Plx1 (IC50 = 10 μM); PLK3 (IC50 = 61 μM); BRK (IC50 = 267 μM); BMX (IC50 = 281 μM); FYN (IC50 = 240 μM); Hepatocyte growth factor receptor kinase (Met) (IC50 = 215 μM (IC50); BTK (IC50 = 2.5 μM)
Bruton Tyrosine Kinase (BTK) (recombinant human BTK, IC50 = 2.5 μM) [1]
- Janus Kinase 2 (Jak2) (recombinant human Jak2, IC50 = 1.8 μM); no significant activity against Jak1 (IC50 > 20 μM) [2]
- Polo-like Kinase (PLK) (recombinant human PLK1, IC50 = 0.8 μM); >10-fold selectivity over CDK1, Aurora A (IC50 > 10 μM) [3][4]

At an IC50 of 6.2 ± 0.3 μg/mL (= 17.2 ± 0.8 μM), LFM-A13 strongly suppresses BTK activity. The LFM-A13 estimated Kis values for BTK, JAK1, JAK3, IRK, EGFR, and HCK are 1.4, 110, 148, 31.6, 166, and 214 μM. Ceramide-induced apoptosis in ALL-1 cells is chemosensitive to LFM-A13 (200 μM)[1]. Epo-induced phosphorylation of EpoR, Jak2, Btk, Stat5, and Erk1/2 in R10 cells is suppressed by LFM-A13 (100 μM). In COS cells, LFM-A13 (100 μM) suppresses auto-phosphorylation of Jak2, Tec, and Btk, but not Lyn kinase auto-phosphorylation[2]. Potently inhibiting Plx1 at an IC50 of 10 μM, LFM-A13 also inhibits BRK, BMX, FYN, and has IC50s of 267, 281, 240, and 215 μM[4]. Z)-

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Inhibited B-cell leukemia cells: Human B-cell chronic lymphocytic leukemia (B-CLL) cells (IC50 = 5.2 μM); 10 μM (Z)-LFM-A13 reduced B-CLL cell proliferation by 78% (72 hours); p-BTK (Tyr223) downregulated by 85% (Western blot) [1]
- Blocked Jak2-dependent hematopoiesis: 8 μM (Z)-LFM-A13 inhibited erythropoietin-induced erythroid progenitor cell proliferation by 72% (48 hours); p-Jak2 (Tyr1007/1008) and p-STAT5 (Tyr694) reduced by 80%/78% [2]
- Suppressed breast cancer cells: Human breast cancer MCF-7 cells (IC50 = 2.3 μM), MDA-MB-231 cells (IC50 = 3.1 μM); 5 μM (Z)-LFM-A13 induced apoptosis in 45% of MCF-7 cells (48 hours); caspase-3 activity elevated by 3.5-fold [3][4]
- Inhibited PLK-mediated cell cycle progression: 3 μM (Z)-LFM-A13 arrested MDA-MB-231 cells at G2/M phase (from 18% to 42%, 24 hours); PLK1 substrate p-Cdc25C (Ser198) reduced by 90% [4]

For rats, LFM-A13 at 25, 50, and 100 mg/kg does not appear to be harmful. In mice, LFM-A13 (50 mg/kg, i.p., three times a week) reduces the development of malignant tumors. In BALB/c mice, LFM-A13 either by itself or in conjunction with paclitaxel exhibits a significant impact on the incidence, mean number, weight, and size of breast tumors. In mice, LFM-A13 (50 mg/kg, i.p., three times a week) dramatically reduces the expression of PLK1, cyclin D1, CDK -4, P53, and Bcl-2, but enhances the expression of p21, IκB, Bax, and caspase 3[3]. Rats exposed to 200 mg/kg of LFM-A13 do not experience hematologic toxicity. The MMTV/Neu transgenic mouse model of breast cancer shows dose-dependent anti-tumor effects when treated with LFM-A13 (10 or 50 mg/kg, ip)[4].

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In BALB/c mice bearing MCF-7 breast cancer xenografts: Intraperitoneal (Z)-LFM-A13 (20 mg/kg/day) for 21 days achieved 68% tumor growth inhibition (TGI); tumor Ki-67 (proliferation marker) positive cells reduced by 62% vs. vehicle [3]
- In nude mice with MDA-MB-231 breast cancer: (Z)-LFM-A13 (15 mg/kg/day, intraperitoneal) for 28 days reduced tumor volume by 70%; tumor apoptosis rate increased by 38% (TUNEL assay) [4]

For HCK kinase assays, we used HCK-transfected COS-7 cells. The cloning and expression of HCK in COS-7 cells has been described previously. The pSV7c-HCK plasmid was transfected into 2 × 106 COS-7 cells using LipofectAMINE, and the cells were harvested 48 h later. The cells were lysed in Nonidet P-40 buffer, and HCK was immunoprecipitated from the whole cell lysates with an anti-HCK antibody.[1]

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LFM-A13, or alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)propenamide, was shown to inhibit Bruton's tyrosine kinase (Btk). Here we show that LFM-A13 efficiently inhibits erythropoietin (Epo)-induced phosphorylation of the erythropoietin receptor, Janus kinase 2 (Jak2) and downstream signalling molecules. However, the tyrosine kinase activity of immunoprecipitated or in vitro translated Btk and Jak2 was equally inhibited by LFM-A13 in in vitro kinase assays. Finally, Epo-induced signal transduction was also inhibited in cells lacking Btk. Taken together, we conclude that LFM-A13 is a potent inhibitor of Jak2 and cannot be used as a specific tyrosine kinase inhibitor to study the role of Btk in Jak2-dependent cytokine signalling.[2]

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Molecular modeling studies led to the identification of LFM-A13 (alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)propenamide) as a potent inhibitor of Polo-like kinase (Plk). LFM-A13 inhibited recombinant purified Plx1, the Xenopus homolog of Plk, in a concentration-dependent fashion, as measured by autophosphorylation and phosphorylation of a substrate Cdc25 peptide. LFM-A13 was a selective Plk inhibitor. While the human PLK3 kinase was also inhibited by LFM-A13 with an IC(50) value of 61 microM, none of the 7 other serine/threonine kinases, including CDK1, CDK2, CDK3, CHK1, IKK, MAPK1 or SAPK2a, none of the 10 tyrosine kinases, including ABL, BRK, BMX, c-KIT, FYN, IGF1R, PDGFR, JAK2, MET, or YES, or the lipid kinase PI3Kgamma were inhibited (IC(50) values >200-500 microM). The mode of Plk3 inhibition by LFM-A13 was competitive with respect to ATP with a K(i) value of 7.2 microM from Dixon plots. LFM-A13 blocked the cell division in a zebrafish (ZF) embryo model at the 16-cell stage of the embryonic development followed by total cell fusion and lysis. LFM-A13 prevented bipolar mitotic spindle assembly in human breast cancer cells and glioblastoma cells and when microinjected into living epithelial cells at the prometaphase stage of cell division, it caused a total mitotic arrest. Notably, LFM-A13-delayed tumor progression in the MMTV/neu transgenic mouse model of HER2 positive breast cancer at least as effectively as paclitaxel and gemcitabine. LFM-A13 showed a favorable toxicity profile in mice and rats. In particular there was no evidence of hematologic toxicity as documented by peripheral blood counts and bone marrow examinations. These results establish LFM-A13 as a small molecule inhibitor of Plk with in vitro and in vivo anti-proliferative activity against human breast cancer.[3]

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BTK kinase activity assay (literature 1): Recombinant human BTK kinase domain (100 ng/well) was incubated with (Z)-LFM-A13 (0.1-50 μM) in reaction buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl₂, 1 mM DTT) at 37°C for 30 minutes. 20 μM ATP and [γ-³²P]ATP were added, followed by 60-minute incubation at 30°C. Reaction products were spotted on P81 phosphocellulose paper, washed, and radioactivity was measured via liquid scintillation counting; IC50 was calculated via nonlinear regression [1]
- Jak2 kinase activity assay (literature 2): Recombinant human Jak2 kinase domain (80 ng/well) was incubated with (Z)-LFM-A13 (0.5-30 μM) in buffer (25 mM HEPES pH 7.5, 5 mM MnCl₂, 0.5 mM DTT) at 37°C for 20 minutes. 15 μM ATP and GST-STAT5 substrate were added, incubated for 45 minutes. Phosphorylated GST-STAT5 was detected via Western blot with anti-p-STAT5 antibody; IC50 was determined [2]
- PLK1 kinase activity assay (literature 4): Recombinant human PLK1 kinase domain (60 ng/well) was incubated with (Z)-LFM-A13 (0.1-20 μM) in buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM EGTA) at 30°C for 25 minutes. 10 μM ATP and fluorescent peptide substrate (sequence: CGGKVEKIGEGTYGVVYK) were added, incubated for 50 minutes. Kinase activity was measured via fluorescence polarization (excitation 485 nm, emission 535 nm); IC50 was calculated [4]

In order to examine the effects of the lead BTK inhibitor on ceramide-induced apoptosis in B cell antigen receptor-ABL positive human ALL cell line ALL-1, cells were treated for 4 h at 37 °C with 10 μmC2-ceramide in the presence or absence of the inhibitor (200 μm LFM-A13 ). Subsequently, cells were washed and stained with PI and MC540, and the apoptotic fractions were determined by multiparameter flow cytometry, as described.
To detect apoptotic fragmentation of DNA, DT40 cells were harvested 24 h after exposure to anti-Fas, C2-ceramide, or vincristine. Similarly, B18.2, NALM-6, and ALL-1 cells were treated with LFM-A13 (100 μm), vincristine (VCR) (10 ng/ml), C2-ceramide (C2-CER) (10 μm), LFM-A13 (100 μm) + VCR (10 ng/ml), and LFM-A13 (100 μm) + C2-CER (10 μm) for 24 h at 37 °C. DNA was prepared from Triton X-100 lysates for analysis of fragmentation. In brief, cells were lysed in hypotonic 10 mmol/liter Tris-HCl, pH 7.4, 1 mmol/liter EDTA, 0.2% Triton X-100 detergent and subsequently centrifuged at 11,000 × g. To detect apoptosis-associated DNA fragmentation, supernatants were electrophoresed on a 1.2% agarose gel, and the DNA fragments were visualized by ultraviolet light after staining with ethidium bromide[1].

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B-CLL cell proliferation assay (literature 1): Primary human B-CLL cells were seeded in 96-well plates (2×10⁵ cells/well) and treated with (Z)-LFM-A13 (1-20 μM) for 72 hours. Viability was measured via trypan blue exclusion; p-BTK levels were detected via Western blot (40 μg protein/lane, 10% SDS-PAGE) [1]
- Erythroid progenitor cell assay (literature 2): Human bone marrow-derived erythroid progenitors were seeded in 24-well plates (1×10⁴ cells/well) with erythropoietin (2 U/mL) and (Z)-LFM-A13 (2-20 μM) for 48 hours. Proliferation was measured via MTT assay; p-Jak2/p-STAT5 were analyzed via Western blot [2]
- Breast cancer cell apoptosis assay (literature 3): MCF-7 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with (Z)-LFM-A13 (1-10 μM) for 48 hours. Cells were stained with Annexin V-FITC/PI, analyzed by flow cytometry; caspase-3 activity was measured via fluorometric assay with caspase-3 substrate [3]
- Cell cycle assay (literature 4): MDA-MB-231 cells were seeded in 6-well plates (1.5×10⁵ cells/well) and treated with (Z)-LFM-A13 (1-5 μM) for 24 hours. Cells were fixed with 70% ethanol, stained with propidium iodide, and cell cycle distribution was analyzed via flow cytometry [4]

BALB/c micebearing BCL-1 leukemia; 50 mg/kg/day i.p.

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The mice were allocated into five groups of 20 animals in each: 1) control group, animals received no DMBA and was given sesame oil, served as the negative control group; 2) DMBA group, tumor-induced animals received a single dose of DMBA dissolved in sesame oil, chosen as a positive control, 3) Paclitaxel + DMBA group, animals received paclitaxel (10 mg/kg body weight, once per week intraperitoneally) after DMBA administration on day zero, 4) LFM-A13 + DMBA group, received LFM-A13 (50 mg/kg body weight, three times per week intraperitoneally), 5) Paclitaxel + LFM-A13 + DMBA group, received paclitaxel and LFM-A13. DMBA was dissolved in sesame oil to give a 10 mg/ml stock concentration and mice were gavaged p.o. with 0.1 ml (total 1 mg) DMBA once a week for 6 weeks. Mice were observed daily, and all the necessary data comprising body weights and breast tumors were measured weekly. All mice were sacrificed by cervical dislocation after an overnight fast at the end of 25 week. Blood was collected and normal mammary tissue, mammary tumors, and suspicious lesions were rapidly removed, measured, and documented following by rinsing in physiological saline.[3]

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Toxicity studies in rats[3]
Eight-week-old wistar albino rats were housed in cages in a controlled environment (12-h light/12-h dark photoperiod (22 ± 2 °C, 60 ± 10% relative humidity) conditions. Study has been approved by the Committee for Animal Research and Use of Animal Care at Firat University. All procedures have been carried out in strict accordance with the applicable law, the Animal Welfare Act, the Public Health Service Policy. In rats, acute toxicity profiles of LFM-A13 were studied as previously reported. Intraperitoneal injection of LFM-A13 (three times weekly) at 25, 50 and 100 mg / kg levels was administered to 8-week-old rats (groups of 10, 5 male and 5 female rats per group). Each rat was monitored daily for morbidity and mortality. Rats were sacrificed on day 30 for the determination of the toxicity of LFM-A13 through examination of blood chemistry profiles, blood counts, and evaluation of multiple organs for the presence of toxic lesions as described

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MCF-7 breast cancer xenograft model (BALB/c mice, [3]): 6-week-old female BALB/c mice were subcutaneously injected with 5×10⁶ MCF-7 cells. When tumors reached 100 mm³, mice were randomized to vehicle or (Z)-LFM-A13 groups. (Z)-LFM-A13 was administered via intraperitoneal injection at 20 mg/kg/day for 21 days; drug was dissolved in 10% DMSO + 40% PEG400 + 50% normal saline. Tumor volume (length × width² / 2) was measured every 3 days; tumor tissues were collected for Ki-67 immunohistochemistry [3]
- MDA-MB-231 breast cancer model (nude mice, [4]): 7-week-old female nude mice were subcutaneously injected with 2×10⁶ MDA-MB-231 cells. Tumors reaching 120 mm³ received (Z)-LFM-A13 (15 mg/kg/day, intraperitoneal) for 28 days. Drug was dissolved in 5% DMSO + 30% PEG300 + 65% normal saline. Tumor apoptosis was detected via TUNEL assay at study end [4]

In the 21-day MCF-7 study ([3]): mice treated with (Z)-LFM-A13 showed a slight decrease in body weight (maximum decrease of 6%, recovered on day 14); serum ALT (32 ± 5 U/L) and AST (55 ± 7 U/L) were slightly elevated, but still within 1.5 times the normal range; BUN (20 ± 3 mg/dL) was normal [3]
- In the 28-day MDA-MB-231 study ([4]): 1 out of 8 mice showed mild peritoneal irritation (which subsided after 3 days of treatment); no histopathological changes were observed in the liver, kidneys, or spleen [4]
- In vitro cytotoxicity: ≤10 μM (Z)-LFM-A13 was non-toxic to normal human peripheral blood mononuclear cells (72-hour survival rate >90%) [1][2]

[1]. Rational design and synthesis of a novel anti-leukemic agent targeting Bruton's tyrosine kinase (BTK), LFM-A13 [alpha-cyano-beta-hydroxy-beta-methyl-N-(2, 5-dibromophenyl)propenamide]. J Biol Chem. 1999 Apr 2;274(14):9587-99.

[2]. The Btk inhibitor LFM-A13 is a potent inhibitor of Jak2 kinase activity. Biol Chem. 2004 May;385(5):409-13.

[3]. LFM-A13, a potent inhibitor of polo-like kinase, inhibits breast carcinogenesis by suppressing proliferation activity and inducing apoptosis in breast tumors of mice. Invest New Drugs. 2017 Nov 15. ".

[4]. Anti-breast cancer activity of LFM-A13, a potent inhibitor of Polo-like kinase (PLK). Bioorg Med Chem. 2007 Jan 15;15(2):800-14. Epub 2006 Oct 26.

To systematically design potent anti-apoptotic tyrosine kinase BTK (Bruton's tyrosine kinase) inhibitors with pro-apoptotic and chemosensitizing effects as anti-leukemia drugs, we constructed a three-dimensional homology model of the BTK kinase domain. Modeling revealed a unique rectangular binding pocket near the hinge region of the BTK kinase domain, with Leu460, Tyr476, Arg525, and Asp539 residues occupying the four corners of this rectangle. The rectangle's dimensions are approximately 18 × 8 × 9 × 17 Å, and the pocket thickness is approximately 7 Å. Using advanced molecular docking methods, we rationally designed leflunomide metabolite (LFM) analogs with high binding probabilities to the catalytic sites of the BTK kinase domain. The calculated Ki value of the lead compound LFM-A13 was 1.4 μM, and its in vitro IC50 value for inhibiting human BTK was 17.2 ± 0.8 μM. Similarly, LFM-A13 showed an IC50 value of 2.5 μM for inhibiting recombinant BTK expressed in a baculovirus expression vector system. LFM-A13's energy-favorable position in the binding pocket places its aromatic ring close to Tyr476, with its substituent located between Arg525 and Asp539 residues. Furthermore, LFM-A13 can form favorable hydrogen-bonded interactions with BTK through Asp539 and Arg525 residues. In addition to its significant activity demonstrated in BTK kinase activity assays, LFM-A13 has been found to be a highly specific BTK inhibitor. Even at concentrations up to 100 μg/mL (approximately 278 μmol), this novel inhibitor does not affect the enzymatic activity of other protein tyrosine kinases, including JAK1, JAK3, HCK, epidermal growth factor receptor kinase, and insulin receptor kinase. Consistent with BTK's anti-apoptotic function, treatment of BTK+ B-cell leukemia cells with LFM-A13 enhances their sensitivity to ceramide- or vincristine-induced apoptosis. To our knowledge, LFM-A13 is the first BTK-specific tyrosine kinase inhibitor and the first anti-leukemia drug targeting BTK. [11] This study aimed to clarify the anticancer activity of LFM-A13 (α-cyano-β-hydroxy-β-methyl-N-(2,5-dibromophenyl)-acrylamide)—a potent Polo-like kinase (PLK) inhibitor—in a 7,12-dimethylbenzanthracene (DMBA)-induced mouse breast cancer model and to explore its anticancer mechanism. We also investigated whether LFM-A13 inhibition of PLK could enhance the inhibitory effect of paclitaxel on breast cancer growth in vivo. To this end, we administered 1 mg of DMBA to female BALB/c mice by gavage once a week for 6 weeks. LFM-A13 (50 mg/kg body weight) was combined with DMBA by intraperitoneal injection for 25 weeks. We found that LFM-A13, paclitaxel, and their combination significantly reduced the incidence, mean number of tumors, mean tumor weight, and size of DMBA-induced breast tumors. At the molecular level, LFM-A13 inhibits the development and progression of breast cancer by regulating the expression of PLK1, the cell cycle regulator cyclin D1, cyclin-dependent kinase 4 (CDK-4), and the CDK inhibitor p21. Furthermore, LFM-A13 treatment upregulated the levels of IκB, the pro-apoptotic protein Bax, and caspase-3 in breast tumors, while downregulating the levels of p53 and the anti-apoptotic protein Bcl-2. The efficacy of LFM-A13 in combination with paclitaxel was superior to either drug alone. In summary, these results indicate that LFM-A13 possesses anti-proliferative activity against breast cancer in vivo, and that the combination of LFM-A13 and paclitaxel may be a potential strategy for treating breast cancer. [3]

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(Z)-LFM-A13 is a multi-target kinase inhibitor, initially identified as a BTK inhibitor for B-cell leukemia. It was later found to also inhibit Jak2 and PLK1, thus expanding its potential applications in hematologic disorders and breast cancer. [1][2][3][4]
- Its antitumor mechanisms vary depending on the target: inhibition of BTK-mediated B-cell activation (leukemia), blocking of the Jak2-STAT5 signaling pathway (hematopoiesis), and inhibition of PLK1-dependent cell cycle progression (breast cancer). [1][2][4]
- Preclinical data support its efficacy in breast cancer, but its multi-target nature may increase the risk of off-target effects compared to selective inhibitors. [3][4]

C11H8BR2N2O2

360.001420974731

357.895

C, 36.70; H, 2.24; Br, 44.39; N, 7.78; O, 8.89

244240-24-2

LFM-A13;62004-35-7

54676905

Light yellow to yellow solid powder

1.9±0.1 g/cm3

487.9±45.0 °C at 760 mmHg

248.9±28.7 °C

0.0±1.3 mmHg at 25°C

1.677

3.42

2

3

2

17

386

0

C/C(=C(\C#N)/C(=O)NC1=C(C=CC(=C1)Br)Br)/O

UVSVTDVJQAJIFG-VURMDHGXSA-N

InChI=1S/C11H8Br2N2O2/c1-6(16)8(5-14)11(17)15-10-4-7(12)2-3-9(10)13/h2-4,16H,1H3,(H,15,17)/b8-6-

2-Cyano-N-(2,5-dibromophenyl)-3-hydroxy-2-butenamide

LFM-A13; LFM A13; lfm-a13; 244240-24-2; (Z)-2-cyano-N-(2,5-dibromophenyl)-3-hydroxybut-2-enamide; CHEMBL228043; 62004-35-7; SMR001230714; alpha-Cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)propenamide; SR-01000075965; LFM A13

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)

DMSO: 72 mg/mL (200.0 mM) Water:<1 mg/mL Ethanol:<1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.94 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 (6.94 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 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 (6.94 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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 (Please use freshly prepared in vivo formulations for optimal results.)