PRMT5 (IC50 = 6.2 nM)
GSK3326595 (EPZ-015938) targets protein arginine methyltransferase 5 (PRMT5) [1]

GSK3326595 (10-100 nM, 24-72 h) inhibits the ACE2-RBD interaction, which in turn prevents SARS-CoV-2 spike pseudovirus infection of HEK-293 cells and A549 cells [1]. Peritoneal macrophages are polarized toward the M1 type that is induced by IFN-γ when exposed to GSK3326595 (100 nM, 12 h) [3]. MCL cell death is induced by GSK3326595 (0.15-10 μM, 72 h) [4].

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Type I protein arginine methyltransferases (PRMTs) catalyze asymmetric dimethylation of arginines on proteins. Type I PRMTs and their substrates have been implicated in human cancers, suggesting inhibition of type I PRMTs may offer a therapeutic approach for oncology. The current report describes GSK3368715 (EPZ019997), a potent, reversible type I PRMT inhibitor with anti-tumor effects in human cancer models. Inhibition of PRMT5, the predominant type II PRMT, produces synergistic cancer cell growth inhibition when combined with GSK3368715. Interestingly, deletion of the methylthioadenosine phosphorylase gene (MTAP) results in accumulation of the metabolite 2-methylthioadenosine, an endogenous inhibitor of PRMT5, and correlates with sensitivity to GSK3368715 in cell lines. These data provide rationale to explore MTAP status as a biomarker strategy for patient selection[2].

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Researchers reveal that the PRMT5-specific inhibitor GSK3326595 is able to dramatically reduce ACE2 binding with RBD. Moreover, we discovered that meR671-ACE2 plays an important role in ACE2 binding with Spike1 of the SARS-CoV-2 Omicron, Delta, and Beta variants; and we found that GSK3326595 strongly attenuates ACE2 interaction with Spike1 of the SARS-CoV-2 Omicron, Delta, and Beta variants. Finally, SARS-CoV-2 pseudovirus infection assays uncovered that PRMT5-mediated meR671-ACE2 is essential for SARS-CoV-2 infection in human cells, and pseudovirus infection experiments confirmed that GSK3326595 can strongly suppress SARS-CoV-2 infection of host cells. These findings suggest that as a clinical phase II drug for several kinds of cancers, GSK3326595 is a promising candidate to decrease SARS-CoV-2 infection by inhibiting ACE2 methylation and ACE2-Spike1 interaction[4].

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1. In mantle cell lymphoma (MCL) cell lines with ATM and/or TP53 mutations, treatment with GSK3326595 induced accumulation of γH2AX (a marker of DNA damage), quantified via ImageJ; downregulated expression of DDR pathway genes (AR, DNAPK, NHEJ1, RAD51) measured by real-time qPCR; altered MDM4 mRNA splicing detected by RT-PCR; restored wild-type p53 expression and function, upregulated p53 target genes (MDM2, p21, PUMA) via real-time qPCR, and induced apoptosis analyzed by flow cytometry. In primary MCL cells from relapsed/refractory (R/R) patients, GSK3326595 (two-fold serial dilution, 3-day treatment) reduced cell viability measured by CellTiter-Glo [1]
2. In thioglycollate-elicited peritoneal macrophages treated with GSK3326595 (100 μM, 12 h) and stimulated with IFN-γ (100 ng/mL), the M1/M2 gene marker ratio was increased; mRNA expression of M1 markers (TNF-α, IL-1β, iNOS, MHCII, IP-10) was upregulated, and IP-10 protein secretion was elevated (measured by immunoassay); no significant change in iNOS (M1) and ARG1 (M2) protein expression was observed [3]
3. In HEK-293T and A549 cells overexpressing ACE2, GSK3326595 (100 nM, 48 h) inhibited PRMT5-mediated symmetric dimethylation of ACE2 at R671 (meR671-ACE2) detected by co-IP and western blot; reduced ACE2 binding with SARS-CoV-2 RBD and Spike1 proteins of Omicron, Delta, and Beta variants (assessed by IP and western blot); in vitro methylation assays showed GSK3326595 blocked PRMT5-catalyzed ACE2 methylation at R671; GSK3326595 (10-100 nM) dose-dependently reduced SARS-CoV-2 pseudovirus infection in ACE2-overexpressing HEK-293T and A549 cells [4]
4. In JHH-7 hepatocellular carcinoma (HCC) cells, GSK3326595 (100-1000 nM, 24 h) upregulated Cdkn1b/p27 (cyclin-dependent kinase inhibitor 1B) mRNA and protein levels (measured by qPCR and western blot); inhibited cell proliferation in a dose-dependent manner (assessed by CCK-8 assay); knockdown of PRMT5 (via shRNA) mimicked the antiproliferative effect of GSK3326595, while overexpression of PRMT5 reversed it; GSK3326595 (300 nM) upregulated MHC II-related gene expression in primary hepatocytes and JHH-7 cells (measured by qPCR) [5]

In LDL receptor knockout mice, GSK3326595 (5 mg/kg, Intraperitoneal injection, three times a week for nine weeks) raises liver triglyceride levels without changing atherosclerosis [3]. Enhances resistance to programmed cell death protein 1 (PD-1) immune checkpoint therapy (ICT) in myeloid neoplasia transgene-initiated (MYC-ON) small efficacy in murine hepatocellular carcinoma (HCC) [5]. GSK3326595 is administered orally, once daily, for two weeks.

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Given the important role of inflammation and metabolism in atherosclerotic cardiovascular disease, here we examined the role of PRMT5 in atherosclerosis using the specific PRMT5 inhibitor GSK3326595. Cultured thioglycollate-elicited peritoneal macrophages were exposed to GSK3326595 or DMSO control and stimulated with either 1 ng/mL LPS or 100 ng/mL interferon-gamma for 24 h. Furthermore, male low-density lipoprotein (LDL) receptor knockout mice were fed an atherogenic Western-type diet and injected intraperitoneally 3×/week with a low dose of 5 mg/kg GSK3326595 or solvent control for 9 weeks. In vitro, GSK3326595 primed peritoneal macrophages to interferon-gamma-induced M1 polarization, as evidenced by an increased M1/M2 gene marker ratio. In contrast, no difference was found in the protein expression of iNOS (M1 marker) and ARG1 (M2 marker) in peritoneal macrophages of GSK3326595-treated mice. Also no change in the T cell activation state or the susceptibility to atherosclerosis was detected. However, chronic GSK3326595 treatment did activate genes involved in hepatic fatty acid acquisition, i.e. SREBF1, FASN, and CD36 (+59%, +124%, and +67%, respectively; p < 0.05) and significantly increased hepatic triglyceride levels (+50%; p < 0.05). PRMT5 inhibition by low-dose GSK3326595 treatment does not affect the inflammatory state or atherosclerosis susceptibility of Western-type diet-fed LDL receptor knockout mice, while it induces hepatic triglyceride accumulation. Severe side effects in liver, i.e. development of non-alcoholic fatty liver disease, should thus be taken into account upon chronic treatment with this PRMT5 inhibitor.[3]

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1. In NSG mice bearing Granta-519 or Maver-1 MCL xenografts (ATM/TP53-mutated), daily oral administration of GSK3326595 (100 mg/kg) significantly reduced tumor volume (two-way ANOVA, P<0.05) and tumor weight (two-tailed t-test, P<0.05) compared to vehicle; western blot of tumor tissues showed reduced PRMT5 and H4R3me2s expression, and increased p53 and p21 expression. In PRMT5-knockout Granta-519 xenografts, tumor growth was robustly inhibited. In PDX models of MCL with TP53 mutations or relapsed after CD19 CAR-T therapy, GSK3326595 (100 mg/kg, daily) significantly reduced tumor size and weight, with corresponding changes in PRMT5/p53/p21 expression [1]
2. In male LDL receptor knockout mice fed a Western-type diet and treated with GSK3326595 (5 mg/kg, intraperitoneal injection, 3×/week for 9 weeks), no significant change in atherosclerotic lesion area, lesional macrophage content, or plaque collagen content was observed (Oil Red O, MOMA, Masson's Trichrome staining); no alteration in T cell activation state (CD4+/CD8+ T cell subsets in blood/peritoneum/spleen via flow cytometry) was detected; however, hepatic triglyceride levels increased by 50% (P<0.05), and hepatic genes involved in fatty acid acquisition (SREBF1, FASN, CD36) were upregulated by 59%, 124%, and 67% respectively (P<0.05) [3]
3. In MYC-overexpressing transgenic mice with spontaneous HCC, GSK3326595 (50 mg/kg, daily oral administration for 12 weeks) reduced surface tumor number, liver weight, and serum ALT/AST levels (P<0.05); treatment with 50 or 100 mg/kg GSK3326595 for 2 weeks (after 8 weeks of MYC activation) similarly reduced tumor burden, decreased hepatic SDMA levels (P<0.01), and upregulated p27 expression. In combination with anti-PD-1 antibody (200 μg, intraperitoneal injection every 3 days for 2 weeks), GSK3326595 (25/50 mg/kg daily) further reduced tumor volume/weight in MYC-driven HCC mice; in Hepa1-6 and H22 HCC xenografts, combination of GSK3326595 (50 mg/kg daily) and anti-PD-1 enhanced antitumor efficacy and increased tumor-infiltrating lymphocytes [5]

High Throughput Screen[2]
Type I PRMT inhibitors were found through screening Epizyme’s proprietary HMT-biased library (Mitchell et al., 2015). In summary, compound was incubated with PRMT1 for 30 minutes at room temperature (384-well plate) and reactions were initated upon the addition of SAM and peptide. Final assay conditions were 0.75 nM PRMT1 (NP_001527.3, GST-PRMT1 amino acids 1-371), 200 nM 3H-SAM (specific activity 80 Ci/mmol), 1.5 μM SAM , and 20 nM peptide (Biotin-Ahx-RLARRGGVKRISGLI-NH2, 21st Century Biochemicals) in 20 mM bincine (pH 7.6), 1mM TCEP, 0.005% bovine skin gelatin, 0.002% Tween-20 and 2% DMSO. Reactions were quenched by the addition of SAM (400 μM final). Terminated reactions were transferred to a Streptavidin-coated Flashplate, incubated for at least 1 hour and then the plate was washed with 0.1% Tween-20 using a Biotek ELx405 plate washer. The quantity of 3H-peptide bound to the Flashplate was measured using a PerkinElmer TopCount plate reader.

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PRMT Biochemical Assays[2]
All assays were performed with compound or DMSO prestamped (49x, 2% final) in 96 well plates. Assays for PRMT1, PRMT3, PRMT6 and PRMT8 used H4 1-21 peptide and a buffer comprised of 50 mM Tris (pH 8), 0.002% Tween-20, 0.5 mM EDTA and 1 mM DTT. Briefly, Flag-his-tev-PRMT8 (61-394) was expressed in a baculovirus expression system and purified using Ni-NTA agarose affinity chromatography and Superdex 200 gel filtration chromatography. For all assays, final Adenosyl-L-Methionine (SAM) concentration listed contains a mixture of unlabeled SAM and 3H-SAM All reactions were quenched upon the addition of SAH (0.5 mM final).[2]
For competition studies, substrate was added to the compound plate followed by the addition of enzyme. For SAM competition studies, final assay concentrations consisted of 2 nM PRMT1, 40 nM peptide and titrating SAM (50-8000 nM). For peptide competition studies, final assay concentrations consisted of 2 nM PRMT1, 1000 nM and titrating peptide (1.6-1000 nM). Reactions were incubated at room temperature for 18 minutes prior to quench.[2]
For time dependence studies, enzyme/SAM mix was added to the compound plate and incubated for 3-60 minutes prior to addition of the peptide. For no preincubation assay, peptide was added to the compound plate followed by enzyme/SAM mix to initiate the reaction. Final PRMT1 assay concentrations were 0.5 nM PRMT1, 40 nM peptide and 1100 nM SAM. Reactions were incubated at room temperature for 20 minutes prior to quench.

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In vitro methylation assay: Purified GST-tagged ACE2-WT (601-744 aa) or ACE2-R671K (601-744 aa) proteins were incubated with Myc-PRMT5 and SAM (1 μM/μl); SDMA methylation of ACE2 was detected by western blot using anti-SDMA antibody, and total protein levels were visualized by Coomassie Blue staining. GSK3326595 was added to the reaction system to evaluate its inhibition of PRMT5-mediated ACE2 methylation [4]

Western Blot Analysis[4]
Cell Types: HEK-293T cells, A549 cells
Tested Concentrations: 10 nM, 25 nM, 50 nM, 100 nM
Incubation Duration: 48 h
Experimental Results: Strongly inhibited ACE2-RBD interaction at low concentration. Inhibited SARS-CoV -2 Omicron and other variants Spike1 binding with ACE2. Inhibits SARS-CoV-2 spike pseudovirus infection host cells.

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Cell Cytotoxicity Assay[1]
Cell Types: MCL cells
Tested Concentrations: 0.15 μM, 0.3 μM, 0.6 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM
Incubation Duration: 72 h
Experimental Results: Resulted in modest growth inhibition in MCL cells.

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1. MCL cell viability assay: MCL cell lines (Granta-519, Maver-1, Z-138, JVM-2) were treated with two-fold serial dilutions of GSK3326595 for 6 days; primary MCL cells from R/R patients were treated with two-fold serial dilutions of GSK3326595 for 3 days; cell viability was measured using CellTiter-Glo assay [1]
2. DNA damage detection: MCL cell lines were treated with GSK3326595, then fixed and stained with anti-γH2AX antibody for immunofluorescence (IF); γH2AX foci were quantified using ImageJ (scale bars = 20 μm) [1]
3. Apoptosis analysis: MCL cell lines were treated with GSK3326595 or LLY-283, then stained with apoptosis-specific dyes and analyzed by flow cytometry [1]
4. Macrophage polarization assay: Thioglycollate-elicited peritoneal macrophages were treated with GSK3326595 (100 μM) or DMSO for 12 h, then stimulated with LPS (1 ng/mL) or IFN-γ (100 ng/mL) for 24 h; mRNA expression of M1/M2 markers was measured by qPCR, and IP-10 protein secretion was detected by immunoassay; ex vivo peritoneal macrophages from treated mice were analyzed for iNOS/ARG1 protein expression by flow cytometry [3]
5. ACE2 methylation and binding assay: HEK-293T/A549 cells were transfected with Flag-ACE2, Myc-PRMT5, and GFP-RBD; cells were treated with GSK3326595 (100 nM) for 48 h; co-IP was performed using anti-Flag/anti-GFP antibodies, and western blot was used to detect ACE2 SDMA modification, PRMT5-ACE2 interaction, and ACE2-RBD binding [4]
6. HCC cell proliferation assay: JHH-7 cells were treated with GSK3326595 at concentrations of 100 nM, 300 nM, 500 nM, 1000 nM for 24 h; cell proliferation was assessed by CCK-8 assay. For PRMT5 knockdown/overexpression experiments, JHH-7 cells were infected with lentiviral shPRMT5 or pcDNA-PRMT5, then cultured and analyzed for cell proliferation and gene/protein expression [5]
7. MHC II gene expression assay: Primary hepatocytes and JHH-7 cells were treated with GSK3326595 (300 nM); total RNA was extracted, and MHC II-related gene expression was measured by real-time qPCR [5]

Animal/Disease Models: LDL receptor knockout mice[3]
Doses: 5 mg/kg
Route of Administration: intraperitoneal (ip)injection
Experimental Results: Did not alter atherosclerosis susceptibility. Increased hepatic triglyceride levels without changing the hyperlipidemia extent. Activated genes involved in fatty acid acquisition.

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Animal/Disease Models: myelocytomatosis transgene turned on mice[5]
Doses: 25 mg/kg, 50 mg/kg
Route of Administration: Oral
Experimental Results: Dramatically suppressed tumor growth at 50 mg/kg. demonstrated better therapeutic efficacy at 25 mg/kg.

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1. MCL xenograft model: NSG mice were subcutaneously implanted with Granta-519 or Maver-1 MCL cells; once tumors were established, mice were randomly divided into vehicle and GSK3326595 groups (100 mg/kg, daily oral administration); tumor volume was measured every few days and plotted against treatment time (n=5); at the endpoint, tumors were excised and weighed, and western blot was performed on tumor tissues. For PRMT5 KO models, Granta-519 cells with PRMT5 knockout were implanted into NSG mice, and tumor growth was monitored similarly [1]
2. MCL PDX model: PDX models were established from MCL patients with TP53 mutations or relapsed after CD19 CAR-T therapy; PDX mice were treated with GSK3326595 (100 mg/kg, daily oral administration, n=5); tumor size was measured at different time points, and tumor weight/protein expression were analyzed at the endpoint [1]
3. Atherosclerosis model: Male LDL receptor knockout mice were fed a Western-type diet for 9 weeks; GSK3326595 was dissolved in DMSO-based solvent and administered via intraperitoneal injection at a dose of 5 mg/kg, 3 times per week; body weight, plasma cholesterol/triglyceride levels, and tissue triglyceride levels (BAT, WAT, liver) were measured; aortic root cryosections were stained to assess atherosclerotic lesions [3]
4. HCC transgenic model: MYC-ON transgenic mice (spontaneous HCC) were treated with GSK3326595 (50 mg/kg or 100 mg/kg, daily oral administration) for 2 weeks (after 8 weeks of MYC activation) or 12 weeks; liver tumor number, weight, and serum ALT/AST levels were measured; MRI was used to assess tumor volume in some groups (4-week treatment, 50 mg/kg dose). For combination therapy, mice received GSK3326595 (25/50 mg/kg daily) plus anti-PD-1 antibody (200 μg, intraperitoneal injection every 3 days for 2 weeks) [5]
5. HCC xenograft model: Hepa1-6 and H22 HCC cells were subcutaneously grafted into C57BL/6J mice; mice were treated with GSK3326595 (50 mg/kg daily oral administration) plus anti-PD-1 antibody (200 μg, intraperitoneal injection every 3 days for 1 week); tumor volume/weight and tumor-infiltrating immune cells were analyzed [5]

1. In LDL receptor knockout mice treated with GSK3326595 (5 mg/kg, 3 times a week for 9 weeks), no obvious toxicity was observed, but liver triglyceride levels increased by 50% (P<0.05), indicating possible hepatic lipid metabolism disorder; no changes in body weight or plasma cholesterol/triglyceride levels were detected [3]. 2. In MYC-driven HCC mice treated with GSK3326595 (50/100 mg/kg daily), serum ALT/AST levels decreased (indicating improved liver function) [5].

[1]. Exploiting PRMT5 as a target for combination therapy in mantle cell lymphoma characterized by frequent ATM and TP53 mutations. Blood cancer journal, 2023, 13(1): 27.

[2]. Anti-tumor activity of the type I PRMT inhibitor, GSK3368715, synergizes with PRMT5 inhibition through MTAP loss. Cancer cell, 2019, 36(1): 100-114. e25.

[3]. PRMT5 inhibition induces pro‐inflammatory macrophage polarization and increased hepatic triglyceride levels without affecting atherosclerosis in mice. Journal of Cellular and Molecular Medicine, 2023, 27(8): 1056-1068.

[4]. GSK3326595 is a promising drug to prevent SARS-CoV-2 Omicron and other variants infection by inhibiting ACE2-R671 di-methylation. Journal of Medical Virology, 2023, 95(1): e28158.

[5]. Myelocytomatosis-protein arginine N-methyltransferase 5 Axis defines the tumorigenesis and immune response in hepatocellular carcinoma. Hepatology, 2021, 74(4): 1932-1951.

Pemrametostat is an orally administered, selective small-molecule protein arginine methyltransferase 5 (PRMT5) inhibitor with potential antiproliferative and antitumor activity. Although its mechanism of action is not fully elucidated, oral administration of Pemrametostat binds to the substrate recognition site of PRMT5, inhibiting its methyltransferase activity. This reduces the levels of monomethylated and dimethylated arginine residues in histones H2A, H3, and H4, and regulates the expression of genes involved in various cellular processes, including cell proliferation. Therefore, this drug may increase the expression of antiproliferative genes and/or decrease the expression of proliferative genes, leading to a slowing of the growth of rapidly proliferating cells, including cancer cells. PRMT5 is an arginine methyltransferase that catalyzes the formation of ω-N monomethylarginine (MMA) and symmetric dimethylarginine (sDMA) on histones and various other protein substrates, and is overexpressed in a variety of tumors.

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1. GSK3326595 is a specific PRMT5 inhibitor; PRMT5 expression is upregulated in relapsed/refractory mantle cell lymphoma (R/R MCL) with ATM/TP53 mutations and is associated with poor prognosis; combined with PRMT5 targeting (using GSK3326595) and ATR/CDK4 inhibitors, it has shown synergistic antitumor effects in MCL [1]
2. GSK3326595 inhibits PRMT5-mediated ACE2-R671 dimethylation, thereby reducing ACE2 N-glycosylation and its binding to SARS-CoV-2 Spike1 proteins (including Omicron/Delta/Beta variants), thereby inhibiting viral infection; GSK3326595 is a phase II clinical drug for multiple cancers and is also a preventive drug for SARS-CoV-2 Potential candidate drugs for infection [4]
3. In MYC-driven HCC, PRMT5 is a direct target gene of MYC; GSK3326595 inhibits PRMT5, upregulates p27 (inhibits cell proliferation) and promotes MHC II expression/lymphocyte infiltration (enhances anti-tumor immunity); GSK3326595 combined with anti-PD-1 can improve the efficacy of "immune cold" hepatocellular carcinoma [5]

C24H32N6O3

452.55

452.253

C, 63.70; H, 7.13; N, 18.57; O, 10.61

1616392-22-3

1848944-46-6 (succinate); 1616392-22-3

90241742

Typically exists as white to light yellow solids at room temperature

1.3±0.1 g/cm3

760.3±60.0 °C at 760 mmHg

413.6±32.9 °C

0.0±2.7 mmHg at 25°C

1.626

2.88

3

7

7

33

656

1

O([H])[C@@]([H])(C([H])([H])N([H])C(C1=C([H])C(=NC([H])=N1)N([H])C1([H])C([H])([H])C([H])([H])N(C(C([H])([H])[H])=O)C([H])([H])C1([H])[H])=O)C([H])([H])N1C([H])([H])C2=C([H])C([H])=C([H])C([H])=C2C([H])([H])C1([H])[H]

JLCCNYVTIWRPIZ-NRFANRHFSA-N

InChI=1S/C24H32N6O3/c1-17(31)30-10-7-20(8-11-30)28-23-12-22(26-16-27-23)24(33)25-13-21(32)15-29-9-6-18-4-2-3-5-19(18)14-29/h2-5,12,16,20-21,32H,6-11,13-15H2,1H3,(H,25,33)(H,26,27,28)/t21-/m0/s1

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

EPZ-015938; EPZ 015938; EPZ015938; GSK3326595; 1616392-22-3; GSK-3326595; Pemrametostat; 6-[(1-acetylpiperidin-4-yl)amino]-N-[(2S)-2-hydroxy-3-(1,2,3,4-tetrahydroisoquinolin-2-yl)propyl]pyrimidine-4-carboxamide; EPZ015938; (S)-6-((1-acetylpiperidin-4-yl)amino)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamide; GSK-3326595A; Pemrametostat; GSK3326595; GSK 3326595; GSK-3326595;

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 (5.52 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 2: ≥ 2.5 mg/mL (5.52 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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Solubility in Formulation 3: 2.08 mg/mL (4.60 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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 4: 2.08 mg/mL (4.60 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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 5: ≥ 2.08 mg/mL (4.60 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 6: 2.2mg/mL in10% DMSO : 40% PEG300 : 5% Tween80 + : 45% saline

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