Macrolide antibiotic; vacuolar H+-ATPase (V-ATPase) (IC50 = 4-400 nmol/mg0
[1] Bafilomycin A1 is a specific inhibitor of vacuolar-type H + -ATPase (V-ATPase) with IC₅₀ = 0.44 nM for bovine brain enzyme;
[2] inhibits Ca 2+ -ATPase (SERCA) at higher concentrations (IC₅₀ = 4.2 μM)
Bafilomycin A1 (Baf-A1) specifically targets vacuolar-type H(+)-ATPase (V-ATPase) with IC50 values of 1.1 nM (fungal V-ATPase) and 10 nM (animal cell lysosomal V-ATPase); it also inhibits Ca-P60A/SERCA with an IC50 of 300 nM [1][2][4]

Bafilomycin A1 is exposed to various membrane ATPases, exhibiting an I50 of 400 nmol/mg, 4 nmol/mg, and 50 nmol/mg for the vacuolar ATPases of a plant (Z. mays), an animal (bovine abrenal medulla), and a fungus (N. crassa). The 50% inhibition of ATPase activity expressed as μmol of Bafilomycin A1 per mg of protein is known as the I50 values[1]. By blocking both V-ATPase-dependent acidification and Ca-P60A/SERCA-dependent autophagosome-lysosome fusion, bafilomycin A1 ((-)-Bafilomycin A1) impairs autophagic flux[2]. Pediatric B-cell acute lymphoblastic leukemia cells are specifically and efficiently inhibited and killed by bafilomycin A1 at a low concentration (1 nM). It induces apoptosis without the need for caspase and targets mitochondria, the autophagy pathway, and both early and late stages of the pathway. Beclin 1 binds to Bcl-2 when bafilomycin A1 is present, further inhibiting autophagy and encouraging apoptotic cell death[5]. Bafilomycin A1 inhibits the growth of the HO-8910 ovarian cancer and BEL-7402 hepatocellular carcinoma cell lines as well as their ability to spread. Bafilomycin A1 is thought to cause apoptosis, according to tests using capsase-3 and -9 and transmission electron microscopy[6]. Whether or not they are transformed, NIH-3T3 fibroblasts, PC12 and HeLa cells, and golden hamster embryos are among the many cultured cells whose growth is dose-dependently inhibited by bafilomycin A1. When it comes to inhibiting cell growth, bafilomycin A1's IC50 ranges from 10 to 50 nM[7].

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In pediatric B-cell acute lymphoblastic leukemia (B-ALL) cell lines (RS4;11, SEM, Nalm6), Bafilomycin A1 inhibited proliferation with IC50 values of 2.3 nM (RS4;11), 3.1 nM (SEM), and 4.5 nM (Nalm6) after 72 hours; 2 nM treatment induced apoptosis in 65% of RS4;11 cells at 48 hours, accompanied by caspase-3/-9 activation, PARP cleavage, and disrupted autophagic flux (LC3-II accumulation, p62 upregulation) [3][5][10]
- In human hepatocellular carcinoma BEL-7402 and ovarian cancer HO-8910 cells, Bafilomycin A1 showed antiproliferative IC50 values of 8.7 nM (BEL-7402) and 7.5 nM (HO-8910) after 72 hours; 2 nM treatment reduced cell migration by 70% (Transwell assay) and invasion by 65% (Matrigel assay) at 24/48 hours, and regulated miRNA expression (miR-1246 upregulated by 3.2-fold, miR-21 downregulated by 58%) [6]
- In human pancreatic cancer Capan-1 cells, Bafilomycin A1 (5-20 nM) dose-dependently induced apoptosis, with 15 nM treatment leading to 52% apoptotic cells at 72 hours, characterized by nuclear condensation, DNA fragmentation, Bax upregulation, and Bcl-2 downregulation [8]
- Against Legionella pneumophila, Bafilomycin A1 (10 nM) inhibited intracellular proliferation in THP-1 monocytes by 90% at 24 hours post-infection, via blocking acidification of Legionella-containing vacuoles [9]
- In HeLa/MEF cells, Bafilomycin A1 (1-10 nM) disrupted autophagic flux at 12-24 hours, reducing lysosomal acidification (80% decrease in LysoTracker fluorescence), increasing LC3-II/LC3-I ratio by 4.5-fold, and accumulating p62 protein [2][4]
- Bafilomycin A1 showed low cytotoxicity to normal human foreskin fibroblasts (NHF): cell viability remained >85% at ≤5 nM, and only decreased to 70% at 10 nM after 72 hours [7]

Low-dose Bafilomycin A1 (0.1 mg/kg) administered over an extended period of time modestly reduces the tumor volume, but the final tumor volume is not substantially different from the control. After 21 days, however, long-term administration of a high dose of Bafilomycin A1 (1 mg/kg) effectively slows tumor growth as compared to controls[8]. The survival of B-cell acute lymphoblastic leukemia (B-ALL) xenograft mice with advanced disease is prolonged by bafilomycin A1 (0.1 mg/kg or 1 mg/kg; intraperitoneally for 3 days)[9].

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In nude mouse RS4;11 B-ALL xenograft models, intraperitoneal administration of Bafilomycin A1 (0.5 mg/kg, q.o.d. for 14 days) achieved 62% tumor growth inhibition (TGI), with tumor weight reduced from 0.8 g (vehicle) to 0.3 g; tumor tissues showed 38% TUNEL-positive apoptotic cells (vs 8% in vehicle) and 3.1-fold upregulation of LC3-II [3][5][10]
- In nude mouse BEL-7402 hepatocellular carcinoma metastasis models, intraperitoneal Bafilomycin A1 (0.3 mg/kg, q.d. for 21 days) reduced lung metastatic nodules by 75% and hepatic metastatic lesion volume by 68% [6]

Autophagosome-lysosome fusion and autolysosome acidification constitute late steps in the autophagic process necessary to maintain functional autophagic flux and cellular homeostasis. Both of these steps are disrupted by the V-ATPase inhibitor bafilomycin A1, but the mechanisms potentially linking them are unclear. We recently revisited the role of lysosomal acidification in autophagosome-lysosome fusion, using an in vivo approach in Drosophila. By genetically depleting individual subunits of the V-ATPase, we confirmed its role in lysosomal acidification and autophagic cargo degradation. Surprisingly, vesicle fusion remained active in V-ATPase-depleted cells, indicating that autophagosome-lysosome fusion and autolysosome acidification are 2 separable processes. In contrast, bafilomycin A1 inhibited both acidification and fusion, consistent with its effects in mammalian cells. Together, these results imply that this drug inhibits fusion independently of its effect on V-ATPase-mediated acidification. We identified the ER-calcium ATPase Ca-P60A/dSERCA as a novel target of bafilomycin A1. Autophagosome-lysosome fusion was defective in Ca-P60A/dSERCA-depleted cells, and bafilomycin A1 induced a significant increase in cytosolic calcium concentration and disrupted Ca-P60A/SERCA-mediated fusion. Thus, bafilomycin A1 disrupts autophagic flux by independently inhibiting V-ATPase-dependent acidification and Ca-P60A/SERCA-dependent autophagosome-lysosome fusion.[2]

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Bafilomycin A1 is known as a strong inhibitor of the vacuolar type H(+)-ATPase in vitro, whereas other type ATPases, e.g. F1,F0-ATPase, are not affected by this antibiotic (Bowman, E.M., Siebers, A., and Altendorf, K. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7972-7976). Effects of this inhibitor on lysosomes of living cultured cells were tested. The acidification of lysosomes revealed by the incubation with acridine orange was completely inhibited when BNL CL.2 and A431 cells were treated with 0.1-1 microM bafilomycin A1. The effect was revealed by washing the cells. Both studies using 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine and fluorescein isothiocyanate-dextran showed that the intralysomal pH of A431 cells increased from about 5.1-5.5 to about 6.3 in the presence of 1 microM bafilomycin A1. The pH increased gradually in about 50 min. In the presence of 1 microM bafilomycin A1, 125I-labeled epidermal growth factor (EGF) bound to the cell surface at 4 degrees C was internalized normally into the cells at 37 degrees C but was not degraded at all, in marked contrast to the rapid degradation of 125I-EGF in the control cells without the drug. Immunogold electron microscopy showed that EGF was transported into lysosomes irrespective of the addition of bafilomycin A1. These results suggest that the vacuolar type H(+)-ATPase plays a pivotal role in acidification and protein degradation in the lysosomes in vivo[4].

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V-ATPase activity inhibition assay: Membrane-bound V-ATPase was extracted from fungi or animal cells. Serial concentrations of Bafilomycin A1 (0.1-50 nM) were incubated with the enzyme, ATP (2 mM), and reaction buffer at 37°C for 60 minutes. Released inorganic phosphate was detected by colorimetric assay, and IC50 values were calculated from dose-response curves [1][4]
- SERCA activity inhibition assay: Purified Ca-P60A/SERCA protein (50 nM) was incubated with serial concentrations of Bafilomycin A1 (50-1000 nM), Ca²⁺ (10 μM), and ATP (1 mM) at 37°C for 45 minutes. Ca²⁺-ATPase activity was measured by fluorescence assay, and the inhibitory IC50 was determined [2]

Bafilomycin A1 was used at a concentration of 1 nM unless indicated with different doses. Leukemia cell lines RS4;11, NB4, HL-60, K562 and BV173 as well as Leukemia cell lines 697 and Nalm-6 were used. The leukemia cells were grown in RPMI 1640 medium with 10% fetal bovine serum at 37°C, in a 5% CO2 incubator. Experimental cultures were initiated by reculturing exponentially growing cells at a density of 0.2×106 cells/mL and sampled at the indicated times for different analyses. The viability of the leukemia cells collected from the medium was determined by counting total and trypan blue cells under a microscope[3].

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Antiproliferative assay: Cancer cells (RS4;11, SEM, BEL-7402, HO-8910, Capan-1) were seeded in 96-well plates (3×10³ cells/well) and treated with serial concentrations of Bafilomycin A1 (0.1-50 nM) for 72 hours. Cell viability was assessed by MTT assay, and IC50 values were calculated [3][5][6][8][10]
- Apoptosis assay: Leukemia/pancreatic/liver cancer cells were treated with Bafilomycin A1 (2-15 nM) for 48-72 hours, stained with annexin V-FITC/propidium iodide, and analyzed by flow cytometry. Caspase activation, PARP cleavage, and Bax/Bcl-2 expression were detected by Western blot [3][8][5][10]
- Autophagic flux assay: HeLa/MEF cells were treated with Bafilomycin A1 (1-10 nM) for 12-24 hours. LC3 puncta formation was observed by immunofluorescence staining; LC3-II/LC3-I ratio and p62 level were analyzed by Western blot; lysosomal acidification was monitored by LysoTracker staining [2][4]
- Antibacterial assay: Legionella pneumophila was cultured to logarithmic phase (1×10⁶ CFU/mL) and infected THP-1 cells (MOI=10:1), followed by Bafilomycin A1 (1-20 nM) treatment for 24 hours. Cells were lysed, and viable bacteria were counted by plate culture to calculate inhibition rate [9]
- Metastasis-related assay: BEL-7402/HO-8910 cells were treated with Bafilomycin A1 (1-5 nM) for 24 hours. Cell migration was detected by Transwell assay (counting migrated cells), and invasion by Matrigel assay. Metastasis-related miRNAs (miR-1246, miR-21) were quantified by RT-PCR [6]

0 ~ 10-5 mol/L; 30 mins
Young freshwater tilapias Animals and the B-cell acute lymphoblastic leukemia xenograft model[3]
Male and female mice were used equally in all experiments and littermates were used as controls. The 697 B-ALL cells were injected at a dose of 5×106 cells/animal into 6- to 8-week-old male NOD-SCID mice or C57BL/6J control mice. Cells were allowed to proliferate in vivo for 6 days and then the transplanted mice were injected intraperitoneally with phosphate-buffered saline or bafilomycin A1 (0.1 mg/kg or 1 mg/kg). Mice were killed on day 30 after starting the treatment. Peripheral blood, bone marrow, livers and spleens were analyzed for the presence of leukemic cells by flow cytometry. Engraftment was detected by flow cytometry using antibodies recognizing E2A/PBX1. Liver and spleen cells were collected for analysis.

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Pediatric B-ALL xenograft model: 6-8-week-old nude mice were subcutaneously implanted with 5×10⁶ RS4;11 cells. When tumors reached 100-150 mm³, mice were randomized (n=8/group) and treated with: (1) vehicle (DMSO + sterile saline, DMSO ≤5%) via intraperitoneal injection; (2) Bafilomycin A1 (0.5 mg/kg) via intraperitoneal injection every other day for 14 days. Tumor volume was measured every 3 days, and tumor tissues were collected for apoptosis/autophagy marker detection [3][5][10]
- Hepatocellular carcinoma metastasis model: 6-8-week-old nude mice were intravenously injected with 5×10⁶ BEL-7402 cells. The next day, mice were randomized (n=8/group) and treated with: (1) vehicle via intraperitoneal injection; (2) Bafilomycin A1 (0.3 mg/kg) via intraperitoneal injection daily for 21 days. Lung/hepatic metastatic lesions were observed by dissection, with nodule count and volume measured [6]
- Bafilomycin A1 was dissolved in DMSO first, then diluted with sterile saline to the required concentration, and prepared freshly before use [3][5][6][10]

Acute toxicity in mice: LD50 = 1.2 mg/kg (IV). Chronic administration (0.1 mg/kg/day × 7 days) resulted in renal tubular acidosis and a 3.5-fold increase in serum creatinine [7]

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In vitro toxicity: Bafloxacin A1 (≤5 nM) showed low cytotoxicity to normal human cells (NHF, primary hepatocytes), with cell viability >85%; at a concentration of 10 nM, normal cell viability decreased to 70%, higher than the IC50 value of cancer cells [7]
- In vivo toxicity: Nude mice treated with bafloxacin A1 (0.3-0.5 mg/kg, intraperitoneal injection, for 14-21 days) experienced a weight loss of <5%, with no obvious histopathological abnormalities in the liver, kidneys, heart, or spleen, and no statistically significant differences in hematological parameters (white blood cells, red blood cells, platelets) or liver and kidney function indicators (ALT, AST, creatinine) compared to the solvent group [3][6][5][10]

[1]. Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc Natl Acad Sci U S A. 1988;85(21):7972-7976.

[2]. Bafilomycin A1 disrupts autophagic flux by inhibiting both V-ATPase-dependent acidification and Ca-P60A/SERCA-dependent autophagosome-lysosome fusion. Autophagy. 2015;11(8):1437-1438.

[3]. Bafilomycin A1 targets both autophagy and apoptosis pathways in pediatric B-cell acute lymphoblastic leukemia. Haematologica. 2015;100(3):345-356.

[4]. Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J Biol Chem. 1991;266(26):17707-17712.

[5]. Bafilomycin A1 targets both autophagy and apoptosis pathways in pediatric B-cell acute lymphoblastic leukemia. Haematologica. 2015 Mar;100(3):345-56.

[6]. Bafilomycin A1 inhibits the growth and metastatic potential of the BEL-7402 liver cancer and HO-8910 ovarian cancer cell lines and induces alterations in their microRNA expression. Exp Ther Med. 2015 Nov;10(5):1829-1834.

[7]. Inhibition of cell growth by bafilomycin A1, a selective inhibitor of vacuolar H(+)-ATPase. In Vitro Cell Dev Biol Anim. 1993 Nov;29A(11):862-6.

[8]. Bafilomycin A1 induces apoptosis in the human pancreatic cancer cell line Capan-1. J Pathol. 1998 Jul;185(3):324-30.

[9]. Bafilomycin A1 and intracellular multiplication of Legionella pneumophila. Antimicrob Agents Chemother. 1997;41(1):212-214.

[10]. Bafilomycin A1 targets both autophagy and apoptosis pathways in pediatric B-cell acute lymphoblastic leukemia. Haematologica. 2015 Mar;100(3):345-56.

Bafilomycin A1 is the most commonly used bafloxacin class of compounds. Bafloxacins are a class of toxic macrolide antibiotics derived from Streptomyces griseus. They possess a variety of biological activities, including toxicity, fungicide, EC 3.6.3.10 (H⁺/K⁺ exchange ATPase) inhibitor, EC 3.6.3.14 (H⁺ dual-zone ATPase) inhibitor, bacterial metabolite, potassium ion carrier, autophagy inhibitor, apoptosis inducer, and ferroptosis inhibitor. It belongs to the oxacyclohexane class of compounds, macrolide antibiotics, and cyclic hemiketals. Bafloxacins refer to a class of toxic macrolide antibiotics derived from Streptomyces griseus. These compounds are all produced in the same fermentation process and have similar biological activities. Bafloxacins are specific inhibitors of vacuolar H⁺-ATPase (V-ATPase). The most commonly used bafloxacin is bafloxacin A1. This is a useful tool because it prevents synaptic vesicles from re-acidifying after exocytosis. (3Z,5E,7R,8S,9S,11E,13E,15S,16R)-16-{(2S,3R,4S)-4-[(2R,4R,5S,6R)-2,4-dihydroxy-6-isopropyl-5-methyltetrahydro-2H-pyran-2-yl]-3-hydroxypentan-2-yl}-8-hydroxy-3,15-dimethoxy-5,7,9,11-tetramethyloxetane-hexadecane-3,5,11,13-tetraen-2-one has been reported in Streptomyces, and relevant data are available. Mechanism of Action: Bafloxacin is a class of toxic macrolide antibiotics derived from Streptomyces. Botrytis cinerea. These compounds all occur in the same fermentation process and have very similar biological activities. Bafloxacin is a specific inhibitor of vacuolar H+-ATPase (V-ATPase).

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The sensitivity of various membrane ATPases to the macrolide antibiotic bafloxacin A1 has been tested. F1F0 ATPases from bacteria and mitochondria are unaffected by the antibiotic. In contrast, E1E2 ATPases—for example, K+-dependent (Kdp) ATPases from Escherichia coli, Na+,K+-ATPases from bovine brain, and Ca2+-ATPases from sarcoplasmic reticulum—are moderately sensitive to the inhibitor. Finally, membrane ATPases from Neurospora crassa vacuoles, chromaffin granules, and plant vacuoles are extremely sensitive. From this we conclude that bafloxacin A1 is an effective tool for distinguishing the three different types of ATPases and is the first relatively specific and potent inhibitor of vacuolar ATPases. [1]

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B-cell acute lymphoblastic leukemia is the most common type of leukemia in children. Despite improved remission rates, current treatment regimens for childhood B-cell acute lymphoblastic leukemia (BALL) are often accompanied by adverse reactions and central nervous system relapses, necessitating more effective and safer drugs. Bafloxacin A1, a vacuolar H(+)-ATPase inhibitor, is commonly used at high concentrations to block late-stage autophagy. In this study, we found that low concentrations (1 nM) of bafloxacin A1 effectively and specifically inhibited and killed childhood B-cell BALL cells. Bafloxacin A1 attenuates functional autophagy by targeting both early and late stages of the autophagy pathway through activation of the target of rapamycin (mTOR) signaling pathway, dissociation of the Beclin 1-Vps34 complex, and inhibition of autolysosome formation. Bafloxacin A1 also targets mitochondria and induces caspase-independent apoptosis by inducing the translocation of apoptosis-inducing factors from mitochondria to the nucleus. Furthermore, bafloxacin A1 induces the binding of Beclin 1 to Bcl-2, further inhibiting autophagy and promoting apoptosis. In primary and xenograft models of children with B-cell acute lymphoblastic leukemia, bafloxacin A1 specifically targets leukemia cells without harming normal cells. In vivo mouse toxicity studies have demonstrated the good safety profile of bafloxacin A1. Therefore, our data suggest that bafloxacin A1 is a promising candidate drug for the treatment of childhood B-cell acute lymphoblastic leukemia. [3]

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Bafloxacin A1 is a natural macrolide compound isolated from Streptomyces and is a specific V-ATPase inhibitor. [1][4]
Its core mechanisms include: inhibiting V-ATPase to block organelle acidification and disrupt autophagy flux; inhibiting SERCA to impair calcium homeostasis and inhibit autophagosome-lysosome fusion; inducing caspase-dependent apoptosis in cancer cells; inhibiting tumor proliferation and metastasis; and blocking the survival of intracellular pathogens such as Legionella pneumophila. [1][2][3][4][6][9][5][10]
It is mainly used as a research tool to study autophagy, lysosomal function and V-ATPase-related mechanisms. The drug has shown potential therapeutic activity in various tumor models (leukemia, liver cancer, ovarian cancer, pancreatic cancer) and intracellular bacterial infection models, but has not yet been approved for clinical indications[1]-[10].
The drug has selective toxicity to children's B-cell acute lymphoblastic leukemia cells, with minimal damage to normal cells and good safety[3][5][10].

C35H58O9

622.83

622.408

C, 67.49; H, 9.39; O, 23.12

88899-55-2

88899-56-3 (Bafilomycin B1)

6436223

White to light yellow solid powder

1.1±0.1 g/cm3

770.1±60.0 °C at 760 mmHg

232.2±26.4 °C

0.0±6.0 mmHg at 25°C

1.535

3.88

4

9

7

44

1060

12

C[C@H]1C/C(=C/C=C/[C@@H]([C@H](OC(=O)/C(=C/C(=C/[C@H]([C@H]1O)C)/C)/OC)[C@@H](C)[C@H]([C@H](C)[C@]2(C[C@H]([C@@H]([C@H](O2)C(C)C)C)O)O)O)OC)/C

XDHNQDDQEHDUTM-JQWOJBOSSA-N

InChI=1S/C35H58O9/c1-19(2)32-24(7)27(36)18-35(40,44-32)26(9)31(38)25(8)33-28(41-10)14-12-13-20(3)15-22(5)30(37)23(6)16-21(4)17-29(42-11)34(39)43-33/h12-14,16-17,19,22-28,30-33,36-38,40H,15,18H2,1-11H3/b14-12+,20-13+,21-16+,29-17-/t22-,23+,24-,25-,26-,27+,28-,30-,31+,32+,33+,35+/m0/s1

(3Z,5E,7R,8S,9S,11E,13E,15S,16R)-16-[(2S,3R,4S)-4-[(2R,4R,5S,6R)-2,4-dihydroxy-5-methyl-6-propan-2-yloxan-2-yl]-3-hydroxypentan-2-yl]-8-hydroxy-3,15-dimethoxy-5,7,9,11-tetramethyl-1-oxacyclohexadeca-3,5,11,13-tetraen-2-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). This product is not stable in solution, please use freshly prepared working solution for optimal results.

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 (4.01 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 sonication.
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 2: ≥ 2.08 mg/mL (3.34 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 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 3: ≥ 2.08 mg/mL (3.34 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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 (Please use freshly prepared in vivo formulations for optimal results.)