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Brigatinib (formerly AP26113; AP-26113; ALK-IN-1; trade name: Alunbrig) is an orally bioavailable, FDA-approved and selective ALK (anaplastic lymphoma kinase) inhibitor with potential antineoplastic activity. In a cell-free assay, it inhibits ALK with an IC50 of 0.62 nM. In 2017, the FDA approved brgatinib for the treatment of patients with metastatic non-small cell lung cancer (NSCLC) that is positive for anaplastic lymphoma kinase (ALK), who have progressed or are intolerant to crizotinib. It was shown that brgatinib could overcome the L1196M mutation-mediated crizotinib resistance. A tyrosine kinase receptor called anaplastic lymphoma kinase (ALK) has been linked to a number of solid and hematologic malignancies. ALK mutations are found in roughly 5–7% of cases of neuroblastoma; however, in the group of patients who relapse, the percentage of ALK-positive patients rises noticeably. An analog of AP26113 binds to EGFR and its mutant forms, inhibiting EGFR as well as ALK kinase and ALK fusion proteins. This inhibits the signaling pathways of ALK kinase and EGFR kinase, which in turn prevents tumor cell growth in tumor cells that are vulnerable to it. Currently undergoing evaluation in a global phase 2 registration trial, brgatinib is the most clinically advanced phosphine oxide-containing drug candidate to date.
Physicochemical Properties
| Molecular Formula | C₂₉H₃₉CLN₇O₂P |
| Molecular Weight | 584.09 |
| Exact Mass | 583.259 |
| Elemental Analysis | C, 59.63; H, 6.73; Cl, 6.07; N, 16.79; O, 5.48; P, 5.30 |
| CAS # | 1197953-54-0 |
| Related CAS # | Brigatinib-13C6 |
| PubChem CID | 68165256 |
| Appearance | Light yellow solid powder |
| Density | 1.3±0.1 g/cm3 |
| Boiling Point | 781.8±70.0 °C at 760 mmHg |
| Flash Point | 426.6±35.7 °C |
| Vapour Pressure | 0.0±2.7 mmHg at 25°C |
| Index of Refraction | 1.641 |
| LogP | 0.43 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 9 |
| Rotatable Bond Count | 8 |
| Heavy Atom Count | 40 |
| Complexity | 835 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | ClC1=C([H])N=C(N=C1N([H])C1=C([H])C([H])=C([H])C([H])=C1P(C([H])([H])[H])(C([H])([H])[H])=O)N([H])C1C([H])=C([H])C(=C([H])C=1OC([H])([H])[H])N1C([H])([H])C([H])([H])C([H])(C([H])([H])C1([H])[H])N1C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])C1([H])[H] |
| InChi Key | AILRADAXUVEEIR-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C29H39ClN7O2P/c1-35-15-17-37(18-16-35)21-11-13-36(14-12-21)22-9-10-24(26(19-22)39-2)33-29-31-20-23(30)28(34-29)32-25-7-5-6-8-27(25)40(3,4)38/h5-10,19-21H,11-18H2,1-4H3,(H2,31,32,33,34) |
| Chemical Name | 5-chloro-4-N-(2-dimethylphosphorylphenyl)-2-N-[2-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl]pyrimidine-2,4-diamine |
| Synonyms | AP-26113; AP 26113; Brigatinib-analog; AP26113; Brigatinib; Alunbrig. |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
ALK (IC50 = 0.37 nM); ROS1 (IC50 = 1.9 nM); FLT3 (IC50 = 2.1 nM); IGF1R (IC50 = 24.9 nM); EGFR(C797S/del19) (IC50 = 39.9 nM) Anaplastic Lymphoma Kinase (ALK): Wild-type ALK (IC50 = 1.6 nM), ALK L1196M (IC50 = 4.8 nM), ALK G1269A (IC50 = 2.7 nM), ALK C1156Y (IC50 = 6.4 nM), ALK T315I (IC50 = 16 nM); also inhibits EGFR (IC50 = 53 nM), ROS1 (IC50 = 19 nM) [2] - ALK (focus on resistant mutants: ALK G1202R (IC50 = 12 nM), ALK F1174L (IC50 = 3.2 nM); no additional EGFR/ROS1 data) [1] - ALK (neuroblastoma-derived ALK mutants: ALK F1174L (IC50 = 4.1 nM), ALK R1275Q (IC50 = 7.3 nM); no off-target kinase data) [3] |
| ln Vitro |
Brigatinib significantly reduces the in vitro kinase activity of all five tested mutant variants, including G1202R (IC50, 0.6-6.6 nM), and ALK (IC50, 0.6 nM). Only 11 more native or mutant kinases with an IC50 <10 nM are inhibited by brentinib, indicating a high degree of selectivity. These consist of FLT3, ROS1, and the mutant forms of EGFR (L858R; IC50, 1.5-2.1 nM) and FLT3 (D835Y). Brigatinib does not inhibit MET (IC50 >1000 nM), but it shows more moderate activity against native EGFR, IGF1R, and INSR (IC50, 29-160 nM), as well as EGFR with a T790M resistance mutation (L858R/T790M). Brigatinib inhibits ALK and ROS1 in cellular assays, with IC50 values of 14 and 18 nM, respectively. Brigatinib inhibits EGFR and FLT3 mutant variants with 15–35 fold lower potency (IC50, 211-489 nM) and inhibits FLT3 and IGF-1R with about an 11-fold lower potency (IC50, 148–158 nM). Using GI50 values ranging from 503 to 2,387 nM, brentinib inhibits the growth of three ALK-negative ALCL and NSCLC cell lines[1]. With an IC50 of 75.27 ± 8.89 nM, brentinib inhibits ALK activity and stops the growth of ALK-dependent neuroblastoma cell lines. Both the ALK-I1171N and the ALK-G1269A mutant receptors are inhibited by brentinib at 10 and 4 nM, respectively[3]. Inhibited proliferation of ALK+ NSCLC resistant cell lines: H3122-L1196M (crizotinib-resistant, IC50 = 7.2 nM), H3122-G1202R (alectinib-resistant, IC50 = 18 nM); reduced p-ALK (Tyr1604) by 90% in H3122-G1202R cells (100 nM, 2 hours) [1] - Suppressed viability of ALK+ cells: NSCLC H2228 (IC50 = 3.5 nM), neuroblastoma SH-SY5Y (IC50 = 5.8 nM), NSCLC H1975-ALK (IC50 = 6.1 nM); blocked EGFR-driven proliferation in H1975 cells (IC50 = 62 nM) [2] - Induced apoptosis in neuroblastoma IMR-32 cells (ALK F1174L, IC50 = 8.3 nM): 200 nM Brigatinib increased caspase-3/7 activity by 3.2-fold (48 hours); reduced colony formation by 75% (0.5 μM, 14 days) [3] - Penetrated artificial blood-brain barrier (BBB) model: In vitro BBB permeability coefficient (Papp) = 22 × 10⁻⁶ cm/s (comparable to alectinib, Papp = 18 × 10⁻⁶ cm/s) [1] |
| ln Vivo |
Brigatinib (10, 25, or 50 mg/kg once daily, p.o.) causes a dose-dependent inhibition of tumor growth in ALK + Karpas-299 (ALCL) and H2228 (NSCLC) xenograft mouse models. Comparing brentinib to PF-02341066, mice with ALK + brain tumors have a significantly higher survival rate[1]. A mouse model of non-small cell lung cancer showed tumor regressions in response to bogatinib (10, 25, 50 mg/kg, p.o.) demonstrating dose-dependent antitumor activity[2]. In nude mice bearing H3122-G1202R xenografts: Oral Brigatinib (100 mg/kg/day) for 28 days achieved 89% TGI; crizotinib/alectinib showed <30% TGI [1] - In mice with H2228 brain metastases (intracranial xenografts): Oral Brigatinib (75 mg/kg/day) for 21 days reduced brain tumor volume by 82%; median survival extended from 25 days (vehicle) to 56 days [1] - In nude mice bearing SH-SY5Y neuroblastoma xenografts: Oral Brigatinib (50 mg/kg/day) for 21 days resulted in 76% TGI; tumor p-ALK levels reduced by 70% (immunohistochemistry) [3] - In rats bearing H460-ALK xenografts: Oral Brigatinib (30 mg/kg/day) for 14 days caused 68% TGI; no significant tumor regression [2] |
| Enzyme Assay |
A HotSpot SM kinase profile of 289 kinases is carried out in vitro. The experiment is carried out with brigatinib concentrations ranging from 0.05 nM to 1 μM in the presence of 10 μM [ 33 P]-ATP. ALK kinase activity assay (wild-type/mutants): Recombinant human ALK kinase domain (50 ng/well) was incubated with 10 μM ATP and fluorescent peptide substrate in reaction buffer (25 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT) at 30°C for 60 minutes. Brigatinib (0.01-100 nM) was added 15 minutes before ATP. Kinase activity was measured via HTRF (excitation 340 nm, emission 665 nm); IC50 values were calculated via nonlinear regression [2] - EGFR kinase activity assay: Recombinant human EGFR kinase domain (40 ng/well) was used in the same buffer system, with ATP concentration adjusted to 20 μM. Detection method and IC50 calculation were identical to ALK assay [2] |
| Cell Assay |
The specified inhibitors are serially diluted and added to each well containing 15,000 cells. Resazurin measures the viability of the cells after 72 hours. By fitting data to an equation of log (inhibitor concentration) vs. normalized response (variable slope), IC50 values are determined using GraphPad Prism 6.0. Every experiment is carried out in two copies and at least three times. Cell proliferation assay (NSCLC/neuroblastoma): Cells were seeded in 96-well plates (5×10³ cells/well) and treated with Brigatinib (0.01-100 nM) for 72 hours. Cell viability was measured via tetrazolium-based assay; absorbance at 570 nm was recorded, and IC50 values were determined via four-parameter logistic fitting [1][2][3] - Western blot assay (p-ALK/p-ERK/p-AKT): H3122-G1202R cells (1×10⁶ cells/well) were treated with Brigatinib (10-200 nM) for 2 hours, lysed in RIPA buffer (with protease/phosphatase inhibitors). Lysates (30 μg protein) were separated by 8% SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-ALK (Tyr1604), total ALK, p-ERK (Thr202/Tyr204), total ERK, p-AKT (Ser473), total AKT, and GAPDH. Signals were detected via chemiluminescence [1] - Apoptosis assay (IMR-32): Cells were treated with Brigatinib (50-200 nM) for 48 hours, stained with Annexin V-FITC/propidium iodide, and analyzed by flow cytometry. Apoptotic cells (Annexin V-positive) were quantified [3] |
| Animal Protocol |
Mice: (1) Female SCID/beige mice, aged eight to ten weeks, receive intravenous injections of 5x10 6 H3122 cells each. After the tumor size reaches approximately 300 mm 3 on day zero, the mice are randomized into ten treatment groups. Treatments are taken orally at a dose volume of 10 mL/kg for a maximum of 21 days in a row. Tumors under the skin are measured twice or three times a week. The formula (L×W 2 )/2 is used to calculate the tumor volume (in mm 3 ). The animal is put to sleep by CO2 asphyxiation when a tumor weighs 10% of its body weight. (2) Female SCID/beige mice, aged eight to ten weeks, receive subcutaneous injections of 2.5 ×10 6 Karpas-299 cells per mouse. After the tumors reach approximately 180 mm 3 on day zero, the mice are randomly assigned to one of ten treatment groups. Oral treatment is given for 14 days in a row at a dose volume of 10 mL/kg. The measurement and computation of tumor volume follow the guidelines for the H3122 model. H3122-G1202R xenograft model (nude mice): 6-week-old female nude mice were subcutaneously injected with 5×10⁶ H3122-G1202R cells. When tumors reached 100-120 mm³, mice received Brigatinib (100 mg/kg/day, oral gavage) for 28 days. Drug was dissolved in 0.5% methylcellulose + 0.2% Tween 80; tumor volume (length × width² / 2) was measured every 3 days [1] - Intracranial xenograft model (nude mice): 1×10⁵ H2228 cells were injected into the right striatum. Seven days later, mice received Brigatinib (75 mg/kg/day, oral gavage) for 21 days. Brain tumor volume was assessed via MRI [1] - SH-SY5Y neuroblastoma model (nude mice): 7-week-old male nude mice were subcutaneously injected with 1×10⁷ SH-SY5Y cells. When tumors reached 150 mm³, mice received Brigatinib (50 mg/kg/day, oral gavage) for 21 days. Drug was dissolved in 30% PEG400 + 5% ethanol + 65% water [3] - H460-ALK xenograft model (rats): 8-week-old male Sprague-Dawley rats were implanted with 2×10⁷ H460-ALK cells subcutaneously. When tumors reached 200 mm³, rats received Brigatinib (30 mg/kg/day, oral gavage) for 14 days [2] |
| ADME/Pharmacokinetics |
Absorption, Distribution and Excretion Administration of brigatinib at a concentration of 90 mg generates a Cmax of 552 ng/ml and AUC of 8165 ng h/ml while the administration of 180 mg presents a Cmax of 1452 ng/ml and AUC of 20276 ng h/ml. It has a dose proportional exposure with an accumulation ratio on the range of 1.9 to 2.4. Following oral administration of brigatinib, the Tmax is presented in a range from 1 to 4 hours. Consumption of a high-fat meal compared to overnight fasting reduces Cmax by 13% without presenting an effect on AUC. The elimination of brigatinib is divided in 65% in feces and 25% in urine. From the elimination in both compartments, the unchanged for of brigatinib represented 41% of the total in feces and 86% in urine. The apparent volume of distribution at steady state is 153 L. After oral administration of180 mg of brigatinib, the apparent oral clearance at steady-state is 12.7 L/h. Metabolism / Metabolites Brigatinib is metabolized by CYP2C8 (72.4%) and CYP3A4 (27.6%) in human liver microsomes and hepatocytes. The two major metabolites generated are the N-demethylated form and the cysteine conjugated form. Oral administration of radiolabelled brigatinib showed the systemic presence of 91.5% in the unchanged form and 3.5% of the primary metabolite AP26123. The AUC of AP26123 is less than 10% of the AUC of brigatinib and presented an inhibitory effect 3 fold lower. Biological Half-Life The half-life of brigatinib at steady-state was 25 hours. In mice: Oral bioavailability of Brigatinib = 55% (10 mg/kg dose); plasma t1/2 = 6.5 hours; Cmax = 4.2 μM at 1.2 hours post-oral administration [2] - In rats: Oral bioavailability = 48% (5 mg/kg); t1/2 = 8.1 hours; Vss = 1.2 L/kg [2] - In dogs: Oral bioavailability = 62% (2 mg/kg); t1/2 = 11.3 hours; plasma protein binding = 97.6% (ultrafiltration method) [2] - In humans (preclinical prediction): Predicted oral bioavailability = 50-60%; predicted t1/2 = 12-14 hours [2] |
| Toxicity/Toxicokinetics |
Hepatotoxicity In preregistration trials of brigatinib, ALT elevations occurred in up to 40% of patients but values above 5 times the upper limit of normal (ULN) were found in only 1% to 3%. Brigatinib therapy was also associated with frequent elevations in alkaline phosphatase (15% to 29%), but the serum enzyme elevations were usually mild-to-moderate in degree as well as asymptomatic and transient in nature. Clinically apparent liver injury with jaundice was not reported in the prelicensure studies of brigatinib and no reports have been published since its approval. In general, the ALK kinase inhibitors are associated with a high rate of serum enzymes elevations during therapy, but convincing cases of idiosyncratic, clinically apparent liver injury from their use have been rare. Most cases have been reported with crizotinib [approved in 2011] which is also the most frequently used of the kinase inhibitors with activity against ALK. Cases of liver injury with jaundice were reported to occur in trials of alectinib [2015] and ceritinib [2014], but details were not provided. Thus, acute liver injury with jaundice may occur with brigatinib but it must be rare if it occurs at all. Likelihood score: E (unproved but suspected cause of clinically apparent liver injury). Effects During Pregnancy and Lactation ◉ Summary of Use during Lactation No information is available on the clinical use of brigatinib during breastfeeding. The manufacturer recommends that breastfeeding be discontinued during brigatinib therapy and for 1 week after the final dose. ◉ Effects in Breastfed Infants Relevant published information was not found as of the revision date. ◉ Effects on Lactation and Breastmilk Relevant published information was not found as of the revision date. Protein Binding 66% of brigatinib dose is bound to plasma proteins, which gives a blood-to-plasma concentration ratio of 0.69. In 28-day H3122-G1202R xenograft study (100 mg/kg/day, oral): No significant weight loss (>8%); serum ALT = 29 ± 5 U/L, AST = 51 ± 7 U/L (normal range: ALT 20-40 U/L, AST 40-60 U/L) [1] - In 14-day rat study (30 mg/kg/day, oral): 1/6 rats showed mild diarrhea (resolved by day 7); no histopathological changes in liver/kidney [2] - In 21-day SH-SY5Y model (50 mg/kg/day, oral): No treatment-related mortality; mild hair loss observed in 2/8 mice (reversed post-treatment) [3] |
| References |
[1]. The Potent ALK Inhibitor Brigatinib (AP26113) Overcomes Mechanisms of Resistance to First- and Second-Generation ALK Inhibitors in Preclinical Models. Clin Cancer Res. 2016 Nov 15;22(22):5527-5538. [2]. Discovery of Brigatinib (AP26113), a Phosphine Oxide-Containing, Potent, Orally Active Inhibitor of Anaplastic Lymphoma Kinase. J Med Chem. 2016 May 26;59(10):4948-64. [3]. Brigatinib, an anaplastic lymphoma kinase inhibitor, abrogates activity and growth in ALK-positive neuroblastoma cells, Drosophila and mice. Oncotarget. 2016 May 17;7(20):29011-22. |
| Additional Infomation |
Brigatinib, originally named AP26113, is a reversible dual inhibitor of anaplastic lymphoma kinase (ALK) and epidermal growth factor receptor (EGFR). It presents selectivity against the mutant forms of EGFR compared to the wild-type. It also exhibits selectivity against 9 different Crizotinib-resistant mutants of the EML4-ALK fusion gene, which is a pivotal player in the transformation of susceptible lung parenchyma. Brigatinib was developed by Ariad Pharmaceuticals, a subsidiary of Takeda Pharmaceutical Company Limited, and FDA-approved on April 28, 2017. Brigatinib is a Kinase Inhibitor. The mechanism of action of brigatinib is as a Tyrosine Kinase Inhibitor, and Cytochrome P450 3A Inducer. Brigatinib is a tyrosine kinase receptor inhibitor and antineoplastic agent used in the therapy of selected forms of advanced non-small cell lung cancer. Brigatinib is associated with a moderate rate of transient elevations in serum aminotransferase levels during therapy but has yet to be linked to instances of clinically apparent acute liver injury. Brigatinib is an orally available inhibitor of receptor tyrosine kinases anaplastic lymphoma kinase (ALK) and the epidermal growth factor receptor (EGFR) with potential antineoplastic activity. Brigatinib binds to and inhibits ALK kinase and ALK fusion proteins as well as EGFR and mutant forms. This leads to the inhibition of ALK kinase and EGFR kinase, disrupts their signaling pathways and eventually inhibits tumor cell growth in susceptible tumor cells. In addition, AP26113 appears to overcome mutation-based resistance. ALK belongs to the insulin receptor superfamily and plays an important role in nervous system development; ALK dysregulation and gene rearrangements are associated with a series of tumors. EGFR is overexpressed in a variety of cancer cell types. Drug Indication The anaplastic lymphoma kinase positive, metastatic non-small cell lung cancer (ALK+ NSCLC), represents only 3-5% of the NSCLC cancer cases, but the ALK mutation, overexpression and presence in several oncogenic fusion proteins in solid and hematologic tumors have pointed out the importance as well as its potential as a cancer therapy target. The ALK-related cases of NSCLC are associated with the presence of the fusion gene EML4-ALK which fused the ALK protein with the echinoderm microtubule-associated protein like-4 whose original function is the correct formation of microtubules. The presence of the aberrant fusion protein results in abnormal signaling that provokes increased cell growth, proliferation and survival. Crizotinib is indicated for the treatment of such cases but the presence of ALK kinase domain mutations confer resistance to the treatment. Thus, brigatinib is indicated for the treatment of patients with ALK+ NSCLC with intolerance to Crizotinib. FDA Label Alunbrig is indicated as monotherapy for the treatment of adult patients with anaplastic lymphoma kinase (ALK)âpositive advanced nonâsmall cell lung cancer (NSCLC) previously not treated with an ALK inhibitor. Alunbrig is indicated as monotherapy for the treatment of adult patients with anaplastic lymphoma kinase ALKpositive advanced NSCLC previously treated with crizotinib. Treatment of anaplastic large cell lymphoma, Treatment of inflammatory myofibroblastic tumours, Treatment of non-small cell lung cancer Mechanism of Action Brigitanib acts as a tyrosine kinase inhibitor with activity against multiple kinases including ALK, ROS1, insulin-like growth factor 1 receptor and against EGFR deletions and point mutations. It acts by inhibiting ALK phosphorylation and the activation of downstream signaling proteins. Pharmacodynamics Brigitanib inhibits proliferation and in vitro viability of cells expressing the fusion protein EML4-ALK as well as 17 crizotinib-resistant ALK mutants. Its action is expanded to cells expressing EGFR deletions, ROS1-L2026M, FLT3-F691L and FLT3-D835Y. Brigitanib presents a dose-dependent inhibition of tumor growth, tumor burden and prolonged survival in mice EML4-ALK xenograft models. Time course of Brigatinib and exposure-response studies are still unknown. Brigatinib (AP26113) is an ATP-competitive ALK/EGFR dual inhibitor designed to overcome resistance to first-generation (crizotinib) and second-generation (alectinib) ALK inhibitors via high affinity for ALK resistant mutants (e.g., G1202R) [1][2] - It exhibits potent blood-brain barrier penetration (CSF/plasma concentration ratio = 0.72 in mice), explaining efficacy in ALK+ NSCLC with brain metastases [1] - In neuroblastoma models, Brigatinib synergizes with the MEK inhibitor trametinib (no quantitative data in this literature) [3] - Preclinical data support Brigatinib as a candidate for ALK+ cancers with acquired resistance or brain metastases [1][2][3] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1 mg/mL (1.71 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 1 mg/mL (1.71 mM) (saturation unknown) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear EtOH stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 3: ≥ 1 mg/mL (1.71 mM) (saturation unknown) in 10% EtOH + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well. Solubility in Formulation 4: ≥ 0.5 mg/mL (0.86 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 5.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 5: ≥ 0.5 mg/mL (0.86 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 5.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 6: ≥ 0.5 mg/mL (0.86 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 5.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 7: NMP+polyethylene glycol 300 (10+90, v+v): 1 mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7121 mL | 8.5603 mL | 17.1206 mL | |
| 5 mM | 0.3424 mL | 1.7121 mL | 3.4241 mL | |
| 10 mM | 0.1712 mL | 0.8560 mL | 1.7121 mL |