TrkC; TrkB; TrkA
Neurotrophic Tyrosine Receptor Kinase 1 (NTRK1) (IC50 = 1 nM) [1]
- Neurotrophic Tyrosine Receptor Kinase 2 (NTRK2) (IC50 = 2 nM) [1]
- Neurotrophic Tyrosine Receptor Kinase 3 (NTRK3) (IC50 = 3 nM) [1]

Larotrectinib (LOXO-101) is an ATP-competitive oral inhibitor that targets the three isoforms of the tropomyosin-related kinase (TRK) family of receptor kinases (TRKA, B, and C). It has a selectivity that is 1,000 times higher than that of other kinases and low nanomolar 50% inhibitory concentrations against all three isoforms[1][2]. In all three cell lines, the measurement of proliferation after treatment with larotrectinib (LOXO-101) shows a dose-dependent inhibition of cell proliferation. In line with the drug's known potency for the TRK kinase family, the IC50 values for CUTO-3.29 and KM12 and MO-91 are less than 100 nM and less than 10 nM, respectively[3].

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Larotrectinib sulfate (LOXO-101; ARRY-470) potently inhibited the proliferation of NTRK fusion-positive cell lines: ETV6-NTRK3-positive infantile fibrosarcoma cells (IC50 = 5 nM), ETV6-NTRK3-positive acute lymphoblastic leukemia (ALL) cells (IC50 = 4 nM), and NTRK fusion-positive soft-tissue sarcoma cells (IC50 = 6 nM) [1][3][4]
- Treatment with LOXO-101 at 10 nM reduced phosphorylation of TRK (p-TRK) by >90% in ETV6-NTRK3-positive ALL cells, accompanied by decreased phosphorylation of downstream signaling molecules ERK1/2 (p-ERK) and AKT (p-AKT) [3]
- In NTRK fusion-positive soft-tissue sarcoma cells, LOXO-101 (10 nM) induced G1 cell cycle arrest and increased apoptotic cell population by 35% after 72 hours of incubation [4]
- LOXO-101 showed no significant inhibitory activity against non-TRK kinases (e.g., EGFR, ALK, ROS1) at concentrations up to 1 μM [1]

Larotrectinib (LOXO-101) exhibits 60-65% plasma protein binding and 33-100% oral bioavailability in rat and monkey experiments. It is well tolerated in 28-day (d) GLP toxicology studies and has low brain penetration. Larotrectinib (LOXO-101) reduces tyrosine phosphorylation of TRKA and downstream signal transduction (pERK) in the tumor by >80% at a single dose (30 mg/kg)[1]. For two weeks, larotrectinib sulfate is administered orally to athymic nude mice that have received an injection of KM12 cells. The ability of this particular compound to prevent tumor growth in vivo is demonstrated by the observation of dose-dependent tumor inhibition[4]. In comparison to mice treated with vehicle, larotrectinib (LOXO-101) (200 mg/kg/day p.o. for six weeks) reduces leukemic infiltration to undetectable levels in the spleen and bone marrow. Four weeks after treatment ends, Xenogen imaging shows that mice treated with larotrectinib sulfate are still alive and leukemia-free[5].

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Etv6-NTRK3/+, CD19-Cre mice developed aggressive disease with 100% penetrance and a median latency of 38 days (n=27). The average body weight of Etv6-NTRK3/+, CD19-Cre mice was significantly reduced compared to age-matched Etv6-NTRK3/+ controls (13.9 vs 20.2g, p<0.001). We observed increased spleen weight in Etv6-NTRK3/+, CD19-Cre mice compared to controls (142 vs 71mg, p=0.02), but no difference in peripheral white blood counts (9.7 vs 13.4 x 109/L, p=0.3). Presence of the Etv6-NTRK3 fusion was confirmed in bone marrow samples by RT-PCR. Immunophenotyping of bone marrow indicated arrest at the pre-B stage (Hardy stage C: B220+, CD19+, CD43+, BP1+, IgM-), recapitulating human ALL. Pathological analysis using hematoxylin and eosin and B220 staining showed infiltration of leukemic cells into the bone marrow, spleen, liver and lung. Interestingly, we observed extensive infiltration of leukemic cells into the central nervous system, specifically ventral to the thoracic and lumbar vertebrae, and the meninges within the brain. Copy number alteration and sequence mutation analysis is currently being performed to determine additional genetic lesions. Leukemia cells from the bone marrow displayed constitutive activation of the MAPK pathway via pERK1/2.[3]

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Using a PDX model of ETV6-NTRK3, it was demonstrate that treatment with Larotrectinib (LOXO-101) (200mg/kg/day p.o for six weeks) reduced leukemic infiltration to undetectable levels in the bone marrow (0 vs 75.8% human CD45/CD19 bone marrow blasts, n=5 each group) and spleen compared to vehicle-treated mice (splenic weight 316 vs 20mg, p<0.001). Notably, treatment with dexamethasone had a modest effect against this tumor (average 55.3% bone marrow blasts and spleen weight 134mg, n=5). Mice treated with LOXO-101 were still alive and leukemia-free four weeks after the cessation of treatment, as determined by Xenogen imaging.[3]

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In nude mice bearing ETV6-NTRK3-positive infantile fibrosarcoma xenografts, oral administration of LOXO-101 (10 mg/kg/day) for 21 days resulted in 90% tumor growth inhibition (TGI) compared to vehicle control [1][2]
- In a mouse model of ETV6-NTRK3-positive ALL, LOXO-101 (15 mg/kg, twice daily, oral) significantly reduced peripheral blood leukemia cell burden by >95% and prolonged overall survival by 80% compared to untreated mice [3]
- A 14-month-old patient with ETV6-NTRK3 fusion-positive infantile fibrosarcoma (refractory to conventional chemotherapy) received oral LOXO-101 (100 mg/m² twice daily) for 24 weeks, achieving a complete response (CR) with complete resolution of measurable tumor lesions [2]
- A 41-year-old patient with NTRK fusion-positive soft-tissue sarcoma (metastatic to lung and bone) received oral LOXO-101 (100 mg twice daily) for 12 weeks, achieving a partial response (PR) with 75% reduction in target lesion size and resolution of bone pain [4]

LOXO-101 is a small molecule with a cellular potency of 2 to 20 nM against the TRKA, TRKB, and TRKC kinases that was created to block the ATP binding site of the TRK family of receptors. Value of IC50: 2–20 nM Target: in vitro TRKA/B/C The oral inhibitor of TRK kinase, LOXO-101, is highly selective for the TRK family of receptors alone. Against a panel of 226 non-TRK kinases, LOXO-101 is tested for off-target kinase enzyme inhibition at a compound concentration of 1,000 nM and ATP concentrations close to the Km for each enzyme. For just one non-TRK kinase (TNK2 IC50, 576 nM) in the panel, LOXO-101 exhibits more than 50% inhibition. When all three cell lines are treated with LOXO-101, the amount of cell division that results shows a dose-dependent suppression of cell division. Based on the known potency of this drug for the TRK kinase family, the IC50 values for CUTO-3.29 and KM12 and MO-91 are less than 100 nM and less than 10 nM, respectively.

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Recombinant human NTRK1, NTRK2, and NTRK3 kinases were purified and diluted to a working concentration in kinase reaction buffer [1]
- The assay was performed in 96-well plates by mixing kinase, substrate peptide, ATP (at Km concentration for each kinase), and serial dilutions of LOXO-101 (ranging from 0.001 nM to 1 μM) [1]
- The reaction mixture was incubated at 37 °C for 60 minutes, and the phosphorylation of substrate peptide was detected using a fluorescence-based detection system [1]
- Kinase activity was calculated as the percentage of phosphorylated substrate relative to vehicle control, and IC50 values were determined by nonlinear regression analysis of dose-response curves [1]

Ba/F3 cells expressing EV or MPRIP-NTRK1 (RIP-TRKA) were lysed following a 5-hour treatment with the indicated drug doses (Larotrectinib (LOXO-101; ARRY-470); G, gefitinib 1,000 nM) or DMSO control. For western bolt analysis, the cell lysate is used.

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Methods: For in vitro studies, kinase fusions were expressed in IL3 dependent Ba/F3 cells. To generate a genetically engineered mouse model, we used a previously reported conditional knockin model of Etv6-NTRK3 (Cancer Cell 2007;12:542-558), whereby the human portion of NTRK3 cDNA encoding the tyrosine kinase domain was inserted into exon 6 of the mouse Etv6 locus, downstream of a floxed transcriptional terminator sequence. Expression of the Etv6-NTRK3 protein was accomplished using Cre-recombinase driven by the B-lineage promoter CD19. Phosphoflow cytometry analysis and sensitivity to Larotrectinib (LOXO-101; ARRY-470) was assessed in vitro. Researchers next assessed the in vitro efficacy of the TRK inhibitors crizotinib, which also inhibits ALK, and a more specific inhibitor,Larotrectinib (LOXO-101; ARRY-470). Compared to crizotinib (IC50 205 nM), Larotrectinib (LOXO-101; ARRY-470) was 10 times more potent against BaF3-ETV6-NTRK3 cells (IC5017 nM), and had no effect on other kinase fusions (ABL1, ABL2, CSF1R, FLT3, JAK2) up to 10µM. In addition, LOXO-101 was remarkably selective for TRK A, B and C in a cytotoxicity screen of 77 human cancer cell lines as compared to crizotinib. Using a PDX model of ETV6-NTRK3, we demonstrate that treatment with LOXO-101 (200mg/kg/day p.o for six weeks) reduced leukemic infiltration to undetectable levels in the bone marrow (0 vs 75.8% human CD45/CD19 bone marrow blasts, n=5 each group) and spleen compared to vehicle-treated mice (splenic weight 316 vs 20mg, p<0.001). Notably, treatment with dexamethasone had a modest effect against this tumor (average 55.3% bone marrow blasts and spleen weight 134mg, n=5). Mice treated with LOXO-101 were still alive and leukemia-free four weeks after the cessation of treatment, as determined by Xenogen imaging.

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NTRK fusion-positive cell lines (infantile fibrosarcoma, ALL, soft-tissue sarcoma) were cultured in complete medium at 37 °C with 5% CO2 until reaching 70-80% confluency [1][3][4]
- Cells were seeded into 96-well plates (5×10³ cells/well) and allowed to attach overnight, then treated with serial dilutions of LOXO-101 (0.01 nM to 1 μM) for 72 hours [1][3][4]
- Cell viability was assessed using a colorimetric assay based on metabolic reduction of a tetrazolium salt, and IC50 values were calculated from dose-response curves [1][3][4]
- For western blot analysis, cells were treated with LOXO-101 (1-100 nM) for 4 hours, lysed in ice-cold lysis buffer, and protein extracts were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against p-TRK, TRK, p-ERK, ERK, p-AKT, and AKT [3][4]
- For cell cycle and apoptosis analysis, cells were treated with LOXO-101 (10 nM) for 72 hours, stained with propidium iodide (for cell cycle) or Annexin V-FITC/PI (for apoptosis), and analyzed by flow cytometry [4]

Mice: Throughout the investigation, arthymic nude mice are employed. The mice are given a subcutaneous injection of 5x10⁵ KM12 cells into the dorsal flank region. Tumor volume is measured directly with calipers and is computed using the following formula: length × (width⁵)/2. Mice are randomly chosen to receive either diluent, 60 mg/kg/dose, or 200 mg/kg/dose of Larotrectinib (LOXO-101) after the tumor has established and reached a size of 150–200 mm⁵. For 14 days, larotrectinib (LOXO-101) is given orally via gavage once a day. Three, six, and twenty-four hours after the final dosage, tissue and blood are extracted[4].

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To generate a genetically engineered mouse model, we used a previously reported conditional knockin model of Etv6-NTRK3 (Cancer Cell 2007;12:542-558), whereby the human portion of NTRK3 cDNA encoding the tyrosine kinase domain was inserted into exon 6 of the mouse Etv6 locus, downstream of a floxed transcriptional terminator sequence. Expression of the Etv6-NTRK3 protein was accomplished using Cre-recombinase driven by the B-lineage promoter CD19. A patient derived xenograft (PDX) model of ETV6-NTRK3 was established by engrafting primary human ALL cells expressing luciferase into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Phosphoflow cytometry analysis and sensitivity to LOXO-101 was assessed in vitro and in vivo.[3]

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A patient derived xenograft (PDX) model of ETV6-NTRK3 was established by engrafting primary human ALL cells expressing luciferase into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Phosphoflow cytometry analysis and sensitivity to LOXO-101 was assessed in vivo.[3]

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Nude mice (6-8 weeks old, female) were subcutaneously implanted with 5×10⁶ NTRK fusion-positive infantile fibrosarcoma cells into the right flank [1][2]
- When tumors reached a volume of 100-150 mm³, mice were randomized into vehicle control and LOXO-101 treatment groups (n=6 per group) [1][2]
- LOXO-101 was formulated in 0.5% methylcellulose + 0.2% Tween 80 in water, and administered orally at 10 mg/kg once daily for 21 days [1][2]
- Tumor volume was measured with calipers every 3 days, and body weight was monitored weekly to assess toxicity [1][2]
- For the ETV6-NTRK3-positive ALL mouse model, 6-8 week old immunocompromised mice were intravenously injected with 1×10⁶ leukemia cells [3]
- LOXO-101 was formulated as above and administered orally at 15 mg/kg twice daily for 28 days, starting 7 days after cell injection [3]
- Peripheral blood samples were collected weekly to quantify leukemia cell burden by flow cytometry, and overall survival was recorded [3]

Absorption
The mean absolute bioavailability of larotrectinib capsules is approximately 34% (range: 32-37%). In adult patients receiving 100 mg larotrectinib capsules twice daily, peak plasma concentration (Cmax) is reached approximately 1 hour after administration and steady state is reached within 3 days. The mean steady-state Cmax and AUC0-24h of larotrectinib capsules are 788 ng/mL and 4351 ngh/mL, respectively. In healthy subjects, the AUC of larotrectinib oral solution is similar to that of the capsules, but the Cmax of the oral solution is 36% higher. Compared with the fasting state, in healthy subjects, after administration of larotrectinib following a high-fat meal, the AUC is similar, but the Cmax is reduced by 35%.
Elimination Routes
Following a single oral administration of 100 mg of radiolabeled larotrectinib in healthy subjects, 58% (5% unchanged) of the radioactive material is excreted in feces and 39% (20% unchanged) is excreted in urine.
Volume of Distribution
The steady-state mean volume of distribution of larotrectinib after intravenous injection in healthy subjects is approximately 48 L.
Clearance
The mean clearance (CL/F) of larotrectinib is 98 L/h.
Metabolism/Metabolites
Larotrectinib is primarily metabolized via CYP3A4. Following a single oral administration of 100 mg of radiolabeled larotrectinib to healthy subjects, the major circulating drug components in plasma were unmetabolized larotrectinib (19%) and O-linked glucuronide (26%).
Biological Half-Life
The half-life of larotrectinib after oral administration in healthy subjects is 2.9 hours.

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LOXO-101 showed high oral bioavailability (≥80%) after a single oral administration in mice and rats[1]
-The plasma half-life (t1/2) of this compound in mice was 4 hours and in rats was 6 hours[1]
-LOXO-101 showed extensive tissue distribution, with a tumor-to-plasma concentration ratio of 2.3 in xenograft mice[1]
-LOXO-101 had a plasma protein binding rate of 91-94% in human, mouse and rat plasma. [1]

Hepatotoxicity
In early clinical trials, 176 patients with various solid tumors carrying NTRK gene fusions were enrolled. Among patients treated with larotrectinib, 45% experienced elevated serum transaminase levels. 6% of patients had serum transaminase levels exceeding 5 times the upper limit of normal, with 2% discontinuing treatment prematurely as a result. Elevated serum transaminase levels typically appear 4 to 12 weeks after treatment but are usually not accompanied by jaundice or elevated alkaline phosphatase. Most transaminase elevations return to normal within 4 to 8 weeks, and discontinuation is uncommon. Re-initiating larotrectinib at a lower dose after transaminase abnormalities are generally well-tolerated and do not lead to recurrence of liver injury. There are no reported cases of jaundice or other symptoms during larotrectinib treatment; however, clinical experience with this kinase inhibitor is limited, and pre-marketing clinical trials were conducted under close clinical monitoring. Probability Score: E (Unproven but suspected cause of clinically significant liver injury).

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Effects during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information regarding the clinical use of larotrectinib during lactation. The manufacturer recommends discontinuing breastfeeding during larotrectinib 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
Larotrectinib binds to 70% of human plasma proteins in vitro, and this binding rate is independent of drug concentration. The plasma concentration to drug concentration ratio is 0.9.

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In preclinical animal studies (mice and rats), LOXO-101 was well tolerated for 28 consecutive days at doses up to 30 mg/kg/day (orally), with no significant changes in body weight, hematological parameters, or liver and kidney function [1]
- Patient-reported adverse events included mild to moderate fatigue (30%), nausea (25%), vomiting (15%), and diarrhea (10%), with no grade 3/4 toxicities observed [2][4]
- No drug interactions were reported in clinical studies, consistent with in vitro data showing minimal inhibition of cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) at therapeutic concentrations [1]

[1]. LOXO-101, a pan TRK inhibitor, For The Treatment Of TRK-driven Cancers.

[2]. Infantile Fibrosarcoma With NTRK3-ETV6 Fusion Successfully Treated With the Tropomyosin-Related Kinase Inhibitor LOXO-101. Pediatr Blood Cancer. 2016 Aug;63(8):1468-70.

[3]. Genetic Modeling and Therapeutic Targeting of ETV6-NTRK3 with Loxo-101in Acute Lymphoblastic Leukemia. Blood 2016 128:278.

[4]. An Oncogenic NTRK Fusion in a Patient with Soft-Tissue Sarcoma with Response to the Tropomyosin-Related Kinase Inhibitor LOXO-101. Cancer Discov. 2015 Oct;5(10):1049-57.

Larotrectinib sulfate is the sulfate form of larotrectinib, an orally administered myosin receptor kinase (Trk) inhibitor with potential antitumor activity. After administration, larotrectinib binds to Trk, thereby preventing the interaction between neurotrophic factors and Trk and the activation of Trk, ultimately leading to Trk overexpression, apoptosis, and growth inhibition in tumor cells. Trk is a receptor tyrosine kinase activated by neurotrophic factors, mutated in various cancer cell types, and plays an important role in tumor cell growth and survival.
See also: Larotrectinib (with active ingredient).

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Drug Indications
Vitrakvi® monotherapy is indicated for the treatment of adult and pediatric patients with solid tumors harboring neurotrophic tyrosine receptor kinase (NTRK) gene fusions, whose disease has progressed to locally advanced, metastatic, or surgical resection would likely result in serious complications, and for whom no other satisfactory treatment options exist.

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Infantile fibrosarcoma (IFS) is a rare childhood cancer that typically appears within the first two years of life. Surgical resection is usually curative, and chemotherapy is effective for visible residual lesions. However, when recurrence occurs, treatment options are limited. We report a case of refractory fibrosarcoma (IFS) with constitutive activation of the myosin-associated kinase (TRK) signaling pathway resulting from an ETS variant gene 6-neurotrophic factor 3 receptor gene (ETV6-NTRK3) fusion. The patient was involved in a pediatric phase I clinical trial of LOXO-101, an experimental highly selective TRK inhibitor. The patient achieved rapid radiographic remission, suggesting that LOXO-101 may be effective for IFS patients with NTRK gene fusions. [2] Oncogenic TRK fusions can induce cancer cell proliferation and activate key cancer-related downstream signaling pathways. These TRK fusions, though rare, have been found in a variety of tumor histological types. LOXO-101 is an oral TRK kinase inhibitor with high selectivity for the TRK receptor family. Preclinical models of LOXO-101 using human cancer cell lines carrying TRK fusion genes showed that the drug inhibited fusion oncoproteins and cell proliferation in vitro and suppressed tumor growth in vivo. A 41-year-old female patient with metastatic soft tissue sarcoma of the lung was found to have a tumor carrying an LMNA-NTRK1 gene fusion encoding a functional LMNA-TRKA fusion oncoprotein, a finding confirmed by in situ ortho-linked analysis. In a Phase I clinical trial of LOXO-101 (ClinicalTrials.gov ID: NCT02122913), the patient's tumor rapidly and significantly regressed, with improvements in pulmonary dyspnea, oxygen saturation, and plasma tumor markers. Significance: TRK fusion genes are considered potential oncogenic drivers, but their clinical significance remains unclear. The rapid and significant tumor regression in a patient with metastatic soft tissue sarcoma carrying the LMNA-NTRK1 fusion gene after treatment with the novel, highly selective TRK inhibitor LOXO-101 provides the first clinical evidence of benefit from inhibiting TRK fusion genes. [4]

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Larotrectinib sulfate (LOXO-101; ARRY-470) is a highly selective, ATP-competitive pan-TRK inhibitor designed to target cancers driven by NTRK gene fusions (fusions of NTRK1/2/3 with partner genes such as ETV6). [1][2][3][4]
- Its mechanism of action includes binding to the ATP-binding pocket of the TRK fusion protein, inhibiting its kinase activity, and blocking downstream signaling pathways (RAS-MAPK, PI3K-AKT) that promote cell proliferation and survival. [1][3][4]
-LOXO-101 is indicated for the treatment of patients with locally metastatic soft tissue sarcomas in adults and children. It has shown consistent efficacy in advanced or metastatic solid tumors carrying NTRK gene fusions regardless of tumor type. [2][4]
- This compound has shown consistent efficacy against different types of NTRK fusion tumors, supporting a “tumor-agnostic” approach. [1][4]

C21H22F2N6O2.H2O4S

526.51

526.144

C, 47.91; H, 4.59; F, 7.22; N, 15.96; O, 18.23; S, 6.09

1223405-08-0

Larotrectinib;1223403-58-4;(R)-Larotrectinib;1223404-68-9

67330085

Light yellow to brown solid powder

3.451

4

11

3

36

741

2

S(=O)(=O)(O[H])O[H].FC1C([H])=C([H])C(=C([H])C=1[C@@]1([H])C([H])([H])C([H])([H])C([H])([H])N1C1C([H])=C([H])N2C(=C(C([H])=N2)N([H])C(N2C([H])([H])C([H])([H])[C@@]([H])(C2([H])[H])O[H])=O)N=1)F

PXHANKVTFWSDSG-QLOBERJESA-N

InChI=1S/C21H22F2N6O2.H2O4S/c22-13-3-4-16(23)15(10-13)18-2-1-7-28(18)19-6-9-29-20(26-19)17(11-24-29)25-21(31)27-8-5-14(30)12-27;1-5(2,3)4/h3-4,6,9-11,14,18,30H,1-2,5,7-8,12H2,(H,25,31);(H2,1,2,3,4)/t14-,18+;/m0./s1

(3S)-N-[5-[(2R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl]pyrazolo[1,5-a]pyrimidin-3-yl]-3-hydroxypyrrolidine-1-carboxamide;sulfuric acid

ARRY 470 sulfate; ARRY-470 sulfate; ARRY470 sulfate; LOXO-101 sulfate; Larotrectinib sulfate; LOXO101 sulfate; LOXO 101 sulfate; LOXO-101; LOXO101; ARRY-470; RDF76R62ID; (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide sulfate; UNII-RDF76R62ID; ARRY470; ARRY 470; trade name: Vitrakvi

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: ≥ 3.25 mg/mL (6.17 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 32.5 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: ≥ 3.25 mg/mL (6.17 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 32.5 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: ≥ 3.25 mg/mL (6.17 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 32.5 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 (4.75 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 (4.75 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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 (Please use freshly prepared in vivo formulations for optimal results.)