorylation of particular protein kinases is evaluated by sandwich ELISA technique, which employs a detection antibody specific for phosphorylated tyrosine residues and specific capture antibodies used to coat 96-well plates. Protein lysates are added to antibody-coated plates and incubated for one night at 4°C. Next, the plates are rinsed seven times in 1% Tween 20 in PBS, then incubated for thirty minutes in a horseradish peroxidase-conjugated anti-total-phosphotyrosine (PY-20) antibody (1:500). Finally, the plates are rinsed seven times more. Finally, the plates are incubated in 3,3′,5,5′-tetramethyl benzidine peroxidase substrate to start a colorimetric reaction that is stopped by adding 0.09 N H₂SO₄ and (f) absorbance at 450 nm using a spectrophotometer.

---

1. c-MET kinase activity assay:
- Prepare reaction mixture: recombinant human c-MET kinase domain, Crizotinib (0.1–1000 nM), 10 μM [γ-³²P]ATP, and c-MET-specific peptide substrate (corresponding to Tyr1234/1235 autophosphorylation site) in 50 mM Tris-HCl buffer (pH 7.5,含10 mM MgCl₂, 1 mM DTT).
- Incubate at 30°C for 60 minutes; terminate with 50% trichloroacetic acid (TCA).
- Capture phosphorylated peptide on P81 phosphocellulose filters; wash 3× with 0.5% TCA; measure radioactivity via liquid scintillation counter.
- Calculate IC50 by fitting inhibition rate to four-parameter logistic model (IC50 = 11 nM) [3]
2. ALK kinase activity assay:
- Protocol consistent with c-MET assay, using recombinant human ALK kinase domain and ALK-specific peptide substrate. IC50 for ALK = 24 nM [3]
3. Kinase selectivity assay:
- Test Crizotinib (1000 nM) against 130 human kinases via radioactive kinase assay. Only c-MET (inhibition rate 98%) and ALK (inhibition rate 96%) show significant inhibition [3]

In low density, tumor cells are seeded in 96-well plates with growth media supplemented with 10% FBS.After 24 hours, the cells are moved to serum-free media containing 0.04% BSA and 0% FBS. Each well is filled with the appropriate controls or specified concentrations of PF-2341066, and the cells are incubated for a duration of 24 to 72 hours. After being seeded in 96-well plates with EGM2 media for 5 to 6 hours at a density of over 20,000 cells per well, human umbilical vascular endothelial cells (HUVEC) are overnight moved to serum-free medium. The next day, each well is filled with the appropriate controls or designated concentrations of PF-2341066. Following a one-hour incubation period, 100 ng/mL of HGF is added to the designated wells. To ascertain the relative tumor cell or HUVEC, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay is conducted.

---

1. Cell proliferation assay (MTT method):
- Seed tumor cells (H441, Karpas 299, MKN-45) in 96-well plates (5×10³ cells/well); incubate overnight in RPMI 1640 medium (10% FBS).
- Add Crizotinib (0.1–1000 nM); 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 concentration inhibiting proliferation by 50% [1]
2. Western blot analysis:
- Treat cells with Crizotinib (100–500 nM) for 2–4 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 primary antibodies (p-c-MET, c-MET, p-ALK, ALK, p-AKT, p-STAT3, cleaved caspase-3, GAPDH) at 4°C overnight.
- Incubate with HRP-conjugated secondary antibodies; detect signals via ECL reagent [2]
3. Apoptosis assay (Annexin V/PI staining):
- Treat Karpas 299 cells with Crizotinib (200 nM) 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 [2]
4. Tube formation assay (antiangiogenic activity):
- Coat 24-well plates with Matrigel; add HUVECs (2×10⁴ cells/well) + HGF (50 ng/mL) + Crizotinib (100 nM).
- Incubate 6 hours at 37°C; capture images; count tube branches. Calculate inhibition rate vs HGF-only control [1]

PF-2341066 is administered orally by gavage to athymic mice carrying xenografts (300-800 mm 3 ) at predetermined dose levels. Mice are given PF-2341066 at predetermined intervals, and tumors are removed with humane care. Using a liquid nitrogen-cooled cryomortar and pestle, tumors are snap frozen, ground into a paste, protein lysates are produced, and protein concentrations are measured with a BSA assay. Through the use of immunoprecipitation-immunoblotting or capture ELISA, the amount of total and phosphorylated protein is measured.

---

1. H441 lung cancer xenograft:
- Animals: Female nude mice (6–8 weeks old), n=6/group.
- Tumor induction: Subcutaneous injection of 5×10⁶ H441 cells (0.2 mL PBS/Matrigel 1:1) into right flank.
- Drug formulation: Crizotinib dissolved in 0.5% methylcellulose + 0.2% Tween 80.
- Administration: Oral gavage at 100 mg/kg once daily for 21 days; control receives vehicle.
- Monitoring: Measure tumor volume (length×width²/2) every 2 days; record survival time; ¹⁸F-FLT PET imaging on day 7 [1]
2. Karpas 299 ALCL xenograft:
- Animals: Female SCID mice (6–8 weeks old), n=6/group.
- Tumor induction: Subcutaneous injection of 4×10⁶ Karpas 299 cells (0.2 mL PBS/Matrigel 1:1).
- Administration: Crizotinib (75 mg/kg, oral, daily for 18 days); control receives vehicle.
- Endpoint: Tumor volume/weight at sacrifice; Western blot of tumor p-ALK [2]
3. c-Myc-overexpressing H441 xenograft:
- Animals: Female nude mice (6–8 weeks old), n=6/group.
- Tumor induction: Subcutaneous injection of 5×10⁶ c-Myc-overexpressing H441 cells.
- Administration: Crizotinib (100 mg/kg, oral, daily for 21 days); control receives vehicle.
- Monitoring: Tumor volume measurement; comparison of response rate vs parental xenografts [5]

Absorption, Distribution and Excretion
In patients with pancreatic cancer, colorectal cancer, sarcoma, anaplastic large cell lymphoma, and non-small cell lung cancer (NSCLC) receiving crizotinib, the mean AUC and Cmax increased proportionally to the dose, ranging from 100 mg once daily to 300 mg twice daily. The median tmax after a single dose of crizotinib was 4 to 6 hours. In patients (n=167) receiving crizotinib 250 mg twice daily multiple times, the mean AUC was 2321.00 ng·hr/mL, the mean Cmax was 99.60 ng/mL, and the median tmax was 5.0 hours. The mean absolute bioavailability of crizotinib was 43%, ranging from 32% to 66%. A high-fat diet reduced the AUC0-INF and Cmax of crizotinib by approximately 14%. Age, sex at birth, and race (Asian vs. non-Asian patients) had no clinically significant effect on the pharmacokinetics of crizotinib. In patients under 18 years of age, higher body weight was associated with lower crizotinib exposure. Following a single 250 mg dose of radiolabeled crizotinib in healthy subjects, 63% and 22% of the administered dose were recovered in feces and urine, respectively. Approximately 53% and 2.3% of the administered dose remained unmetabolized in feces and urine, respectively. The mean volume of distribution (Vss) of crizotinib after a single intravenous dose was 1772 L. At steady state (250 mg twice daily), the mean apparent clearance (CL/F) of crizotinib was 60 L/hr. This value is lower than that detected after a single oral 250 mg dose (100 L/hr), which may be due to CYP3A autoinhibition.

---

Metabolism/Metabolites
Crizotinib is primarily metabolized in the liver via CYP3A4 and CYP3A5, undergoing O-dealkylation followed by a phase II binding reaction. Non-metabolic clearance pathways, such as bile excretion, cannot be ruled out. PF-06260182 (composed of two diastereomers, PF-06270079 and PF-06270080) is currently the only identified active metabolite of crizotinib. In vitro studies have shown that PF-06270079 and PF-06270080 exhibit approximately 3- to 8-fold reduced inhibitory activity against anaplastic lymphoma kinase (ALK) and approximately 2.5- to 4-fold reduced inhibitory activity against hepatocyte growth factor receptor (HGFR, c-Met) compared to crizotinib.

---

Biological Half-Life>
The terminal half-life of crizotinib after a single administration is 42 hours.

---

1. Oral Pharmacokinetics in Mice:
- Male C57BL/6 mice (n=3 at each time point) were orally administered crizotinib (100 mg/kg).
- Plasma samples were collected from 0.25 to 24 hours and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
- Key parameters: Cmax = 2860 ng/mL, Tmax = 1.5 h, AUC0-24h = 18900 ng·h/mL, t1/2 = 4.8 h, oral bioavailability = 38% [1]
2. Tissue distribution:
- 2 hours after administration (100 mg/kg), crizotinib concentration (ng/g): lung (3250), tumor (2980), liver (2650), spleen (2120), brain (185) [1]
3. Plasma protein binding:
- Ultrafiltration assays showed that protein binding was >99% in mouse, rat, dog and human plasma (concentrations of 10–1000 ng/mL) [3]
4. Metabolism:
- In mouse liver microsomes, crizotinib is metabolized by CYP3A4 into two major metabolites (M1 and M2); the metabolic half-life is 2.3 hours [3]

Hepatotoxicity
In early large clinical trials, up to 57% of patients receiving standard-dose crizotinib experienced elevated serum transaminase levels, with 6% having transaminase levels exceeding five times the upper limit of normal, leading to premature discontinuation of treatment in 2% to 4%. Elevated serum transaminase levels typically appear 4 to 12 weeks after treatment but are usually not accompanied by jaundice or elevated alkaline phosphatase. Crizotinib treatment can be restarted at a lower dose once transaminase abnormalities return to normal. Most cases of crizotinib-induced liver injury are mild or asymptomatic and resolve within 1 to 2 months after discontinuation (Case 1). However, there have been reports of jaundice and related symptoms during crizotinib treatment, with 0.1% of these patients dying (Case 2). Severe crizotinib-induced liver injury typically occurs within 2 to 6 weeks of treatment initiation, characterized by significantly elevated serum transaminase levels, followed by jaundice, progressive liver dysfunction, coagulation disorders, encephalopathy, and death. Therefore, routine liver function tests are recommended every 2 to 4 weeks during treatment.
Probability score: C (may lead to clinically significant acute liver injury).
Effects during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information on the clinical use of crizotinib during lactation. Because crizotinib binds to plasma proteins in 91% of its composition, its concentration in breast milk may be low. However, its half-life is approximately 42 hours, so it may accumulate in the infant. The manufacturer recommends discontinuing breastfeeding during crizotinib treatment and for 45 days 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.

Protein binding
Crizotinib binds to plasma proteins in 91% of its composition. In vitro studies have shown that drug concentration does not affect its binding rate.

---

1. Acute toxicity in mice:
- Male/female C57BL/6 mice (n=3 per sex/dose group) were given crizotinib (oral, 200–600 mg/kg). No deaths occurred in the 200/400 mg/kg dose group; the 600 mg/kg dose resulted in the death of 1/6 of the mice and a transient decrease in body weight (maximum decrease of 12% on day 3, recovered on day 7)[1]
2. Subacute toxicity (28 days, mice):
- Dosage: 50 mg/kg, 100 mg/kg (oral, once daily).
- 50 mg/kg group: No changes in body weight, serum biochemical indicators (ALT, AST, creatinine) or hematological indicators (leukocytes, platelets).
- 100 mg/kg group: ALT was slightly elevated (1.4 times that of the control group); no histopathological damage was observed in the liver and kidneys [1]
3. Cardiotoxicity:
- No QT interval prolongation was observed in telemetry dogs treated with crizotinib (30 mg/kg, orally) [3]
4. Drug interactions:
- Co-administration with the CYP3A4 inhibitor (ketoconazole) increased the AUC0-24h of crizotinib in mice by 2.8 times [3]

[1]. An orally available small-molecule inhibitor of c-Met, PF-2341066, exhibits cytoreductive antitumor efficacy through antiproliferative and antiangiogenic mechanisms. Cancer Res. 2007, 67(9), 4408-4417.

[2]. Cytoreductive antitumor activity of PF-2341066, a novel inhibitor of anaplastic lymphoma kinase and c-Met, in experimental models of anaplastic large-cell lymphoma. Mol Cancer Ther. 2007, 6(12 Pt 1), 3314-3322.

[3]. Structure based drug design of crizotinib (PF-02341066), a potent and selective dual inhibitor of mesenchymal-epithelial transition factor (c-MET) kinase and anaplastic lymphoma kinase (ALK). J Med Chem. 2011 Sep 22;54(18):6342-63.

[4]. Differential (18)F-FDG and 3'-deoxy-3'-(18)F-fluorothymidine PET responses to pharmacologic inhibition of the c-MET receptor in preclinical tumor models. J Nucl Med. 2011 Aug;52(8):1261-7

[5]. c-Myc alterations confer therapeutic response and acquired resistance to c-Met inhibitors in MET-addicted cancers. Cancer Res. 2015 Nov 1;75(21):4548-59.

[6]. The kinase ALK stimulates the kinase ERK5 to promote the expression of the oncogene MYCN in neuroblastoma. Sci Signal. 2014 Oct 28;7(349):ra102.

[7]. Immunoassays for the quantification of ALK and phosphorylated ALK support the evaluation of on-target ALK inhibitors in neuroblastoma. Mol Oncol. 2017 Aug;11(8):996-1006.

[8]. Identifying and Targeting Sporadic Oncogenic GeneticLiu H, et al. Identifying and Targeting Sporadic Oncogenic Genetic Aberrations in Mouse Models of Triple Negative Breast Cancer. Cancer Discov. 2018 Mar;8(3):354-369.

Pharmacodynamics
In a phase I study, 37 patients with refractory to multiple solid tumors received crizotinib at 50 to 300 mg daily or twice daily. Two patients with non-small cell lung cancer (NSCLC) harboring echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) mutations responded to treatment; therefore, subsequent studies focused on advanced ALK-positive patients. In this group, the 6-month progression-free survival rate in the crizotinib treatment group was approximately 72%. The two-year overall survival rate was significantly higher in ALK-mutant positive patients receiving crizotinib compared to those who did not receive crizotinib (54% vs 36%). The use of crizotinib may cause hepatotoxicity, interstitial lung disease (ILD), pneumonia, QT interval prolongation, bradycardia, severe vision loss, embryo-fetal toxicity and gastrointestinal toxicity in children and adolescents with anaplastic large cell lymphoma (ALCL) or children with inflammatory myofibroblastic tumor (IMT).

---

1. Treatment background: crizotinib (Xalkori; PF02341066) is the first dual c-MET/ALK inhibitor approved by the FDA for the treatment of ALK-positive non-small cell lung cancer (NSCLC) and ROS1-positive NSCLC, meeting an unmet need in the field of targeted therapy[3]
2. Mechanism of action: It competitively binds to the ATP-binding pockets of c-MET and ALK, inhibiting their autophosphorylation and downstream pathways (c-MET-PI3K-AKT, ALK-JAK-STAT3). It can also inhibit tumor angiogenesis by blocking c-MET-mediated endothelial cell activation[1]
3. Resistance mechanism: c-Myc overexpression confers acquired resistance to crizotinib in c-MET-dependent cancers by upregulating other survival pathways (such as MAPK)[5]
4. Structural features: Crizotinib has a unique 2-aminopyridine core structure, which gives it high affinity for both c-MET and ALK, and very little off-target binding[3]

C21H22CL2FN5O

450.34

449.118

C, 56.01; H, 4.92; Cl, 15.74; F, 4.22; N, 15.55; O, 3.55

877399-52-5

Crizotinib hydrochloride;1415560-69-8;Crizotinib-d5;1395950-84-1; 877399-53-6

11626560

white to off-white solid powder

1.5±0.1 g/cm3

599.2±50.0 °C at 760 mmHg

316.2±30.1 °C

0.0±1.7 mmHg at 25°C

1.673

4.73

2

6

5

30

558

1

O(C1C(N)=NC=C(C2C=NN(C3CCNCC3)C=2)C=1)[C@@H](C1C(Cl)=CC=C(F)C=1Cl)C

KTEIFNKAUNYNJU-GFCCVEGCSA-N

InChI=1S/C21H22Cl2FN5O/c1-12(19-16(22)2-3-17(24)20(19)23)30-18-8-13(9-27-21(18)25)14-10-28-29(11-14)15-4-6-26-7-5-15/h2-3,8-12,15,26H,4-7H2,1H3,(H2,25,27)/t12-/m1/s1

3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-2-amine

PF-2341066; PF2341066; PF02341066; PF-02341066; PF 2341066; Crizotinib; PF 02341066; US trade name: Xalkori

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: ≥ 1.25 mg/mL (2.78 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 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 2: ≥ 1 mg/mL (2.22 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 10.0 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.

---

View More

Solubility in Formulation 3: ≥ 1 mg/mL (2.22 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 10.0 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 4: 5% DMSO+30% PEG 300+dd H2O: 5 mg/mL

---

Solubility in Formulation 5: 20 mg/mL (44.41 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; Need ultrasonic and warming and heat to 40°C.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

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