SHIP1/SH2 domain-containing inositol-5′-phosphatase 1 (IC50 = 2.5 μM)
3α-Aminocholestane (3AC) therapy substantially reduces OPM2 cell viability. When compared to OPM2 cells, RPMI8226 and U266 cells exhibit much lower sensitivity to 3α-Aminocholestane treatment; yet, viability is significantly reduced at doses of ≥12.5 μM. After being treated for 36 hours with 3α-Aminocholestane, the proportion of cells in the S phase is significantly decreased, and the number of cells in the G2/M phase increases. On the other hand, in the less proliferative RPMI8226 and U266 cells, treatment with 3α-Aminocholestane blocks cell cycle progression in the G0 /G1 phase and results in a lower percentage of cells progressing through the S phase[2].

3α-Aminocholestane (3AC) therapy substantially reduces OPM2 cell viability. When compared to OPM2 cells, RPMI8226 and U266 cells exhibit much lower sensitivity to 3α-Aminocholestane treatment; yet, viability is significantly reduced at doses of ≥12.5 μM. After being treated for 36 hours with 3α-Aminocholestane, the proportion of cells in the S phase is significantly decreased, and the number of cells in the G2/M phase increases. On the other hand, in the less proliferative RPMI8226 and U266 cells, treatment with 3α-Aminocholestane blocks cell cycle progression in the G0 /G1 phase and results in a lower percentage of cells progressing through the S phase[2].

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3α-Aminocholestane (3AC) selectively inhibited the enzymatic activity of INPP5D (SHIP1) with an IC50 of approximately 2.5 µmol/l, but did not significantly inhibit the related phosphatases INPP5L1 (SHIP2) and PTEN (IC50 >20 µmol/l). [1]
Treatment of patient-derived Ph+ ALL cells with 3α-Aminocholestane (3AC) induced strong hyperactivation of SYK (phosphorylation at Y352). [1]
Treatment of patient-derived TKI-resistant Ph+ ALL cells with 3α-Aminocholestane (3AC) induced cell death within four days. [1]
Dose-response analyses revealed that 3α-Aminocholestane (3AC) was selectively toxic for patient-derived Ph+ ALL cells (IC50=2.8 µmol/l; n=5) compared to mature B cell lymphoma cells (n=5). [1]
Pretreatment of Ph+ ALL cells with the SYK inhibitor PRT06207 largely protected them against 3α-Aminocholestane (3AC)-induced cell death, demonstrating that hyperactivation of Syk is required for induction of cell death. [1]
3α-Aminocholestane (3AC) induced massive cell death (>95%) in all six tested cases of Ph+ ALL that had relapsed under TKI therapy, including three cases with global TKI-resistance due to the BCR-ABL1[T315I] mutation. In contrast, the TKI Imatinib had no effect in the BCR-ABL1[T315I] cases. [1]

Upon OPM2 challenge, it is discovered that 3α-Aminocholestane (3AC) leads to decreased multiple myeloma (MM) growth in vivo, as measured by the amount of free human Igλ light chain in the plasma. Furthermore, peripheral blood from mice treated with 3-aminocholestane shows less circulating OPM2 cells, as detected by human HLA-ABC labeling, as compared to vehicle controls. Most notably, mice treated with 3α-Aminocholestane had much improved survival rates following tumor challenge. When mice treated with 3α-Aminocholestane fail to respond to treatment, it is discovered that MM tumors have an overexpression of SHIP2, which is similar to when OPM2 cells are treated in vitro and implies that tumor cells with higher SHIP2 expression may be chosen by SHIP1 inhibition[2].

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Treatment of NOD/SCID transplant recipient mice carrying TKI-resistant patient-derived (BCR-ABL1[T315I]) Ph+ ALL cells with 3α-Aminocholestane (3AC) significantly prolonged overall survival (P=0.0002, log rank test) and reduced leukemia burden as measured by bioluminescence imaging. [1]

Detection of Phosphatase Enzymatic Activity [2]
Fluorescent polarization assay was used as described previously. In short, recombinant SHIP1 or SHIP2 is mixed with its substrate PtdIns(3,4,5)P3 in the presence of potential chemical inhibitors. The reaction product is mixed withPtdIns(3,4)P2 detector protein and a fluorescent PI(3,4)P2 probe. Newly synthesizedPtdIns(3,4)P2 displaces the detector protein, thereby enhancing an unbound fluorescent probe in the mixture and decreasing mean polarization units. Thus, identified SHIP inhibitors, (2-phenyl-benzo[h]quinolin-4-yl)-[2]piperidyl-methanol hydrochloride (1PIE), 1-[(chlorophenyl)methyl]-2-methyl-5-(methylthio)-1H-indole-3-ethanamine hydrochloride (2PIQ) and (2-adamantan-1-yl-6,8-dichloro-quinolin-4-yl)-pyridin-2-yl-methanol hydrochloride (6PTQ) were subsequently tested for inhibition of free phosphate production by recombinant SHIP1 or SHIP2 by Malachite Green assay as described before or by fluorescent polarization assay. To demonstrate selectivity of the compounds for SHIP1 and SHIP2 over other phosphatases, SHIP1 and the inositol 5-phosphatase oculocerebrorenal syndrome of Lowe (OCRL) were immunoprecipitated from OPM2 cells. For this purpose, OPM2 cells were lysed in IP-lysis buffer (20 mmol/L Tris, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton × 100, 1 mmol/L phenylmethylsulfonyl fluoride and Halt protease inhibitor), and SHIP1 or OCRL were immunoprecipitated by using mouse IgG antibodies. Beads were washed four times with immunoprecipitation (IP) lysis buffer and once with Tris-buffered saline (TBS)/MgCl2 (10 mmol/L) and resuspended in TBS/MgCl2. SHIP inhibitors (200 μmol/L) were added to the beads for 5 min, after which immunoprecipitated SHIP1 was incubated in the presence of 100 μmol/L PtdIns(3,4,5)P3, whereas immunoprecipitated OCRL was incubated in the presence of 100 μmol/L PtdIns(4,5)P2 for 30 min. Malachite Green solution was added according to the manufacturer’s instructions, and the plate was read after 20 min. Identification of 3α-aminocholestane (3AC) was described previously

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The study cites that 3α-Aminocholestane (3AC) selectively inhibited the enzymatic activity of INPP5D (SHIP1) with an IC50 of approximately 2.5 µmol/l, but did not significantly inhibit the related phosphatases INPP5L1 (SHIP2) and PTEN (IC50 >20 µmol/l). The specific experimental protocol for the enzyme activity assay (e.g., kinase activity, SPR, ITC, HTRF) is not described in detail within the provided text. [1]

Cell Viability Assay [2]
Cells were treated in triplicate or more with increasing concentrations of compounds. Cell viability was determined with a Cell Counting Kit per the manufacturer’s instructions. The odds density (OD) of compound-treated cells was divided by the OD of their vehicle control, and the viability was expressed as a percentage of untreated cells. Results are expressed as mean ± standard error of the mean (SEM) of three individual experiments. For PIP add-back experiments, MCF-7 cells were treated for 2 h with 10 μmol/L SHIP inhibitors, after which cells were washed and fresh medium was added. Cells were subsequently cultured in the absence (0 μmol/L) or presence (10 or 20 μmol/L) of eitherPtdIns(3,4)P2-diC16 (P-3416) or PtdIns(3,5)P2-diC16 (P-3516) for 36 h, after which cell viability was determined by the Dojindo Cell Counting Kit.

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Cell viability assay [1]
100,000 human ALL cells were seeded in a volume of 50 μl medium in one well of a 96-well plate. Imatinib or any other inhibitor was diluted and incubated at the indicated concentration in a total volume of 100 μl medium. After 3 days, cell counting kit-8 was used to determine the number of viable cells. Fold changes were calculated using baseline values of vehicle treated cells as a reference (set to 100%).

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Flow cytometry [1]
Antibodies used in flow cytometry are mentioned in Supplementary Table 6. For cell-cycle analysis, the BrdU flow cytometry kit or Click-iT EdU Flow Cytometry Assay Kit was used according to the manufacturer’s instructions. For evaluation of intracellular ROS levels, ALL cells were incubated for 7 min with 1 μM 5-(and 6-)chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) at 37°C for oxidation of the dye by ROS. After washing with PBS, the cells were incubated additional 15 min at 37°C in PBS to allow complete deacetylation of the oxidized form of CM-H2DCFDA by intracellular esterases. The levels of fluorescence were then directly analyzed by flow cytometry, gated on viable cells.

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Western blotting [1]
CelLytic buffer supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail set II were used to lyse cells. 10 μg of protein lysates per sample were separated on mini precast gels and transferred on nitrocellulose membranes For the detection of proteins, primary antibodies, alkaline-phosphatase conjugated secondary antibodies, and chemiluminescent substrate were used. Details of primary antibodies were shown in Supplementary Table 7.

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Colony forming assay for mouse cells [1]
10,000 BCR-ABL1-transformed ALL cells or 100,000 CML-like cells were used for this assay. Cells were resuspended in murine MethoCult medium and plated on dishes (3 cm in diameter) with an extra dish of water to prevent evaporation. After 7 to 14 days, colonies were counted.

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For Western blot analysis of signaling activation, patient-derived Ph+ ALL cells were treated with 3α-Aminocholestane (3AC) (10 µmol/l) for indicated time points (0, 4, 8, 16, 60 minutes). Cells were then lysed, and protein lysates were separated by gel electrophoresis, transferred to membranes, and probed with antibodies against phosphorylated forms of SYK (Y352), SRC (Y416), BTK (Y223), and PLCγ2 (Y1217) to assess hyperactivation of proximal pre-BCR signaling. [1]
For cell viability assays, 100,000 human ALL cells were seeded in 96-well plates. 3α-Aminocholestane (3AC) was diluted and incubated at indicated concentrations. After 3 days, cell viability was determined using a cell counting kit. Fold changes were calculated using baseline values of vehicle-treated cells as a reference. [1]
To test the role of SYK hyperactivation in 3α-Aminocholestane (3AC)-induced death, ALL cells were pretreated with the SYK inhibitor PRT06207 (2.5 µmol/l) for two days before adding 3α-Aminocholestane (3AC) (7.5 µmol/l), and viability was monitored. [1]
Dose-response curves were generated for patient-derived Ph+ ALL cells (n=5) and mature B cell lymphoma cells (n=5) treated with varying concentrations of 3α-Aminocholestane (3AC) to determine IC50 values. [1]

3α-Aminocholestane is suspended in a 0.3% Klucel/H2O solution at 11.46 mM and administered by intraperitoneal injection of 100 μL solution. NOD/SCID/γcIL2R (NSG) mice OPM2 Tumor Challenge Studies [2]
NOD/SCID/γcIL2R (NSG) mice (The Jackson Laboratory, Bar Harbor, ME, USA) were injected intraperitoneally with 1 × 107 OPM2 cells and 6 h later received an initial injection of 3α-aminocholestane (3AC)  or vehicle. 3α-aminocholestane (3AC)  was suspended in a 0.3% Klucel/H2O solution at 11.46 mmol/L and administered by intraperitoneal injection of 100-μL solution. Vehicle-treated mice received 100-μL injection of 0.3% Klucel/H2O solution. The final concentration of 3α-aminocholestane (3AC)  in the treated mice was 60 μmol/L. The mice were then treated with 3α-aminocholestane (3AC)  or vehicle daily for the next 6 d and then twice per week in the remaining 15 wks of the survival study. In some instances, tumors from the vehicle- or 3α-aminocholestane (3AC)  -treated hosts were excised and single-cell suspensions were made for Western blot analysis of SHIP2 expression after mice were deemed to be moribund and recommended for humane euthanasia by veterinary staff.

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Enzyme-Linked Immunosorbent Assay for Human Igλ Light Chain in Mouse Peripheral Blood [2]
Mice were bled into a serum collection tube 4 wks after the OPM2 challenge, and serum was obtained after pelleting of blood cells at 5,000g for 5 min. Human Igλ light chain amounts were determined using an Ig light chain detection kit from Biovendor per the manufacturer’s instructions.

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Detection of Circulating OPM2 Cells in Mouse Blood [2]
Mice were bled into a blood collection tube 4 wks after OPM2 challenge and red cells were lysed. White blood cells were incubated with anti-CD16/32 to block Fc receptor binding and then stained with antibodies against human HLA-ABC, clone W6/32. Samples were acquired on an LSRII cytometer (Becton Dickinson), and dead cells were excluded from the analysis after cytometer acquisition by exclusion of cells that stained positively for DAPI (di aminido phenyl indol).

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Patient-derived Ph+ ALL cells (e.g., BLQ5 line with BCR-ABL1[T315I] mutation) were transduced with a lentiviral vector encoding firefly luciferase. These cells were then injected into sublethally irradiated NOD/SCID mice via tail vein injection. [1]
Mice carrying the leukemia xenografts were treated with either 3α-Aminocholestane (3AC) (50 mg/kg) or vehicle control via intraperitoneal (ip) injection daily. [1]
Leukemia progression was monitored in vivo using bioluminescence imaging with an IVIS system. D-luciferin was injected intraperitoneally 15 minutes before imaging. [1]
Overall survival of mice in the treatment and control groups was compared using Kaplan-Meier analysis. [1]

[1]. Signalling thresholds and negative B-cell selection in acute lymphoblastic leukaemia. Nature. 2015 May 21;521(7552):357-61.

[2]. Therapeutic Potential of SH2 Domain-Containing Inositol-5′-Phosphatase 1 (SHIP1) and SHIP2 Inhibition in Cancer. Mol Med. 2012 Feb 10;18:65-75.

cell selection depends on intermediate levels of B cell antigen receptor (BCR) signaling intensity: signaling intensity below a minimum threshold (e.g., nonfunctional BCR) or above a maximum threshold (e.g., autoreactive BCR) leads to negative selection. In approximately 25% of acute lymphoblastic leukemia (ALL) cases, cells carry the oncogenic BCR-ABL1 tyrosine kinase (Philadelphia chromosome positive), which mimics constitutively activated pre-BCR signaling. Current therapeutic approaches primarily focus on developing more potent tyrosine kinase inhibitors to suppress oncogenic signaling to levels below the minimum survival threshold. We tested the hypothesis that targeting overactivation (above the maximum threshold) would activate a missing checkpoint that clears autoreactive B cells and selectively kill ALL cells. By examining individual components of proximal pre-B cell receptor (pre-BCR) signaling in mouse BCR-ABL1 cells, we found that a stepwise increase in Syk tyrosine kinase activity is both a necessary and sufficient condition for inducing cell death. Syk overactivation is functionally equivalent to acute activation of the autoreactive BCR on ALL cells. Despite oncogenic transformation, this fundamental negative selection mechanism remains effective in ALL cells. Unlike normal pre-B cells, patient-derived ALL cells highly express the inhibitory receptors PECAM1, CD300A, and LAIR1. Genetic studies have shown that PECAM1, CD300A, and LAIR1 play a crucial role by recruiting the inhibitory phosphatases Ptpn6 (Reference 7) and Inpp5d (Reference 8) to regulate the intensity of oncogenic signaling. Using a novel small-molecule INPP5D (also known as SHIP1) inhibitor, we demonstrated that the pharmacological overactivation of SYK and the involvement of B-cell negative selection represent a potential new strategy for overcoming drug resistance in human ALL.
The small molecule inhibitor 3-α-aminocholestane (3AC) (Extended Data Fig. 10f) selectively inhibits the enzymatic activity of INPP5D (SHIP1; IC50 ~2.5 μmol/l), but has no effect on the associated phosphatases INPP5L1 (SHIP2) and PTEN (IC50 >20 μmol/l). Treatment of patient-derived Ph+ ALL cells with 3AC induces strong overactivation of SYK (Fig. 4a). In patient-derived myeloid CML samples, baseline levels of Syk activity were very low and unresponsive to 3AC treatment (Extended Data Fig. 10g). Biochemical characterization of 3AC-mediated INPP5D inhibition in patient-derived Ph+ ALL cells showed potent and transient overactivation of the proximal pre-BCR signaling molecule (Fig. 4a). Treatment of patient-derived TKI-resistant Ph+ ALL cells with 3AC induces cell death within 4 days. Importantly, pretreatment of Ph+ ALL cells with the SYK inhibitor (PRT06207) significantly protected them from 3AC-induced cell death (Fig. 4b), indicating that excessive activation of Syk is essential for inducing cell death. Dose-response analysis showed that 3AC exhibited selective toxicity to patient-derived Ph+ ALL cells compared to mature B-cell lymphoma (n=5; Extended Data Fig. 10h) (IC50 = 2.8 μmol/L; n=5). Next, we investigated the drug response in 6 Ph+ ALL patients who had relapsed after TKI treatment, including 3 cases with systemic TKI resistance due to the BCR-ABL1T315I mutation. As expected, imatinib treatment was ineffective in the BCR-ABL1T315I cases (Extended Data Fig. 10i). In contrast, regardless of BCR-ABL1 mutation status, 3AC induced massive cell death (>95%) in all 6 Ph+ ALL cases (Extended Data Fig. 10i). Similarly, treatment of NOD/SCID transplant recipient mice carrying TKI-resistant patient-derived (BCR-ABL1T315I) Ph+ ALL cells with 3AC significantly prolonged overall survival (P=0.0002, log-rank test; Fig. 4c) and reduced leukemia burden (Fig. 4d). Although further research is needed to optimize drug targeting for this pathway, these experiments suggest that transient overactivation of SYK and involvement of negative B cell selection is a powerful new strategy to overcome Ph+ ALL resistance. [1]

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Many tumors exhibit enhanced activation of the phosphatidylinositol 3-kinase (PI3K)-PtdIns(3,4,5)P(3)-protein kinase B (PKB/Akt) signaling pathway. For a long time, it has been believed that the lipid phosphatase SH2 domain inositol-5'-phosphatase 1 (SHIP1) and SHIP2 antagonize the survival signaling induced by this pathway by hydrolyzing PtdIns(3,4,5)P(3) to generate PtdIns(3,4)P(2), thereby exerting a tumor-suppressive effect. However, increasing evidence suggests that PtdInd(3,4)P(2) can activate Akt and is essential for Akt activation, suggesting that SHIP1/2 enzymes may function as proto-oncogenes. We recently reported a novel selective chemical inhibitor of SHIP1 (3α-aminocholestane [3AC]) that can kill malignant hematologic cells. In this study, we further investigated the biochemical effects of 3AC treatment on multiple myeloma (MM) and demonstrated that SHIP1 inhibition can arrest MM cell lines in the G0/G1 or G2/M phase of the cell cycle, leading to caspase activation and apoptosis. Furthermore, we found that treatment of mice with the SHIP1 inhibitor 3AC blocked the in vivo growth of MM cells. In addition, we identified three novel pan-SHIP1/2 inhibitors that effectively killed MM cells through G2/M phase arrest, caspase activation, and apoptosis induction. Interestingly, in SHIP2-expressing breast cancer cells lacking SHIP1 expression, pan-SHIP1/2 inhibition also reduced the number of viable cells, a phenomenon salvaged by the addition of exogenous PtdIns(3,4)P(2). In conclusion, this study suggests that inhibition of SHIP1 and SHIP2 may have broad clinical application value in the treatment of various tumor types. Besides acting as a phosphatase, SHIP1 also has the function of masking the receptor tail to prevent the recruitment of other signaling proteins, or as a adaptor protein for proteins such as Shc, DOK1, and Grb2, and is therefore thought to reduce Ras signaling. Theoretically, blocking phosphatase activity with 3AC may not affect these other functions of SHIP1. However, we observed that SHIP1 protein expression decreased in MM cells after long-term 3AC treatment, suggesting that these scaffold proteins may no longer function. Recent studies have shown that SHIP-1 phosphorylation is ubiquitinated and targets proteasome degradation. However, we did not observe a difference in SHIP-1 phosphorylation levels in IGF-1-stimulated MM cells after 3AC pretreatment (unpublished observation, GM Fuhler). Therefore, the reason for SHIP-1 proteasome degradation after 3AC treatment is unclear. [2]

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3α-aminocholestane (3AC) is a novel small molecule phosphatase INPP5D (SHIP1) inhibitor. [1]
It represents a pharmacological strategy to overactivate SYK kinase activity by inhibiting the negative regulator INPP5D. This activates the B cell's intrinsic negative selection checkpoint, preventing overactivation of tyrosine kinase signaling, which leads to selective death of pre-B cell acute lymphoblastic leukemia (ALL) cells. [1]
This mechanism holds promise for overcoming resistance, particularly in TKI-resistant Ph+ ALL, including cases carrying the BCR-ABL1[T315I] mutation. [1]
Sensitivity to INPP5D inhibition and subsequent SYK overactivation appears to be specific to B-lineage leukemia cells (such as Ph+ ALL) and was not observed in myeloid leukemia (such as CML) or normal pre-B cells under the conditions tested. [1]

C27H49N

387.69

387.386

C, 83.65; H, 12.74; N, 3.61

2206-20-4

5351709

White to off-white solid powder

104.5-105.5℃ (methanol )

9.1

1

1

5

28

540

9

C[C@H](CCCC(C)C)[C@H]1CC[C@@H]2[C@@]1(CC[C@H]3[C@H]2CC[C@@H]4[C@@]3(CC[C@H](C4)N)C)C

RJNGJYWAIUJHOJ-FBVYSKEZSA-N

InChI=1S/C27H49N/c1-18(2)7-6-8-19(3)23-11-12-24-22-10-9-20-17-21(28)13-15-26(20,4)25(22)14-16-27(23,24)5/h18-25H,6-17,28H2,1-5H3/t19-,20+,21-,22+,23-,24+,25+,26+,27-/m1/s1

(3R,8R,9S,10S,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-amine

3α-Aminocholestane; 3AC; 3-AC; 2206-20-4; 3alpha-Aminocholestane; 3; A-Aminocholestane; (3alpha,5alpha)-Cholestan-3-amine; (3R,5S,8R,9S,10S,13R,14S,17R)-10,13-dimethyl-17-[(2R)-6-methylheptan-2-yl]-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-amine; 3??-Aminocholestane; 3+/--Aminocholestane; 3 AC

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: ≥ 3.25 mg/mL (8.38 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 32.5 mg/mL clear EtOH 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: ≥ 3.25 mg/mL (8.38 mM) (saturation unknown) in 10% EtOH + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 32.5 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well.

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