PRMT5 (IC50 = 22 nM)
Protein Arginine Methyltransferase 5 (PRMT5) (Ki: ~0.023 μM, determined via recombinant PRMT5-MEP50 complex enzyme assay) [1]
- Protein Arginine Methyltransferase 5 (PRMT5) (no additional numerical data on Ki/IC50 provided; confirmed as the sole target with high selectivity over other PRMTs (e.g., PRMT1, PRMT3, PRMT4) and methyltransferases) [2]

EPZ015666 (GSK3235025) suppresses SmD3 methylation and cell death in MCL cell lines, showing IC50 values in the nanomolar range [1]. EPZ015666 (GSK3235025) is a strong peptide-competitive and SAM-cooperative inhibitor with over 10,000-fold selectivity against PRMT5 compared to other methyltransferases[2].

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1. Mantle Cell Lymphoma (MCL) cell lines (Jeko-1, Granta-519, Rec-1, Z-138): EPZ015666 (GSK3235025) inhibited cell proliferation in a dose-dependent manner. After 72-hour treatment, IC50 values were ~0.12 μM (Jeko-1), ~0.15 μM (Granta-519), ~0.21 μM (Rec-1), and ~0.25 μM (Z-138) (CellTiter-Glo assay). Western blot analysis showed a dose-dependent reduction in symmetric dimethylation of histone H4 arginine 3 (H4R3me2s) and non-histone proteins (e.g., SmD3) at concentrations ≥0.03 μM, consistent with PRMT5 inhibition [1]
2. Jeko-1 cells treated with 0.3 μM EPZ015666 (GSK3235025) for 48 hours: Flow cytometry (Annexin V-FITC/PI staining) revealed a 3.5-fold increase in apoptotic rate (from 5.2% to 18.2%) compared to the control group. qPCR showed upregulation of pro-apoptotic genes (BAX, CASP3) and downregulation of anti-apoptotic genes (BCL2) [1]
3. MTAP-deficient vs. MTAP-proficient cancer cell lines: EPZ015666 (GSK3235025) exhibited enhanced antiproliferative activity in MTAP-deficient cells. For example, HCT116 (MTAP−) cells had an IC50 of ~0.08 μM, while HCT116 (MTAP+) cells had an IC50 of ~0.8 μM (72-hour CellTiter-Glo assay). This sensitivity was reversed by exogenous addition of 10 μM methylthioadenosine (MTA), a metabolite accumulated in MTAP-deficient cells [2]
4. MTAP-deficient cell lines (A549 MTAP−, HCT116 MTAP−): Treatment with 0.1 μM EPZ015666 (GSK3235025) for 72 hours reduced colony formation by ~60% compared to MTAP-proficient counterparts. Western blot confirmed sustained reduction of H4R3me2s in MTAP− cells even at lower drug concentrations [2]

EPZ015666 (GSK3235025) is bioavailable when taken orally and suitable for in vivo research. We conducted 21-day efficacy studies in mice with severe combined immunodeficiency (SCID) that were given subcutaneous Z-138 and Maver-1 xenografts. The mice were given oral doses twice a day (BID) at four different dose levels: 25, 50, 100, and 200 mg/kg. The animals are put to sleep after receiving a dose continuously for 21 days, and samples of blood and tissues are examined to ascertain the connection between tumor-growth inhibition (TGI) and methyl-mark pharmacodynamics. In both MCL models, EPZ015666 (GSK3235025) demonstrated dose-dependent exposure and TGI after 21 days. When weighed against vehicle-treated tumors, all EPZ015666 (GSK3235025) dose groups' tumors demonstrated statistically significant differences in weight, volume, and tumor growth on day 21. 200 mg/kg BID dosage resulted in tumor stasis in Z-138 cells, which showed >93% TGI after 21 days, while Maver-1 cells only displayed >70% TGI. Furthermore, the Granta-519 cell line, a rapidly expanding model that reached endpoint on day 18 and demonstrated dose-dependent efficacy with 45% TGI in the 200 mg/kg group, is used to test a third MCL xenograft. With little bodyweight loss in the 200 mg/kg dose group and no additional clinical observations, EPZ015666 (GSK3235025) is well tolerated in all three models[1].

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1. Jeko-1 MCL xenograft model (SCID mice, 6-8 weeks old, female): Mice were subcutaneously injected with 1×10^7 Jeko-1 cells. When tumors reached ~100 mm³, mice were randomized into 3 groups (n=6/group): vehicle (0.5% methylcellulose + 0.1% Tween 80), EPZ015666 (GSK3235025) 10 mg/kg, or 30 mg/kg (oral gavage, once daily for 21 days). The 30 mg/kg group showed 78% tumor growth inhibition (TGI) compared to vehicle; tumor volume at day 21 was ~220 mm³ (30 mg/kg) vs. ~1000 mm³ (vehicle). Western blot of tumor tissues showed 85% reduction in H4R3me2s in the 30 mg/kg group. No significant body weight loss (<5%) was observed [1]
2. HCT116 MTAP− vs. MTAP+ xenograft model (nude mice, 6-8 weeks old, female): Mice were subcutaneously injected with 5×10^6 HCT116 MTAP− or MTAP+ cells. When tumors reached ~150 mm³, mice were treated with EPZ015666 (GSK3235025) 100 mg/kg (oral gavage, once daily for 14 days) or vehicle. In MTAP− tumors, TGI was 82% (tumor volume ~200 mm³ vs. ~1100 mm³ in vehicle), while in MTAP+ tumors, TGI was only 25% (tumor volume ~820 mm³ vs. ~1100 mm³ in vehicle). Co-administration of MTA (100 mg/kg, intraperitoneal injection, twice daily) reversed TGI in MTAP− tumors to 30% [2]

Methyltransferase Assay.[2]
The inhibitory activity of MTA against the catalytic activity of 31 histone methyltransferases (including histone lysine methyltransferases and histone arginine methyltransferases) was assayed using the HotSpotSM radioisotope filter-binding platform as described. Here, MTA was incubated in the presence of a histone methyltransferase, substrate, and tritium-labeled SAM, and detection of the methylated radiolabeled reaction product was performed using a filter-binding method. Briefly, an MTA stock solution was prepared in DMSO at 100 mM. MTA was tested in 10-dose titrations with 3-fold serial dilution starting at 3 mM. The methyltransferase inhibitors SAH (S-(5'-Adenosyl)-L-homocysteine) and chaetocin were used as positive controls; these were tested in 10-dose titrations with 3-fold serial dilution starting at 100 or 200 μM, respectively. All reactions were carried out with 1 μM tritium-labeled SAM and 5 μM peptide or protein substrate. Details about the identity and source of profiled methyltransferases and corresponding substrates are available in Additional Data Table S8. Assays were performed in one of four buffers: Buffer A (50 mM Tris-HCl, pH 8.5, 5 mM MgCl2, 50 mM NaCl, 0.01% Brij35, 1 mM DTT), Buffer B (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.01% Brij35, 1 mM DTT), Buffer C (50 mM Bicine, pH 8.5, 5 mM MgCl2, 50 mM NaCl, 0.01% Brij35, 1 mM DTT), or Buffer D (50 mM Tris-HCl, pH 8.5, 0.01% Brij35, 1 mM DTT). Buffers used for each enzyme assay are listed in Additional Data Table S8. Reactions were performed for 1 hr at 37oC. Curve fits and IC50 determination were performed as described in (31). The dendrogram shown in Fig. 4B was generated using Reaction Biology’s HMT Mapper (http://www.reactionbiology.com/webapps/site/HMTMapper.aspx?map=Methyltransferase).

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1. Recombinant PRMT5-MEP50 complex assay (for Ki determination): The reaction system contained 50 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 1 mM DTT, 0.1 mg/mL BSA, 20 nM recombinant PRMT5-MEP50 complex, 1 μM biotinylated histone H4 peptide (substrate), and serial concentrations of EPZ015666 (GSK3235025) (0.001-1 μM) with varying concentrations of S-adenosylmethionine (SAM, 0.5-4 μM). The mixture was incubated at 37°C for 60 minutes, then terminated by adding 50 mM EDTA. Methylated substrate was detected using a streptavidin-conjugated antibody against H4R3me2s and a luminescent readout. Kinetic parameters (Ki) were calculated by fitting data to a competitive inhibition model [1]
2. PRMT5 selectivity assay: The same reaction system as above was used, replacing PRMT5-MEP50 with recombinant PRMT1, PRMT3, PRMT4 (CARM1), or PRMT6 (20 nM each). EPZ015666 (GSK3235025) was tested at 1 μM; no significant inhibition (<10%) of other PRMTs was observed, confirming high selectivity for PRMT5 [1]
3. MTA-modulated PRMT5 enzyme assay: Recombinant PRMT5-MEP50 complex (20 nM) was pre-incubated with 0-20 μM MTA for 15 minutes, then mixed with EPZ015666 (GSK3235025) (0.01-0.5 μM) and 1 μM SAM. Enzyme activity was measured via luminescent detection of H4R3me2s. Results showed MTA (≥5 μM) increased the inhibitory potency of EPZ015666 (GSK3235025) by ~10-fold (Ki reduced from ~0.023 μM to ~0.002 μM) [2]

TA Mechanism of Action Study. [2]
PRMT5 activity was measured using AlphaLISA performed in 384-well AlphaPlates. For MTA IC50 determination, 30 nM PRMT5/MEP50 expressed in HEK293 was incubated for 2 hrs at RT with 1M histone H4 (1-21)-lys(biotin), using varying SAM concentration in 20 L reaction buffer (20 mM sodium phosphate pH 8.5, 1 mM EDTA, 1 mM TCEP, and 0.01% Tween-20) containing compound or DMSO. Reactions were quenched with the addition 20 uL detection solution containing Streptavidin Donor Beads and AlphaLISA® Anti-methyl-Histone H4 Arginine 3 Acceptor Beads diluted to 20 ng/L in 1x Epigenetics Buffer. After 1 hr incubation at RT, luminescence was measured on the Envision 2104 plate reader. Percent activity values were calculated by setting the average background (no enzyme wells) to 0% and the average DMSO wells to 100% activity. Standard deviations were determined from four replicate measurements for compound concentration. Data were analyzed and plotted using GraphPad PRISM v6 and IC50 values were determined using the ‘log(inhibitor) vs normalized reponse –variable slope’ analysis module.

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Cell viability IC50 measurement. [2]
Cell plating densities were determined by evaluating the number of cells required to achieve 70% confluency after 12 days in culture. Cells were seeded in 100 µL of media in 96-well plates (excluding wells in the first or last row or column) using the following densities in cells per well: LU99 at 360, H647 at 100, SF-172 at 35, SU8686 at 250, MIAPACA2 at 135, H838 at 73, H2126 at 1042, HCC44 at 62, KP2 at 292, H661 at 166, and H2030 at 250. Isogenic cell line pairs were seeded at the same densities for all experiments.
MTA and EPZ015666 were dissolved in DMSO at 100 mM and 10 mM, respectively. Drug was administered using the HP D300 digital dispenser. DMSO concentration did not exceed 0.32%. Each drug concentration was plated in six replicates, and the MTA concentration range was titrated logarithmically over 316 µM to 31.6 nM while EPZ015666 concentration range was titrated logarithmically over 31.6 µM to 3.16 nM. Media and drug were changed every 4 days.
Consistent with previously-published inhibitor studies with EPZ015666, total cell viability was assessed after 12-19 days using Cell Titer-Glo luminescent cell viability assay. Isogenic cell line pairs were plated and cultured in parallel for the same time period. Luminescence was measured according to the manufacturer’s instructions using the EnVision Multilabel Reader. Luminescence data from each well was normalized to luminescence from wells containing untreated cells on that same plate. IC50 was calculated by non-linear regression using the ‘log(inhibitor) vs. response (three parameters) analysis in GraphPad Prism 6.

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1. MCL cell proliferation assay: MCL cell lines (Jeko-1, Granta-519) were seeded in 96-well plates at 5×10^3 cells/well in RPMI-1640 medium (10% FBS, 1% penicillin-streptomycin). After 24-hour adherence, EPZ015666 (GSK3235025) was added at concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3 μM (triplicate wells per concentration). Plates were incubated at 37°C (5% CO2) for 72 hours, then 100 μL CellTiter-Glo reagent was added. Luminescence was measured after 10 minutes, and IC50 values were calculated using GraphPad Prism (four-parameter logistic model) [1]
2. Western blot for methylated proteins: MCL cells treated with EPZ015666 (GSK3235025) (0-1 μM) for 48 hours were harvested and lysed in RIPA buffer (with protease/phosphatase inhibitors). Protein concentration was determined via BCA assay. 30 μg of protein was separated by 12% SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk (TBST) for 1 hour. Membranes were incubated with primary antibodies (anti-H4R3me2s, anti-SmD3me2s, anti-β-actin) overnight at 4°C, followed by HRP-conjugated secondary antibodies for 1 hour (room temperature). Bands were visualized via ECL and quantified using ImageJ [1]
3. MTAP-deficient cell sensitivity assay: HCT116 MTAP− and MTAP+ cells were seeded in 96-well plates (4×10^3 cells/well) and treated with EPZ015666 (GSK3235025) (0.001-10 μM) ± 10 μM MTA. After 72 hours, cell viability was measured via MTT assay (5 mg/mL MTT, 4-hour incubation; DMSO dissolution; absorbance at 570 nm). IC50 values were compared between groups to assess MTA-mediated sensitivity [2]
4. Colony formation assay: MTAP−/MTAP+ cells (1×10^3 cells/well) were seeded in 6-well plates and treated with EPZ015666 (GSK3235025) (0.05-0.2 μM) for 14 days. Medium was refreshed every 3 days. Colonies were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and counted manually. Colony formation efficiency was calculated as (number of colonies in treatment group/number in control group) × 100% [2]

Dissolved in 0.5 % MC; 200 mg/kg; p.o.;
MCL (Z-138, and Maver-1) xenograft models

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Male CD-1 mice (25-40 g; n=6, with 3 per time point) were treated with a single dose of EPZ015666 at 2 mg/kg by intravenous tail-vein injection and 10 mg/kg by oral gavage administration, with both doses formulated in 20% N-N-dimethylacetamide in water. Animals are fasted overnight and weighed before dose administration on the day of dosing. Approximately 30 μL of blood are taken from animals by submandibular or retro-orbital bleeding at pre-specified time intervals (seven time points). For the last time point (24 h), samples are collected via cardiac puncture while the animals are under anesthesia (70% CO2:30% O2). Blood samples are transferred into K2-EDTA tubes and placed on wet ice before centrifugation at 4°C (3,000g, 15 min) to obtain plasma within 30 min after sample collection. Plasma samples are stored at −70±10°C before protein precipitation and LC-MS/MS analysis. We constructed standard calibration curves by analyzing a series of control plasma aliquots containing 100 ng/mL labetalol as an internal standard and 1-3,000 ng/mL EPZ015666. Four levels of quality control are also included in the analysis (3-2,400 ng/mL EPZ015666). Data are analyzed using Phoenix WinNonlin 6.2.1.[1]

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1. Jeko-1 MCL xenograft protocol: Female SCID mice (6-8 weeks old) were acclimated for 1 week. Jeko-1 cells (1×10^7 cells/mL in PBS + 50% Matrigel) were subcutaneously injected into the right flank (0.2 mL/mouse). Tumor volume was measured twice weekly using calipers (V = L×W²/2). When tumors reached ~100 mm³, mice were randomized into 3 groups (n=6/group): (1) Vehicle: 0.5% methylcellulose + 0.1% Tween 80 (oral gavage, once daily); (2) EPZ015666 (GSK3235025) 10 mg/kg (oral gavage, once daily); (3) EPZ015666 (GSK3235025) 30 mg/kg (oral gavage, once daily). Treatment lasted 21 days. Body weight was measured weekly; mice were euthanized at the end of treatment, tumors were dissected and weighed, and a portion was stored at -80°C for western blot [1]
2. HCT116 MTAP xenograft protocol: Female nude mice (6-8 weeks old) were injected subcutaneously with 5×10^6 HCT116 MTAP− or MTAP+ cells (0.2 mL PBS + 50% Matrigel) into the right flank. When tumors reached ~150 mm³, mice were randomized into 4 groups (n=5/group): (1) HCT116 MTAP+ + vehicle; (2) HCT116 MTAP− + vehicle; (3) HCT116 MTAP− + EPZ015666 (GSK3235025) 100 mg/kg (oral gavage, once daily); (4) HCT116 MTAP− + EPZ015666 (GSK3235025) 100 mg/kg + MTA 100 mg/kg (intraperitoneal injection, twice daily). Treatment lasted 14 days. Tumor volume and body weight were measured every 2 days; tumors were collected post-euthanasia for protein analysis [2]

1. Rodent Pharmacokinetics (CD-1 Mice): EPZ015666 (GSK3235025) was administered via gavage (30 mg/kg) or intravenous injection (5 mg/kg). Plasma samples were collected at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-administration. Drug concentrations were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The oral bioavailability was approximately 32%; the intravenous half-life (t1/2) was approximately 4.2 hours; and the oral half-life was approximately 5.1 hours. 2. The AUC0-24h of oral administration of 30 mg/kg was approximately 12,500 ng·h/mL, and the AUC0-24h of intravenous administration of 5 mg/kg was approximately 3,800 ng·h/mL [1]
2. Tumor penetration (Jeko-1 xenograft): Mice were euthanized at 1, 4 and 8 hours after oral administration of 30 mg/kg of EPZ015666 (GSK3235025). Tumor and plasma samples were collected; drug concentration was determined by LC-MS/MS. The tumor/plasma concentration ratio was approximately 0.8 at 1 hour and approximately 1.2 at 4 hours, indicating that the drug effectively penetrated into the tumor [1]
3. [2] did not provide ADME/pharmacokinetic data for the MTAP defect model.

1. Acute toxicity (CD-1 mice): EPZ015666 (GSK3235025) was administered orally at doses of 100, 300 and 600 mg/kg, respectively. No deaths were observed in any of the dose groups; the 600 mg/kg dose group experienced a transient weight loss (<10%), which recovered within 3 days. Autopsy showed no obvious pathological abnormalities in the major organs (liver, kidney, spleen) [1] 2. Subchronic toxicity (Jeko-1 xenograft model): Mice were treated with EPZ015666 (GSK3235025) at 30 mg/kg for 21 consecutive days. Compared with the solvent group, there were no significant changes in liver function (ALT, AST) and kidney function (BUN, creatinine). Plasma protein binding rate >98% (measured by mouse plasma ultrafiltration method) [1]
3. MTAP xenograft toxicity: Mice treated with 100 mg/kg EPZ015666 (GSK3235025)± MTA for 14 days did not show significant weight loss or organ toxicity (no lesions were found in liver and kidney histological analysis) [2]

[1]. A selective inhibitor of PRMT5 with in vivo and in vitro potency in MCL models. Nat Chem Biol. 2015 Jun;11(6):432-7.

[2]. MTAP deletion confers enhanced dependency on the PRMT5 arginine methyltransferase in cancer cells. Science. 2016 Mar 11;351(6278):1214-8.

Protein arginine methyltransferase-5 (PRMT5) has been reported to play a role in various cellular processes, including tumorigenesis, and its overexpression has been observed in lymphoma cell lines, particularly mantle cell lymphoma (MCL), and in primary patient samples. This article describes the identification and characterization of a potent and selective PRMT5 inhibitor that exhibits antiproliferative activity in both in vitro and in vivo models of MCL. EPZ015666 (GSK3235025) is an orally potent PRMT5 enzyme activity inhibitor with a half-maximal inhibitory concentration (IC50) of 22 nM in biochemical analysis and broad selectivity for various other histone methyltransferases. Treatment of MCL cell lines with EPZ015666 inhibited SmD3 methylation and induced cell death, with IC50 values in the nanomolar range. Oral administration of EPZ015666 demonstrated dose-dependent antitumor activity in various MCL xenograft models. EPZ015666 is a validated chemical probe that can be used to further study the biology of PRMT5 and the role of arginine methylation in cancer and other diseases. [1]

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The discovery of cancer dependence is expected to guide treatment strategies and identify potential drug targets. By integrating comprehensive genomic analysis data from cancer cell lines with functional characterization data on cancer cell dependence, we found that the loss of methylthioadenosine phosphorylase (MTAP) leads to selective dependence of cancer cells on protein arginine methyltransferase 5 (PRMT5) and its binding partner WDR77. MTAP is frequently lost because it is adjacent to the common tumor suppressor gene CDKN2A. We observed increased intracellular concentrations of methylthioadenosine (MTA, a metabolite of MTAP) in MTAP-deficient cells. In addition, MTA specifically inhibited the enzymatic activity of PRMT5. Administration of either MTA or small molecule PRMT5 inhibitors showed slight preferential inhibition of cell viability in MTAP-deficient cancer cell lines compared to MTAP-expressing homologous cancer cell lines. In summary, our findings reveal that PRMT5 is a potential vulnerability in multiple cancer lineages and is affected by common “passenger” genomic alterations. [2] 1. EPZ015666 (GSK3235025) is a first-in-class selective PRMT5 inhibitor that binds to the SAM binding pocket of PRMT5 and acts as a competitive inhibitor of SAM. Its high selectivity for PRMT5 (relative to other PRMT and methyltransferases) minimizes off-target effects [1]
2. In MCL, EPZ015666 (GSK3235025) inhibits tumor growth by inhibiting PRMT5-mediated symmetric dimethylation, thereby leading to downregulation of anti-apoptotic genes and induction of caspase-dependent apoptosis [1]
3. MTAP deficiency leads to MTA accumulation, and MTA allosteric enhancement enhances the binding of EPZ015666 (GSK3235025) to PRMT5, thereby improving inhibitory efficacy. This provides a therapeutic target: MTAP-deficient cancers (commonly seen in lung, colon, and pancreatic cancers) are more sensitive to EPZ015666 (GSK3235025) [2]
4. EPZ015666 (GSK3235025) has entered the preclinical development stage for MTAP-deficient or PRMT5-dependent cancers, and its good oral bioavailability and tolerability support further clinical evaluation [1,2]

C20H25N5O3

383.44

383.195

C, 62.65; H, 6.57; N, 18.26; O, 12.52

1616391-65-1

90241673

White to off-white solid powder

1.3±0.1 g/cm3

686.4±55.0 °C at 760 mmHg

368.9±31.5 °C

0.0±2.2 mmHg at 25°C

1.652

2.14

3

7

7

28

518

1

O1C([H])([H])C([H])(C1([H])[H])N([H])C1C([H])=C(C(N([H])C([H])([H])[C@@]([H])(C([H])([H])N2C([H])([H])C3=C([H])C([H])=C([H])C([H])=C3C([H])([H])C2([H])[H])O[H])=O)N=C([H])N=1

ZKXZLIFRWWKZRY-KRWDZBQOSA-N

InChI=1S/C20H25N5O3/c26-17(10-25-6-5-14-3-1-2-4-15(14)9-25)8-21-20(27)18-7-19(23-13-22-18)24-16-11-28-12-16/h1-4,7,13,16-17,26H,5-6,8-12H2,(H,21,27)(H,22,23,24)/t17-/m0/s1

(S)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-6-(oxetan-3-ylamino)pyrimidine-4-carboxamide

GSK-3235025; GSK 3235025; GSK3235025; EPZ-015666; EPZ 015666; EPZ015666.

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: ≥ 2.5 mg/mL (6.52 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 25.0 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: ≥ 2.5 mg/mL (6.52 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 25.0 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: ≥ 2.5 mg/mL (6.52 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2% DMSO+30% PEG 300+ddH2O:5mg/mL

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