mRNA N6-methyladenosine (m6A) demethylase FTO

Acute myeloid leukemia (AML) cells had an IC50 value of 44.8 μM when treated with FB23 for 72 hours, whereas NB4 and MONOMAC6 AML cells have an IC50 value of 23.6 μM [1]. MYC targets, E2F targets, and G2M checkpoint signaling cascades were significantly inhibited by FB23 treatment, which may help explain how FTO rectification and FTO KD decrease cell cycle and proliferation. Cell display and p53 dye are activated by FB23 administration [1].

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Development of a selective and potent FTO inhibitor (FB23) [1]
Previously, we identified MA as an inhibitor of FTO demethylation over ALKBH5 (Huang et al., 2015). The structural complex of FTO/MA clearly elucidated the principles underlying MA’s selectivity, facilitating a structure-guided design for potent FTO inhibitors. To this end, we applied the following two principles: i) to keep the benzyl carboxylic acid as the key element of MA contributing selectivity for FTO over ALKBH5, and ii) to extend the dichloride-substituted benzene to a deeper pocket that could be fully occupied by a bulky ligand (Figures 1A and S1A). Synthesis of the inhibitors involves introducing a 5-membered heterocyclic ring to MA through cross-coupling chemistry (Figure S1B). Among them, FB23 is much more potent than MA in inhibiting FTO-mediated demethylation, with an IC50 of 0.06 μM (Figure 1B) and thus a 140-fold increase over that of MA (Huang et al., 2015).

To validate the direct binding of FB23 to FTO, we established co-crystal structure of FB23 bound with the FTO protein. The crystal structure was solved by molecular replacement and refined to 2.20 Å resolution (Table S1). The superimposition of structural complexes of FTO bound with dm3T ligand or inhibitor revealed no gross differences in overall protein folding (Figure S1C). The 2Fo-Fc density map contoured to 1.0 sigma (Figure 1C), and the simulated annealing Fo-Fc OMIT density map contoured to 3.0 sigma (Figure S1C), demonstrating that FB23 showed an extraordinary shape complementary with the substrate-binding site, occupying the entire binding pocket. Similar to interactions observed in the FTO/MA complex, the phenyl ring in FB23 bearing carboxyl acid substituent forms hydrophobic interactions with the nucleotide recognition lid, thereby ruling out nonspecific binding to either RNA demethylase ALKBH5 or DNA repair enzymes ALKBH2 and ALKBH3. Hydrogen bonding occurs between the carboxyl group in FB23 and the side chain from the Ser229 residue of FTO directly. In FB23 one chlorine atom directly contacts the guanidinium group in Arg96 of FTO. In addition, extra hydrogen bonding was observed between nitrogen or oxygen in the extended heterocyclic ring of FB23 and the amide backbone of Glu234 of FTO, which likely allows the inhibitor FB23 to show enhanced inhibitory activity on FTO compared to MA. Collectively, the FTO/FB23 structure revealed that FB23 possesses specificity for and improved inhibition of FTO.

We further investigated the interaction between FTO and FB23 Dose-dependent attenuation of signals was observed in Carr-Purcell-Meiboom-Gill (CPMG) Nuclear Magnetic Resonance (NMR) titrations (Figures 1D and S1D), and positive saturation transfer difference (STD) signals were also detected (Figure 1D), which indicates that FTO interferes with the state of FB23. We also performed a Cellular Thermal Shift Assay (CETSA) to further validate their interactions in cellular conditions (Martinez Molina et al., 2013). As expected, the presence of FB23 induced an obvious thermal shift of the FTO protein in NB4 and MONOMAC6 AML cells (Figure 1E). Thus, the NMR titration and CETSA assays further demonstrate that FB23 is a direct FTO inhibitor.

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FB23 exhibits moderate anti-proliferation effects and its derivative (FB23-2) shows significantly improved activity [1]
We next sought to examine the anti-proliferative effect of FB23 on AML cells. However, FB23 only moderately inhibited the proliferation of NB4 and MONOMAC6 cells, with an IC50 of 44.8 μM and 23.6 μM, respectively (Figure 1F). As detected by LC-MS/MS analysis, we found that the intracellular concentration of FB23 is a mere 0.02 nmol/million in NB4 cells and 0.015 nmol/million in MONOMAC6 cells (Figure 1G). Thus, the limited inhibitory effect of FB23 on AML cell proliferation is likely due to the low cellular uptake of FB23.

The structure of the FTO/FB23 complex suggests that the optimization on the carboxylic acid of FB23 would not disturb the affinity and specificity for FTO. To improve the permeability of FB23, we synthesized derivatives of the benzyl carboxylic acid on the basis of the bioisosterism principle. The benzohydroxamic acid, termed as FB23-2 (Figures 1H and S1B), displays significantly improved anti-proliferative activity on NB4 and MONOMAC6 cells with an IC50 of 0.8 – 1.5 μM (Figure 1I), and maintains inhibitory activity on FTO demethylation in vitro (Figure 1J). To establish the absolute configuration, we determined the X-ray crystal structure of FB23-2, which unambiguously shows an intramolecular hydrogen bond between the amino hydrogen and the carbonyl of hydroxamic acid (Figure 1H, right panel). In addition, we analyzed the relative configuration of FB23-2 in solution using the Nuclear Overhauser Effect (NOE), which is a transfer of nuclear spin polarization through space, rather than chemical bonds. The strong NOE correlation between H-1 and H-10 in the NOESY spectrum also supports the intramolecular hydrogen bonding (Figure S1E). With this evidence in hand, the docking of FB23-2 to FTO resulted in an excellent fit of FB23-2 in a position of perfect overlap to the crystallographically determined binding mode of inhibitor FB23 bound to FTO (Figure S1F). Next, we detected the cellular uptake of FB23-2 by LC-MS/MS quantitation (Figure 1K). Of note, FB23-2 was detected around 0.05–0.2 nmol/million cells in MONOMAC6 and NB4 cells, which is several folds higher than the cellular uptake of FB23 (see Figure 1G). Meanwhile, FB23 was also detected in tiny amounts in the FB23-2 treated AML cells, which is likely a hydrolysis product of FB23-2. The increased intracellular concentration of FB23-2 likely contributes to its improved anti-proliferation effect in AML cells.

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FB23 and FB23-2 target similar signaling pathways to FTO KD in AML cells [1]
To investigate which genes and signaling pathways are responsible for the anti-leukemic function of FTO inhibitors, we performed transcriptome-wide RNA-sequencing (RNA-seq) analysis of FTO KD, FB23 treated, or FB23-2 treated NB4 AML cells as well as control cells. Via independent analysis of three different comparisons, we found that FTO KD, FB23 treatment, and FB23-2 treatment all caused the significant suppression of MYC targets, E2F targets, and G2M checkpoint signal cascades, which may contribute to the inhibitory effects of FTO inhibitors and FTO KD on cell cycle and proliferation (Figures 5A and S3A–S3D). In addition, all three treatments consistently activated apoptosis and p53 pathways (Figure 5A). Global gene set enrichment analysis (GSEA) indicated that FTO KD and FB23 or FB23-2 treatment display similar effects on regulating a set of functionally important signaling pathways (Figures S3E–S3G). Notably, the vast majority of pathways (41 out of 43, 95.3%) increased by FTO KD could also be enriched by FB23-2 (Figures 5B and S3H); similarly, the majority of signaling pathways suppressed by FB23-2 are also inhibited by FTO KD (Figures 5C and S3H). These results strongly suggest that FTO inhibitors, especially FB23-2, had the same effect on critical signalling pathways that control cell cycle, cell proliferation, and cell survival in AML cells as FTO KD.

Furthermore, we considered shFTO, FB23, and FB23-2 as a single group and re-analyzed the sequencing data between this group and the control group (including shNS group and DMSO group). Consistently, FTO KD and inhibition stimulated apoptosis and p53 pathway (Figures 5D and S3I, and Table S4); meanwhile, MYC targets, G2M checkpoint, and E2F targets were repressed (Figures 5E and S3I, and Table S4). FB23-2 treatment dramatically down-regulated genes enriched in MYC target V1, MYC target V2, E2F targets, and the G2M checkpoint signatures; meanwhile, genes enriched in apoptosis and the p53 pathway signatures were downregulated in AML cells (Figure 5F).

Pharmacokinetic parameters of a single intraperitoneal dosage of 3 mg/kg FB23 to Sprague Dawley (SD). The Cmax and Tmax values of FB23 are 142.5 ng/mL and 0.4 hr respectively[1].

Effect of FTO inhibitors on COX-1 and COX-2 enzymes[1]
The inhibitions of COX1 and COX2 enzymes by FTO inhibitors FB23 and FB23-2 were evaluated using the COX Fluorescent Inhibitor Screening Assay Kit following the manufacturer’s protocols. Briefly, COX-1 and COX-2 were incubated with test compounds at room temperature for 5 min, respectively, then 10 μl ADHP (10-acetyl-3,7-dihydroxyphenoxazine) was added to the sample and background wells (without COX enzymes). Reactions were initiated by quickly adding 10 μl of Arachidonic Acid and incubated for 2 min at room temperature. An excitation wavelength of 535 nm and an emission wavelength of 595 nm were used to obtain the signals.

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HPLC-based assay of the inhibition of m6A demethylation in RNA[1]
In vitro ssRNA demethylations were performed with some modifications on the reported assay (Huang et al., 2015). The reactions, containing 0.25 μM FTOΔN31 or 3 μM ALKBH5ΔN66, 5 μM 15-mer ssRNA (5′-AUUGUCA(m6A)CAGCAGC-3′), 300 μΜ 2OG, 280 μΜ (NH4)2Fe(SO4)2, 2 mM L-ascorbic acid, and inhibitors at required concentrations in 50 mM Tris-HCl (pH 7.5 – 8.0), were incubated at 25 °C for 30 min. The reactions were terminated by heating for 5 min at 90 °C, and then the mixtures were subjected to digestion by nuclease P1 and alkaline phosphatase. The IC50 values were quantitated based on the inhibitory percentages of m6A demethylation in the presence of inhibitors at indicated concentrations, using nonlinear regression, dose-response fit on GraphPad Prism 5.0™. All reactions were performed in triplicate.

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Crystallization and structure determination of FTO/FB23 complex [1]
Crystallizations were conducted with hanging-drop vapor-diffusion method at 18 °C. 8 mg/ml of FTOΔN31 protein was incubated with 5-folds FB23 and mixed with a reservoir solution containing 100 mM sodium citrate (pH 5.4), 11.5% (w/v) polyethylene glycol 3350, and 8% isopropanol. The crystals were cryo-protected using extra 20% (v/v) glycerol. Diffraction data were collected on the BL18U1 and BL17U1 beamline at the Shanghai Synchrotron Research Facility (SSRF). All X-ray data were processed using HKL2000 programs (Otwinowski and Minor, 1997), and converted to structure factors within the CCP4 program (Collaborative Computational Project, 1994). The structure was solved by molecular replacement in Phaser using the structure of FTO/MA complex (PDB code 4QKN) as the searching model. The model of structural complex FTO/FB23 was computationally refined with the program REFMAC5.

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Nuclear Magnetic Resonance (NMR) titration[1]
Phosphate buffer (20 mM sodium phosphate (pH 7.4), 100 mM NaCl, 5% DMSO) was used for NMR data acquisition on a Bruker Avance III-600 MHz spectrometer equipped with a cryogenically cooled probe at 25 °C. Experimental samples contained 200 μM FB23 and FTO protein at 0 μM, 1 μM, 2 μM, and 3 μM, respectively.

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Cellular thermal shift assay (CETSA)[1]
CETSA was conducted according to the protocol as previously described (Martinez Molina et al., 2013). NB4 and MONOMAC6 cells were collected and lysed in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 2 mM DTT. 50 μM FB23 or DMSO was added to the supernatant and incubated at 25 °C for 25 min. After denaturing at various temperatures for 5 min, samples were centrifuged, and the supernatants were analyzed by western blot. All experiments were performed in triplicate.

Cell proliferation assays 5,000 cells/well NB4, FTO KO NB4, and MONOMAC6 AML cells were seeded and treated with DMSO or FTO inhibitors for 72 hr. The cell proliferations were determined with CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay according to the manufacturer’s instructions. 10,000 cells/well human AML cells (MA9.3ITD, MA9.3RAS, U937, ML2, and MV4-11) and four primary cells from AML patients were seeded and subjected to FTO inhibitor treatment for 96 hr as indicated. 10,000 cells/well MA9 and FLT3/NPM1 primary cells isolated from AML mice and 5,000 cells/well shNS and shFTO NB4 cells were seeded and treated with FTO inhibitors for 24 hr, 48 hr, 72 hr, and 96 hr for proliferation determination.[1]

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Quantitation of FB23 and FB23-2 in AML cells NB4 and MONOMAC6 cells were treated with 10 μM FB23 or FB23-2 for 24 hr, respectively. Viable cells were distinguished with 0.1% trypan blue, counted and then harvested with PBS by several washings. Cells were diluted into 100 μl with 50% (v/v) water/methanol and followed by several shock freeze-thaw cycles. The supernatants were collected for analysis. The Ultimate 3000 system coupled with a TSQ Quantiva mass spectrometer was applied to determine the cellular concentration of compound FB23 and FB23-2. Analytes were separated on a XSELECT™ HSS T3 column (100 mm × 3.0 mm, 2.5 μm; Waters, USA). The mobile phases used for elution were (A) 0.1% (v/v) formic acid/water and (B) 0.1% (v/v) formic acid/acetonitrile. The mass spectrometer was operated in the negative MRM mode. Parent-to-product transitions were m/z 375.1→339.1, 375.1→298.1 for FB23, and m/z 390.3→318.0, 390.3→289.9 for FB23-2, respectively [1].

Pharmacokinetics [1]
Inhibitor FB23-2 was formulated in DMSO at 3 mg/ml. SD rat (male, 7 – 8 weeks old, n = 3) were treated intraperitoneally with 1 ml/kg formulated compound. Blood samples were collected by retro-orbital bleeding at 5 min, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hr after the intraperitoneal administration. Blood was collected into EDTA-containing tubes and plasma was obtained by centrifugation at 2,000 g for 5 min. FB23-2 and its hydrolysis metabolite FB23 concentrations in plasma were quantitated by LC-MS/MS method. Noncompartmental analysis with Phoenix 1.4 (Pharsight, USA) was used for all analytical measurements. Area under the concentration-time curve (AUC) was calculated using trapezoidal method. AUC0−∞ = AUC0-t + Ct/ke, ke is elimination rate constant. Elimination half-life (T1/2) = 0.693/ke, mean residence time (MRT) = AUMC/AUC.

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Quantitation of FB23-2 in plasma [1]
Calibration curve concentrations ranged from 1.00 to 500 ng/ml for FB23-2 and FB23. 50 μl of rat plasma was precipitated by adding 150 μl acetonitrile immediately and vortexed to stabilize FB23-2 at each sample collection. 50 μl of study sample supernatant, 25 μl internal standard solution (probenecid and estrone-3-sulfate: 400/100 nmol/l), and 50.0 μl of 5 mM ammonium acetate solution (containing 0.1% formic acid) were added to a 1.5 ml polypropylene tube, then vortexed and centrifuged at 11,000 × g for 10 min, the supernatant was injected to LC-MS/MS. A LC-30AD liquid chromatographic system coupled to a Triple Quad 5500 mass spectrometer was used for acquiring LC-MS/MS data. Analytes were separated on an Eclipse Plus C18 column (100 mm × 4.6 mm I.D., 3.5 μm;). The mobile phases used for isocratic elution were 25% (A) 5 mM ammonium acetate-formic acid (100/0.1, v/v) and 75% (B) acetonitrile. The flow rate was 0.6 ml/min. The mass spectrometer was operated in the negative MRM mode. The parent-to-product transitions were m/z 390.2→318.0 for FB23-2, m/z 283.9→239.9 for probenecid (internal standard of FB23-2), m/z 375.2→298.2 for FB23, m/z 349.2→269.2 for estrone-3-sulfate (internal standard of FB23). The collision energy was set at −16, −30, −28, and −43 eV. The dwell time for each transition was set at 100 ms.

Microsomal stability assay [1]
The assay was conducted as the previously reported (Di et al., 2006). Briefly, 3 μM FB23-2 was incubated with 0.5 mg/ml rat liver microsomal protein at 37 °C in the presence of 1 mM NADPH cofactor. After incubation for 0, 5, 15, 30 and 60 min, respectively, cold acetonitrile was added to terminate the reactions. The solution was centrifuged, and the supernatants were analyzed using LC–MS/MS method similar with the quantitation of FB23 and FB23-2 in AML cells. The parent-to-product transition of probenecid, internal standard of FB23-2, was m/z 283.9→239.9. The formula for calculation was T1/2 = −0.693/k, and the inherent clearance rate CLint = (0.693/in vitro T1/2) × (incubation volume/mg of microsomal protein) × (mg of microsomal protein/gram of liver) × (gram of liver/kg body weight). Each time-point group includes two repeats.

[1]. Small-Molecule Targeting of Oncogenic FTO Demethylase in Acute Myeloid Leukemia. Cancer Cell. 2019 Apr 15;35(4):677-691.e10.

FTO, an mRNA N6-methyladenosine (m6A) demethylase, was reported to promote leukemogenesis. Using structure-based rational design, we have developed two promising FTO inhibitors, namely FB23 and FB23-2, which directly bind to FTO and selectively inhibit FTO's m6A demethylase activity. Mimicking FTO depletion, FB23-2 dramatically suppresses proliferation and promotes the differentiation/apoptosis of human acute myeloid leukemia (AML) cell line cells and primary blast AML cells in vitro. Moreover, FB23-2 significantly inhibits the progression of human AML cell lines and primary cells in xeno-transplanted mice. Collectively, our data suggest that FTO is a druggable target and that targeting FTO by small-molecule inhibitors holds potential to treat AML.[1]

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Few inhibitors for regulation of RNA methylation have been characterized, which exists in sharp contrast to factors of DNA and histone epigenetics. Here we report that through structure-based rational designs, we have successfully developed more effective small-molecule inhibitors of FTO. The MA-derived inhibitor FB23 displays significantly improved inhibitory activity on FTO demethylation of m6A-RNA in vitro. Next, we optimized the physicochemical property of FB23, thus leading to the identification of FB23-2 with a significantly improved ability to hinder the proliferation of a panel of AML cell lines, and also inhibits primary AML LSCs in PDX mice, thus suggesting that FTO might serve as a potential molecular target in LSCs in order to inhibit leukemogenesis. The discovery of FB23-2 and its anti-proliferative effects on AML would increase the current intense interest in RNA methylation, especially with regard to the pharmacology.[1]

Importantly, we tend to show our inhibitors target FTO and impair its demethylation, and by targeting FTO our inhibitor causes a significant biological impact. We validated that the effects of FTO inhibitors on AML are linked to certain downstream targets, e.g., MYC, CEBPA, RARA, and ASB2 RNA transcripts. It remains unknown whether FB23-2 impairs FTO’s binding to target transcripts in cells, however. The target engagement of current inhibitors needs further explorations with a more depth, which could show the potential for these inhibitors to help propel the field of epitranscriptomics forward.

In summary, we provide here a proof-of-concept that small-molecule targeting of oncogenic FTO demethylase may be an effective therapeutic strategy for the treatment of AML. Our study demonstrates the feasibility of attenuated FTO demethylation for the induction of differentiation of AML cells. This effect is likely achieved through specifically regulating expression of critical genes and signalling pathways as a result of elevated m6A levels in mRNA transcripts of these genes that are induced by FTO inhibitors. As FTO-mediated demethylation has also been linked to a variety of cancer types, our findings may have a broad impact on cancer therapy by targeting epitranscriptomic RNA methylation.

C18H14CL2N2O3

377.22136259079

376.04

C, 57.31; H, 3.74; Cl, 18.80; N, 7.43; O, 12.72

2243736-35-6

138393314

White to off-white solid powder

5.7

2

5

4

25

469

0

ClC1C=C(C=C(C=1NC1C=CC=CC=1C(=O)O)Cl)C1C(C)=NOC=1C

VUXZATVQMFSUCM-UHFFFAOYSA-N

InChI=1S/C18H14Cl2N2O3/c1-9-16(10(2)25-22-9)11-7-13(19)17(14(20)8-11)21-15-6-4-3-5-12(15)18(23)24/h3-8,21H,1-2H3,(H,23,24)

2-[2,6-dichloro-4-(3,5-dimethyl-1,2-oxazol-4-yl)anilino]benzoic acid

FB23; FB 23; FB23; 2243736-35-6; 2-((2,6-Dichloro-4-(3,5-dimethylisoxazol-4-yl)phenyl)amino)benzoic acid; 2-[[2,6-bis(chloranyl)-4-(3,5-dimethyl-1,2-oxazol-4-yl)phenyl]amino]benzoic acid; Fb23 inhibitor; CHEMBL4572939; SCHEMBL23261118; BDBM589235;FB-23

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 : ~125 mg/mL (~331.37 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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