PPARβ/δ (IC50 = 22.9 μM)

BS-181 suppresses the growth of cancer cells and induces cell cycle arrest; its IC50 values range from 11.5 to 37 μM, and its effects are seen in all tested cell lines. With an apparent IC50 of 15 μM, BS-181 inhibits RB phosphorylation at Ser⁷⁹⁵ and Ser⁸²¹, which is comparable to the IC50 found for P-Ser2 inhibition. MCF-7 cells treated with BS-181 undergo apoptosis and G1 arrest[1]. The growth of normal gastric epithelial RGM-1 cell line and GC cell line is inhibited by BS-181, with inhibitory concentrations (IC50) ranging from 6.5 μM to 17 to 22 μM. In a dose-dependent manner, BS-181 dramatically reduces the ability of cells to migrate and invade[2].

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Synthesis of BS-181and in vitro kinase inhibition [1]
BS-181 was synthesised from dichloropyrazolo[1,5–a]pyrimidine 2 by sequential selective substitution of the C-7 chloride using benzylamine, Boc-protection, palladium-catalysed displacement of the C-5 chloride using di-Boc-1,6-hexanediamine under Buchwald-Hartwig reaction conditions and de-protection in acidic methanol (Supplemental Fig. S1). Inhibition of CDK7 activity was measured by incubation of increasing amounts of BS-181 with purified recombinant CDK7/CycH/MAT1 complex, followed by measurement of free ATP remaining in the reaction using a luciferase assay (PkLight, Cambrex, UK), luciferase activity therefore providing a measure of inhibition of CDK7 activity. BS-181 inhibited CDK7 activity with an IC50 = 21 nM (Table 1; Supplemental Data Fig. S2), whilst the IC50 achieved with roscovitine was 510 nM, in agreement with previous reports for inhibition of CDK7 by roscovitine (13). The IC50 values for inhibition of CDK1/cycB, CDK4/cycD1, CDK5/p35NCK, CDK6/cycD1 and CDK9/cycT by BS-181 were considerably higher than 1 μM, with inhibition of CDK2/cycE having an IC50 = 880 nM, about 40-fold higher than the IC50 for CDK7.

Seventy protein kinases from many different classes, were tested for inhibition by BS-181. Some inhibition of the activities of several kinases was observed using high concentrations (10 μM) of BS-181 (Supplemental Table 5). Performing IC50 measurements for those kinases that showed the greatest inhibition, CDK2/cycA, CK1 and DYRK1A, demonstrated IC50 of 730 nM, 7.36 μM and 2.3 μM, respectively. These data confirm that BS-181 is a highly selective inhibitor of CDK7 activity.

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BS-181 Promotes Cell Cycle Arrest and Inhibits Cancer Cell Growth [1]
To assess the anti-proliferative activity of BS-181, a panel of cell lines representing a range of tumour types, including breast, lung, prostate and colorectal cancer, were treated with increasing concentrations of BS-181 for 72 hours. Determination of proliferation using the sulforhodamine B assay showed that growth was inhibited for all cell lines tested, with IC50 values ranging from 11.5 to 37 μM (Table 2). The growth inhibition observed for BS-181 was similar to that observed for Roscovitine, which gave IC50 values in the range of 8 to 33.5 μM.

Immunoblotting for CDKs and cyclins following BS-181 or Roscovitine treatment for 4 hours showed down-regulation of CDK4 and cyclin D1 (Fig. 2C), with levels of the other CDKs and cyclins remaining unaffected. Additionally, levels of the anti-apoptotic proteins XIAP and Bcl-xL was reduced by BS-181, with Bcl-2 levels being unchanged.

Treatment with low concentrations of BS-181 for 24 hours showed an increase in cells in G1, accompanied by a reduction in cell numbers in S and G2/M (Fig. 3A; Fig. S3). At higher concentrations, however, cells accumulated in the sub-G1, indicative of apoptosis. This was confirmed by Annexin V staining of cells following BS-181 treatment for 24 hours, with 30% and 83% of cells in staining positive for Annexin V with 25 μM and 50 μM BS-181, respectively (Fig. 3B). No significant apoptosis was observed for roscovitine.

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BS-181-inhibited gastric cancer cell proliferation, migration, and invasion [2]
To evaluate the antiproliferative ability of BS-181 in GC, several different cell lines including MKN28, SGC-7901, AGS, and BGC823 were treated with increasing concentrations of BS-181 for 48 hours. CCK-8 assay showed that GC cell growth was inhibited by BS-181, with inhibitory concentration (IC50) ranging from 17 to 22 μM. For normal gastric epithelial RGM-1 cell line, IC50 was 6.5 μM (Table 1). In addition, we investigated the effects of BS-181 on cell migration and invasion ability. As expected, BS-181 significantly inhibited cell migration and invasion ability in a dose-dependent manner (P<0.05, respectively; Figure 1).

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BS-181-induced cell apoptosis and cell cycle arrest in gastric cancer cells [2]
Cell apoptosis was determined using flow cytometry. Significant increases in apoptotic cells were observed in BS-181-treated BGC823 cells compared to control (P<0.05, respectively; Figure 2A). Our results also showed that BS-181-induced cell apoptosis in a dose- and time-dependent manner. Additionally, caspase-3 and Bax expressions have been significantly increased, while Bcl-2 level has been reduced in cells treated with BS-181 compared to control (P<0.05, respectively) (Figure 2B). These results indicated that BS-181 could induce apoptosis in GC cells. Furthermore, the inhibition of CDK7 activity led to a significant reduction of key antiapoptotic protein XIAP and cell cycle regulator cyclin D1 (P<0.05) (Figure 2C). Thus, BS-181 may regulate cell apoptosis and cell cycle progression via downregulating XIAP and cyclin expression in BGC823 cells. In the present study, cell cycle distribution was analyzed by flow cytometry (Figure 3). Treatment of BS-181 showed an increase in cells in G0/G1, accompanied by a reduction of cell population in S and G2/M phases. These results indicated that BS-181-induced cell cycle arrest in the G0/G1 phase and delayed the progression of the cell cycle.

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BS-181-inhibited CDK7 activity in gastric cancer cells [2]
BS-181 was synthesized as a specific CDK7 inhibitor. In this study, we also confirmed that BS-181 is a specific inhibitor of CDK7. As shown in Table 2, BS-181-inhibited CDK7 activity with an IC50 =0.019 μM, while the IC50 achieved with roscovitine was 0.48 μM. In addition, the IC50 values for inhibition of other CDKs by BS-181 were >1 μM, which is much more higher than that of CDK7. In addition, immunoblotting showed that BS-181 inhibited the phosphorylation of the RNA polymerase II CTD at the well-established CDK7 phosphorylation site serine 5 (P-Ser5).

MCF-7 tumor growth is inhibited in nude mice by BS-181 (5 mg/kg, 10 mg/kg, i.p.). Administration of 10 mg/kg BS-181 intravenously (i.v.) and intraperitoneally (i.p.) results in fast clearance[1]. Comparing the treated group to the control, BS-181 (10 mg/kg/d or 20 mg/kg/d, i.p.) significantly and dose-dependently inhibits the growth of the tumor[2].

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In vivo tumour growth inhibition [1]
The maximum tolerated single dose for BS-181 given intraperitoneally (i.p), was determined as 30 mg/kg, with 10 and 20 mg/kg being well tolerated (data not shown). For xenograft tumour growth inhibition studies, therefore, the animals were injected intraperitoneally twice daily with 5 mg/kg or 10 mg/kg, to give total daily doses of 10 mg/kg or 20 mg/kg, over a period of 14 days. Tumour growth was inhibited in a dose-dependent manner, with 25% and 50% reduction in tumour growth, compared with the control group, for 10 mg/kg/day and 20 mg/kg/day doses of BS-181, respectively (Fig. 4A). At these doses there was no apparent toxicity, as judged by lack of significant adverse effects on animal weights (Fig. 4B).

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BS-181-inhibited tumor growth in vivo and increased survival rate [2]
As previously described,7 the maximum tolerated single dose for BS-181 given intraperitoneally was 30 mg/kg/d, and a dose of 10 mg/kg/d or 20 mg/kg/d was well-tolerated in BALB/c-nu mice. In this study, mice received intraperitoneal injection of BS-181 twice daily with 5 mg/kg/d or 10 mg/kg/d to give daily doses of 10 mg/kg or 20 mg/kg, over a period of 2 weeks. In addition, another group of 15 rats received roscovitine (20 mg/kg/d) injection for a total of 14 days. We observed that tumor growth was significantly inhibited by BS-181 in a dose-dependent manner compared to the control group (P<0.05, respectively) (Figure 5A). However, there was no significant difference in mice body weights between groups during a 14-day observation (Figure 5B). This indicated that there was no apparent toxicity at a daily dose of 10 mg/kg or 20 mg/kg. In addition, all animals were kept for another 30 days for survival observation. As seen in Figure 5C, eight of ten mice (80%) died in the control group, five of ten (50%) died in the roscovitine group, while six of ten (60%, 10 mg/kg/d) and three of ten (30%, 20 mg/kg/d) mice died in BS-181-treated groups. The overall difference in survival rate between rats treated with or without BS-181 was significant (P<0.05, respectively).

The purified recombinant CDK7/CycH/MAT1 complex is incubated with increasing concentrations of BS-181 to measure the amount of inhibited CDK7 activity. The amount of free ATP that remains in the reaction is then measured using a luciferase assay, and the luciferase activity provides a measure of the inhibition of CDK7 activity for IC50 calculation.

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In vitro Kinase Assays [1]
The purified recombinant CDK2/cycE (0050-0055-1), CDK4/cycD1 (0142-0143-1), CDK5/p35NCK (0356-0355-1), CDK7/CycH/MAT1 (0366-0360-4) and CDK9/CycT (0371-0345-1) were purchased from Proqinase GmbH. Kinase assays were performed according to manufacturer’s protocols, using substrate peptides purchased from Proqinase GmbH, as described below. A luciferase assay was used to determine ATP remaining at the end of the kinase reaction, which provides a measure of kinase activity, according to the manufacturer’s protocols.

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Kinase assays in vitro [2]
As previously described,7 kinase assays were performed to evaluate the inhibitory effects of BS-181 on CDK activities in vitro. Kinase assays were carried out using substrate peptides purchased from ProQinase GmbH. A luciferase assay was used to determine ATP remaining at the end of the kinase reaction according to the manufacturer’s protocols.

The supplier's introduction states that the Cell Counting Kit (CCK-8 kit) is used to detect cell viability. In a nutshell, 10⁴ BGC823 cells are seeded per well for 48 hours, either with or without BS-181. Next, each well's absorbance is measured at 450 nm (with a reference at 650 nm).

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Cell Growth Assays [1]
All cells were purchased from ATCC and were routinely cultured in DMEM supplemented with 10% fetal calf serum (FCS). Cell growth was assessed using the Sulforhodamine B (SRB) assay, as described.

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Flow Cytometry [1]
MCF-7 cells were seeded (4 × 105) in 6-well plates in DMEM containing 10% FCS, and allowed to adhere for 24 hours, followed by addition of compounds or DMSO and incubation for 24 hours. Cells were trypsinized, centrifuged at 1100 rpm for 5 minutes and re-suspended in 5 ml of ice-cold PBS, centrifuged as above, gently re-suspended in 2 ml ice-cold 70% ethanol and incubated at 4°C for one hour. Cells were washed twice with 5 ml of ice-cold PBS and re-suspended in 100 μl of PBS containing 100μg/ml RNase and 1ml of 50μg/ml propidium iodide in PBS. Following incubation overnight in the dark at 4°C and filtering through 70μm muslin gauze into FACS tubes to remove cell clumps, stained cells were acquired using the RXP cytomics software on a Beckman Coulter Elite ESP and data were analysed using Flow Jo v7.2.5. For dual labeling with propidium iodide and Annexin V, the cells were trypsinised and collected with the culture medium, centrifuged at 10 rpm for 5 minutes and washed twice with 5 ml of ice-cold PBS containing 2% (w/v) BSA. Cells were labeled with Annexin V-FITC using the Annexin V-FITC apoptosis detection kit I, as per the manufacturer’s instructions. Labeled cells were acquired within 1 hour, using the RXP cytomics software on a Beckman Coulter Elite ESP and the data were analysed using Flow Jo v7.2.5. Statistical analysis was performed for three independent experiments, carried out using the unpaired Student’s t-test to determine p-values.

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Immunoblotting [1]
1×106 cells plated in 10-cm plates, were treated with compounds after 24 hours. 4 hours later, cell lysates were prepared by the addition of 500 μl of hot lysis buffer (4% SDS (w/v), 20% glycerol (v/v), 0.1% bromophenol blue (w/v), 0.1 M Tris-HCl pH6.8, 0.2 M DTT, in H2O), pre-heated to 100°C.

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Cell cycle and apoptosis analysis [2]
Cells were seeded on plates in Dulbecco’s Modified Eagle’s Medium containing 10% fetal calf serum. After incubation with BS-181 for 12 hours, 24 hours, 48 hours, and 72 hours at 0 μM, 1 μM, 10 μM, and 20 μM, cells were harvested, rinsed with cold PBS, and fixed with 70% ice-cold ethanol for 30 minutes and then incubated with propidium iodide (PI) for 30 minutes prior to flow cytometry. Cell apoptosis was determined by flow cytometry using Annexin V-FITC/PI double staining assay. Annexin V-positive and PI-negative cells were identified as apoptotic cells. The apoptotic rate was determined using CellQuest software.

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Cell viability assay [2]
Cell viability was detected using Cell Counting Kit according to supplier’s introductions. Briefly, BGC823 cells were seeded at 104 cells per well for 48 hours with or without BS-181. Then, the absorbance was detected at 450 nm (reference at 650 nm) in each well.

The mice receive a subcutaneous injection of 5×10⁶ BGC823 cells (0.1 mL) in their flanks. Tumor sizes are measured twice a week, and volumes are computed with the following formula: tumor size = (length ×width²)/2. Ultimately, thirty mice with tumor volumes ranging from 100 to 200 mm³ are chosen, and they are randomly assigned to three groups. BS-181 is made in the following conditions: 10% dimethyl sulfoxide/50 mM HCl/5% Tween 20/85% saline, as previously mentioned. For 14 days, mice are given BS-181 injection (ip) twice a day at the prescribed doses (10 mg/kg/d for BS-181 or 20 mg/kg/d for Roscovitine). Injectable vehicles are given to control mice. Daily measurements of the tumor volume and animal weights are made during the course of the 14-day treatment. To observe their survival, all rats are also held for an additional 30 days. In mice, BS-181 at a dose of 5 mg/kg or 10 mg/kg is administered intraperitoneally twice a day. Human tumor xenografts [1]
7-week old female nu/nu-BALB/c athymic nude mice were used. Before inoculation of animal with cells, a 0.72 mg 17β-estradiol 60-day release pellet was implanted subcutaneously. 5×106 cells MCF-7 cells were injected subcutaneously in not more than 0.1ml volume into the flank of the animals. Tumor measurements were performed twice per week, and volumes were calculated using the formula 1/2 [length (mm)] × [width (mm)]2. The animals were randomized and when tumors had reached a volume of 100–200 mm3, animals were entered into the different treatment groups and treatment with test drug or vehicle control was initiated. Animals were treated with compound twice daily by i.p. injection for a total of 14 days. The compounds were prepared in the vehicle of 10% DMSO/50mM HCl/5% Tween 20/85% Saline. Control mice were injected with the vehicle. Compounds were administered by exact body weight, with the injection volume being not more than 0.2ml. At the end of the 14-day treatment period, the mice were sacrificed. Throughout the 14-day treatment period animal weights were determined each day and tumor volumes on alternate days.

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Animal preparation and human tumor xenografts [2]
Human tumor xenografts were established as previously described.7 In total, 5×106 BGC823 cells (0.1 mL) were injected subcutaneously into the flank of the mice. Tumor measurements were performed two times per week, and volumes were calculated using the formula: tumor size = (length [mm] × width2 [mm])/2. Finally, 30 mice (tumor volume 100–200 mm3) were selected and randomly assigned into three groups. As previously described, BS-181 was prepared in 10% dimethyl sulfoxide/50 mM HCl/5% Tween 20/85% saline. Mice received BS-181 injection (ip) twice daily at indicated doses (BS-181 [10 mg/kg/d or 20 mg/kg/d] or roscovitine [20 mg/kg/d]) for a total of 14 days. Control mice were injected with vehicles. Animal weights and tumor volume were measured each day throughout the 14-day treatment. In addition, all rats were kept for another 30 days for survival observation. Mice were injected intraperitoneally twice daily with BS-181 at 5 mg/kg or 10 mg/kg.

In vivo pharmacokinetic studies and tumour growth inhibition [1]
The maximum tolerated single dose for BS-181 given intraperitoneally (i.p), was determined as 30 mg/kg, with 10 and 20 mg/kg being well tolerated (data not shown). For xenograft tumour growth inhibition studies, therefore, the animals were injected intraperitoneally twice daily with 5 mg/kg or 10 mg/kg, to give total daily doses of 10 mg/kg or 20 mg/kg, over a period of 14 days. Tumour growth was inhibited in a dose-dependent manner, with 25% and 50% reduction in tumour growth, compared with the control group, for 10 mg/kg/day and 20 mg/kg/day doses of BS-181, respectively (Fig. 4A). At these doses there was no apparent toxicity, as judged by lack of significant adverse effects on animal weights (Fig. 4B).
Intravenous (i.v) and i.p administration of 10 mg/kg BS-181 showed rapid clearance (Supplemental data Fig. S4). The terminal half-lives were 405 and 343 minutes for i.p and i.v administration, respectively, with the measured plasma concentration at 15 minutes of 1,950 (SEM = 203) and 2,530 (SEM = 269) ng/mL, respectively, and bioavailability being 37% for i.p administration of BS-181 (Tables 3 and 4, Supporting Information).

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Pharmacokinetic studies showed rapid clearance of BS-181 administered i.p. or i.v. In the case of i.p. administration, the maximal blood concentration of BS-181 was 1317 ng/mL. Further, bioavailability was only 37%, indicating a need for further refinement of the BS-181 structure to improve stability and bioavailability. As it stands the studies described here indicate that continuous i.v. infusion or repeated administration is needed for further in vivo evaluation. The observed efficacy, despite the low plasma levels (lower than the IC50 for growth inhibition in vitro), could therefore be due, at least in part to more active metabolites generated following i.p adminsitration. Elucidation of the structures of possible metabolites and their activities will be the subject of future studies.[1]

[1]. The development of a selective cyclin-dependent kinase inhibitor that shows antitumor activity. Cancer Res. 2009 Aug 1;69(15):6208-15.

[2]. Selective CDK7 inhibition with BS-181 suppresses cell proliferation and induces cell cycle arrest and apoptosis in gastric cancer. Drug Des Devel Ther. 2016 Mar 16;10:1181-9.

Normal progression through the cell cycle requires the sequential action of cyclin-dependent kinases CDK1, CDK2, CDK4, and CDK6. Direct or indirect deregulation of CDK activity is a feature of almost all cancers and has led to the development of CDK inhibitors as anticancer agents. The CDK-activating kinase (CAK) plays a critical role in regulating cell cycle by mediating the activating phosphorylation of CDK1, CDK2, CDK4, and CDK6. As such, CDK7, which also regulates transcription as part of the TFIIH basal transcription factor, is an attractive target for the development of anticancer drugs. Computer modeling of the CDK7 structure was used to design potential potent CDK7 inhibitors. Here, we show that a pyrazolo[1,5-a]pyrimidine-derived compound, BS-181, inhibited CAK activity with an IC(50) of 21 nmol/L. Testing of other CDKs as well as another 69 kinases showed that BS-181 only inhibited CDK2 at concentrations lower than 1 micromol/L, with CDK2 being inhibited 35-fold less potently (IC(50) 880 nmol/L) than CDK7. In MCF-7 cells, BS-181 inhibited the phosphorylation of CDK7 substrates, promoted cell cycle arrest and apoptosis to inhibit the growth of cancer cell lines, and showed antitumor effects in vivo. The drug was stable in vivo with a plasma elimination half-life in mice of 405 minutes after i.p. administration of 10 mg/kg. The same dose of drug inhibited the growth of MCF-7 human xenografts in nude mice. BS-181 therefore provides the first example of a potent and selective CDK7 inhibitor with potential as an anticancer agent. [1]
In summary we have discovered the most potent CDK7-selective inhibitor to date by computer-aided drug design. BS-181 selectively exhibited nanomolar enzymatic potency and inhibited all cell lines tested at low micromolar concentrations. For the given route of administration (37% bioavailability) the drug demonstrated in vivo activity in human tumor xenografts. BS-181 warrants further pre-clinical and clinical evaluation as a candidate cancer therapeutic.[1]

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Cyclin-dependent kinase (CDK) family members have been considered as attractive therapeutic targets for cancer. In this study, we aim to investigate the anticancer effects of a selective CDK7 inhibitor, BS-181, in gastric cancer (GC) cell line. Human GC cells (BGC823) were cultured with or without BS-181 at different concentrations for 24-72 hours. BS-181 significantly reduced the activity of CDK7 with downregulation of cyclin D1 and XIAP in GC cells. Treatment with BS-181 induced cell cycle arrest and apoptosis. The expression of Bax and caspase-3 was significantly increased, while Bcl-2 expression was decreased in cells treated with BS-181. In addition, the inhibition of CDK7 with BS-181 resulted in reduced rates of proliferation, migration, and invasion of gastric cells. Those results demonstrated the anticancer activities of selective CDK7 inhibitor BS-181 in BGC823 cells, suggesting that CDK7 may serve as a novel therapeutic target or the treatment of GC.
In conclusion, our study demonstrates that BS-181, the selective inhibitor of CDK7, prevents cell growth both in vitro and in vivo, induces the G1 arrest and apoptosis, and inhibits cell migration and invasion of GC. Therefore, BS-181 provides potent and selective CDK7 inhibitor with the potential as an antigastric cancer agent.[2]

C22H32N6.HCL

416.99

416.245

1397219-81-6

BS-181 dihydrochloride;1883548-83-1;BS-181;1092443-52-1

49867928

White to off-white solid

6.044

4

5

11

29

425

0

CC(C1=C(N=C(C=C2NCC3=CC=CC=C3)NCCCCCCN)N2N=C1)C.[H]Cl

NVIJWMOQODWNFN-UHFFFAOYSA-N

InChI=1S/C22H32N6.ClH/c1-17(2)19-16-26-28-21(25-15-18-10-6-5-7-11-18)14-20(27-22(19)28)24-13-9-4-3-8-12-23;/h5-7,10-11,14,16-17,25H,3-4,8-9,12-13,15,23H2,1-2H3,(H,24,27);1H

5-N-(6-aminohexyl)-7-N-benzyl-3-propan-2-ylpyrazolo[1,5-a]pyrimidine-5,7-diamine;hydrochloride

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 mg/mL (7.19 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 30.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 3 mg/mL (7.19 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 30.0 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: ≥ 3 mg/mL (7.19 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 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 1% DMSO +30% polyethylene glycol+1% Tween 80 : 30 mg/mL

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Solubility in Formulation 5: 50 mg/mL (119.91 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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