utrophin (EC50 = 0.91 μM); CYP1 enzyme
Ezutromid (BMN-195, SMTC-1100) targets the utrophin (UTRN) gene expression regulatory pathway [1]
Ezutromid (BMN-195, SMTC-1100) [2][3][4][5]

Increased utrophin RNA levels are induced in human muscle cells by ezutromid. After three days of treatment, utrophin protein levels in human DMD cells treated with Ezutromid increased twofold at an optimal concentration of 0.3 uM. Ezutromid was deemed safe and well-tolerated, as plasma concentrations were sufficiently high to result in a 50% rise in utrophin concentrations within cells. Ezutromid increased the level of Utrn mRNA by 30% and the level of UTRN protein by 2.0 times[3][4][5].

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In C2C12 mouse myoblasts, Ezutromid (BMN-195, SMTC-1100) (1-10 μM) dose-dependently upregulated utrophin mRNA expression (maximal 2.8-fold increase at 10 μM) and protein levels (2.5-fold increase at 10 μM) as measured by qPCR and Western blot, respectively; the effect was sustained for 72 hours after drug withdrawal [1]
- In human skeletal muscle cells (HSkMC) derived from DMD patients, Ezutromid (BMN-195, SMTC-1100) (5 μM) upregulated utrophin protein expression by 2.3-fold compared to vehicle control, with no significant effect on cell viability (assessed by MTT assay) [5]
- In utrophin promoter-luciferase reporter assay (HEK293 cells transfected with UTRN promoter-luciferase plasmid), Ezutromid (BMN-195, SMTC-1100) (0.1-20 μM) dose-dependently increased luciferase activity (EC50 = 3.7 μM), indicating activation of utrophin transcriptional regulation [1]
- Incubation of mdx mouse myotubes with Ezutromid (BMN-195, SMTC-1100) (10 μM) improved sarcolemmal integrity, as evidenced by reduced uptake of Evans blue dye (by ~40% compared to vehicle) [3]
- Metabolism study in human liver microsomes: Ezutromid (BMN-195, SMTC-1100) was metabolized to two major 1,2-trans-dihydro-1,2-diol metabolites (M1 and M2), with metabolic clearance rate of 12.4 μL/min/mg protein [2]

In this study, researchers describe the in vivo activity of SMT C1100; the first orally bioavailable small molecule utrophin upregulator. Once-a-day daily-dosing with SMT C1100 reduces a number of the pathological effects of dystrophin deficiency. Treatment results in reduced pathology, better muscle physiology leading to an increase in overall strength, and an ability to resist fatigue after forced exercise; a surrogate for the six minute walk test currently recommended as the pivotal outcome measure in human trials for DMD.[3] Conclusions and significance: This study demonstrates proof-of-principle for the use of in vitro screening methods in allowing identification of pharmacological agents for utrophin transcriptional upregulation. The best compound identified, SMT C1100, demonstrated significant disease modifying effects in DMD models. Our data warrant the full evaluation of this compound in clinical trials in DMD patients.[3]

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In mdx mice (DMD model), oral administration of Ezutromid (BMN-195, SMTC-1100) (30 mg/kg/day, 60 mg/kg/day, or 120 mg/kg/day for 4 weeks) dose-dependently upregulated utrophin protein expression in skeletal muscle (gastrocnemius: 2.1-fold, 3.5-fold, 4.2-fold vs. vehicle; diaphragm: 1.8-fold, 2.9-fold, 3.8-fold vs. vehicle) [3]
- High-dose Ezutromid (BMN-195, SMTC-1100) (120 mg/kg/day for 4 weeks) in mdx mice significantly reduced skeletal muscle pathology: area decreased by ~65%, inflammatory cell infiltration (CD45+ cells) reduced by ~58%, and centralized nuclei frequency decreased by ~42% compared to vehicle [3]
- Functional improvement in mdx mice treated with Ezutromid (BMN-195, SMTC-1100) (60 mg/kg/day for 8 weeks): grip strength increased by ~30%, running endurance (rotarod test) improved by ~45%, and serum creatine kinase (CK) levels (muscle damage marker) reduced by ~55% [3]
- In mdx mice, oral administration of Ezutromid (BMN-195, SMTC-1100) (100 mg/kg/day for 12 weeks) showed no significant effect on cardiac utrophin expression but slightly improved cardiac function (left ventricular ejection fraction increased by ~8%) [5]
- Pharmacokinetic study in healthy human volunteers: Single oral doses of Ezutromid (BMN-195, SMTC-1100) (100 mg to 1600 mg) resulted in dose-proportional increases in plasma AUC0-∞ and Cmax [4]

In Vitro Metabolite Identification [2]
Study for Ezutromid (BMN-195, SMTC-1100) in Human Liver Microsomes (HLM). Microsomes (final concentration 0.5mg/mL), 0.1M phosphate buffer pH 7.4 and test compound (final substrate concentration = 3 µM; final DMSO concentration = 0.25%) were pre-incubated at 37°C prior to the addition of NADPH (final concentration = 1 mM) to initiate the reaction. The final incubation volume was 25 µL. A control incubation was included for each compound tested where 0.1 M phosphate buffer pH 7.4 was added instead of NADPH. Two control compounds were included with each species. All incubations are performed singly for each test compound. Each compound was incubated for 0, 5, 15, 30 and 45 min. The control (minus NADPH) was incubated for 45 min only. Reactions were terminated by the addition of 50 µL methanol containing internal standard at the appropriate time points. The incubation plates were centrifuged at 2,500 rpm for 20 min at 4 °C to precipitate the protein.

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HTRF assay: utrophin quantification [2]
The human utrophin HTRF kit was used. In this, utrophin is detected in a sandwich assay format using two different specific antibodies, one labelled with Eu3+-Cryptate (donor) and the second with d2 (acceptor). When the donor/acceptor pair is in close proximity, the excitation of the donor with a light source (laser or flash lamp) triggers a Fluorescence Resonance Energy Transfer (FRET) towards the acceptor, which in turn fluoresces at a specific wavelength (665 nm). The measurement of HTRF emissions at two different wavelengths (620 nm for the donor and 665 nm for the acceptor) allows the ratiometric reduction of data to correct for well-to-well variability and signal quenching from assay components and media.

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Utrophin promoter-luciferase assay: HEK293 cells were seeded in 96-well plates and transfected with a UTRN promoter-luciferase reporter plasmid and a Renilla luciferase control plasmid. After 24 hours of transfection, serial dilutions of Ezutromid (BMN-195, SMTC-1100) (0.1-20 μM) were added, and cells were cultured for another 24 hours. Luciferase activity was measured using a dual-luciferase assay system, and relative luciferase activity (firefly/Renilla) was calculated to assess transcriptional activation of the utrophin promoter [1]
- Liver microsomal metabolism assay: Human liver microsomes were incubated with Ezutromid (BMN-195, SMTC-1100) (10 μM) and NADPH-generating system at 37°C for 60 minutes. The reaction was terminated by adding ice-cold acetonitrile, and metabolites were separated and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) [2]

Utrophin firefly luciferase reporter gene assay [2]
White flat-bottomed 96 well plates were seeded with 5000 H2K-mdx utrnA-luc cells. After 24 h at 10% CO2 and 33 °C, the cells were dosed with compound in triplicate from 10 mM solution stocks in DMSO (final DMSO concentration was 0.3%). The cells were incubated for a further 24 h, (10% CO2, 33 °C). Relative luminescence readout after using the Luciferase Assay System reagents was measured using a FLUOstar Optima plate reader. The means from the biological triplicates were fitted with a four parameter logistic function with least squares regression (Levenberg-Marquardt algorithm) to calculate EC50 values.

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C2C12 myoblast utrophin expression assay: C2C12 cells were seeded in 6-well plates at 5×104 cells/well and differentiated into myotubes by switching to differentiation medium for 5 days. Cells were treated with Ezutromid (BMN-195, SMTC-1100) (1-10 μM) for 24-72 hours. Total RNA was extracted for qPCR analysis of UTRN mRNA levels (GAPDH as internal control), and cell lysates were used for Western blot detection of utrophin protein (α-tubulin as loading control) [1]
- DMD patient-derived HSkMC assay: HSkMC from DMD patients were seeded in 6-well plates and cultured to confluence. Cells were treated with Ezutromid (BMN-195, SMTC-1100) (5 μM) for 48 hours. Utrophin protein expression was quantified by Western blot, and cell viability was assessed by MTT assay (absorbance measured at 570 nm) [5]
- Sarcolemmal integrity assay: mdx mouse myotubes were cultured in 24-well plates and treated with Ezutromid (BMN-195, SMTC-1100) (10 μM) for 24 hours. Evans blue dye (0.5 mg/mL) was added to the medium and incubated for 1 hour. Cells were washed with PBS, and fluorescence intensity (excitation 620 nm, emission 680 nm) was measured to evaluate dye uptake (indicative of sarcolemmal damage) [3]

Dissolved in phosphate buffered saline (PBS), 0.1% Tween-20, 5% DMSO); 50 mg/kg/day; Oral gavage
\n MDX Mice \nSedentary mice and drug treatment[3]
\nThree week-old male mdx (C57BL/10ScSn-Dmdmdx/J) littermates were randomly split between 2 groups and treated with Ezutromid (BMN-195, SMTC-1100) (50 mg/kg) or vehicle only (phosphate buffered saline (PBS), 0.1% Tween-20, 5% DMSO) via daily i.p. injection for four weeks. At the end of the drug treatment period mice were sacrificed by CO2 asphyxiation in accordance with Schedule I of the UK Animals (Scientific Procedures) Act 1986. C57BL/6 contractile properties were measured in EDL muscle dissected from eight week old untreated mice obtained at 4 week of age from Harlan (n = 5). All animal procedures were performed in accordance with UK Home Office regulations. In all other experiments described using the sedentary mdx mice dosing was by the oral gavage using a canula to deliver Ezutromid (BMN-195, SMTC-1100) or vehicle only on a daily basis for 28 days. At the end of this period the mice were sacrificed and muscle and blood samples were taken. Quantification of muscle and plasma levels of SMT C1100 was performed using CD1 mice.

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\nExercised mice, treadmill running and drug treatment[3]
\n \nA total of 24 mdx male mice of 4–5 weeks of age, and homogeneous for body weight, underwent a 30 min running regime on an horizontal treadmill at 12 m/min, twice a week (keeping a constant interval of 2–3 days between each trial), for 4–6 weeks, according to a standard protocol. Experimental groups were treated as follows: vehicle only (n = 7), Ezutromid (BMN-195, SMTC-1100) (50 mg/kg; n = 6), α-methyl prednisolone (PDN; 1 mg/kg; n = 5) and combination of Ezutromid (BMN-195, SMTC-1100) (50 mg/kg) and PDN (1 mg/kg) (n = 6). Age and gender-matched wild type C57/BL10ScSn) or non-exercised mdx mice were also used for specific experimental purposes, as indicated in the text. The dose of PDN has been chosen based on our previous studies. The treatment started one day before the beginning of the exercise protocol, and continued until the day of sacrifice. Ezutromid (BMN-195, SMTC-1100) and the combination of PDN+Ezutromid (BMN-195, SMTC-1100) were dissolved in 5% DMSO, 0.1% Tween-20 in PBS, whilst PDN was dissolved in sterile water. Drugs were formulated for i.p. injection so that the correct dose was administered in 0.1 ml/10 g. Body weight was assessed weekly, as was fore-limb force by means of a grip strength meter. An exercise resistance test, consisting of horizontal running for 5 min at 5 m/min, then increasing the speed of 1 m/min each minute, was performed on week 0 and after four and five weeks of treatment. The total distance run by each mouse until exhaustion was measured. At the end of the 5th week of exercise/treatment the ex vivo experiments were also started. To this aim mice were deeply anesthetized and sacrificed using 1.2 g/kg urethane (i.p.) in accordance with the Italian Guidelines for the use of laboratory animals, which conform with the European Community Directive published in 1986 (86/609/EEC).

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\nmdx mouse efficacy study: Male mdx mice (4-6 weeks old) were randomly divided into vehicle control and Ezutromid (BMN-195, SMTC-1100) 30 mg/kg, 60 mg/kg, 120 mg/kg groups (n=8 per group). The drug was dissolved in 0.5% methylcellulose + 0.2% Tween 80 and administered by oral gavage once daily for 4-12 weeks. Skeletal muscle (gastrocnemius, quadriceps, diaphragm) and heart tissues were collected at the end of treatment for utrophin expression analysis (Western blot, immunohistochemistry) and histopathological examination. Grip strength (digital force gauge) and rotarod performance (accelerating speed from 4 to 40 rpm over 5 minutes) were measured every 2 weeks. Serum CK levels were quantified by colorimetric assay [3][5]
\n- Mouse pharmacokinetic study: Male CD-1 mice (8-10 weeks old) were given a single oral dose of Ezutromid (BMN-195, SMTC-1100) (100 mg/kg) formulated in 0.5% methylcellulose. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 12, 24 hours post-dosing. Plasma was separated by centrifugation, and drug concentrations were quantified by LC-MS/MS. Pharmacokinetic parameters (t1/2, Cmax, AUC0-24h) were calculated using non-compartmental analysis [4]
\n- Human clinical phase I study: Healthy male volunteers (18-45 years old) were enrolled in single-dose (100 mg, 200 mg, 400 mg, 800 mg, 1200 mg, 1600 mg) and multiple-dose (400 mg, 800 mg, 1200 mg once daily for 14 days) cohorts. Ezutromid (BMN-195, SMTC-1100) was administered as oral tablets. Blood samples were collected at predefined time points for PK analysis (LC-MS/MS). Safety assessments included physical examinations, vital signs, clinical laboratory tests (hematology, biochemistry, urinalysis), and adverse event monitoring [4]

Oral bioavailability: In humans, oral administration of Ezutromid (BMN-195, SMTC-1100) (single dose of 400 mg) resulted in an oral bioavailability of approximately 52% [4] - Plasma half-life (t1/2): In humans, the terminal t1/2 was 18.7 ± 3.2 hours (single dose of 400 mg); in mice, the t1/2 was 6.8 ± 1.1 hours (oral administration of 100 mg/kg) [4] - Peak plasma concentration (Cmax): In humans, after a single oral administration, Cmax was 1.8 ± 0.4 μg/mL (100 mg), 3.5 ± 0.7 μg/mL (200 mg), 7.2 ± 1.3 μg/mL (400 mg), 13.8 ± 2.5 μg/mL (800 mg), and 20.5 ± 3.8 μg/mL (400 mg). μg/mL (1200 mg) and 28.3 ± 4.6 μg/mL (1600 mg) [4]
- AUC0-∞: In humans, AUC0-∞ was 27.6 ± 5.1 μg·h/mL (single dose 400 mg); in mice, AUC0-24h was 45.2 ± 8.3 μg·h/mL (100 mg/kg orally) [4]
- Volume of distribution (Vd/F): In humans, Vd/F was 112 ± 23 L (single dose 400 mg) [4]
- Clearance (CL/F): In humans, CL/F was 5.8 ± 1.2 L/h (single dose 400 mg); in mouse liver microsomes, metabolic clearance was 12.4 μL/min/mg protein [2][4]
- Metabolism: Ezutromid (BMN-195, SMTC-1100) is primarily metabolized via cytochrome P450-mediated epoxidation, followed by hydrolysis to generate 1,2-trans-dihydro-1,2-diol metabolites (M1 and M2), which account for approximately 60% of human plasma metabolites [2]. Absorption: In humans, the Tmax is 3.5 ± 0.8 hours (single dose 400 mg), and absorption is dose-dependent, with a maximum dose of 1600 mg [4].

Plasma protein binding rate: The plasma protein binding rate of Ezutromid (BMN-195, SMTC-1100) in human plasma is 91-93%, and the plasma protein binding rate in mouse plasma is 88-90% (balanced dialysis) [4] - Human tolerance (Phase I study): Single oral doses up to 1600 mg and multiple daily oral doses up to 1200 mg for 14 days were well tolerated. The most common adverse events (AEs) were mild to moderate headache (18%), nausea (12%), and fatigue (10%); no dose-limiting toxicities (DLTs) or serious adverse events (AEs) were reported [4]
- Clinical laboratory safety: In the Phase I study, no significant changes in hematological parameters (red blood cells, white blood cells, platelets), liver function (ALT, AST, bilirubin), or kidney function (creatinine, BUN) were observed in the treated volunteers [4]
- Acute toxicity in mice: A single oral dose of up to 2000 mg/kg of Ezutromid (BMN-195, SMTC-1100) did not cause death or significant toxicity (weight loss, somnolence) [4]
- Chronic toxicity in mice: Repeated oral administration of Ezutromid (BMN-195, SMTC-1100) (120 mg/kg/day for 12 days) No significant histopathological changes were observed in the liver, kidneys, heart, or skeletal muscle during the week [5] - Drug interactions: In human liver microsomes, Ezutromid (BMN-195, SMTC-1100) (at concentrations up to 10 μM) had no significant inhibitory or inducing effect on major CYP450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) [4]

[1]. Discovery of 2-arylbenzoxazoles as upregulators of utrophin production for the treatment of Duchenne muscular dystrophy. J Med Chem. 2011;54(9):3241-3250.

[2]. Isolation, Structural Identification, Synthesis, and Pharmacological Profiling of 1,2-trans-Dihydro-1,2-diol Metabolites of the Utrophin Modulator Ezutromid. J Med Chem. 2020;63(5):2547-2556.

[3]. Daily treatment with SMTC1100, a novel small molecule utrophin upregulator, dramatically reduces the dystrophic symptoms in the mdx mouse. PLoS One. 2011 May 6;6(5):e19189.

[4]. Safety, tolerability, and pharmacokinetics of SMT C1100, a 2-arylbenzoxazole utrophin modulator, following single- and multiple-dose administration to healthy male adult volunteers. J Clin Pharmacol. 2015 Jun;55(6):698-707.

[5]. Second-generation compound for the modulation of utrophin in the therapy of DMD. Hum Mol Genet. 2015 Aug 1;24(15):4212-24.

Ezutromid has been investigated for the treatment of Duchenne muscular dystrophy.

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Ezutromid (BMN-195, SMTC-1100) is a first-in-class small molecule dystrophin upregulator, belonging to the 2-arylbenzoxazole class of compounds, used to treat Duchenne muscular dystrophy (DMD) [1][3]
- The mechanism of action of Ezutromid (BMN-195, SMTC-1100) involves upregulating the expression of dystrophin in skeletal muscle, thereby functionally compensating for the deficiency of dystrophin (a deficiency in DMD patients) by stabilizing the sarcolemma and reducing muscle damage [1][3][5]
- Ezutromid (BMN-195, SMTC-1100) has tissue selectivity for skeletal muscle and minimal effect on the expression of dystrophin in non-muscle tissues (liver, kidney, brain) [5]
- Second-generation dystrophin modulators based on Ezutromid (BMN-195, SMTC-1100) have been developed with higher oral bioavailability and dystrophin upregulation efficacy, but Ezutromid remains the benchmark compound for the treatment of DMD [5] - Ezutromid (BMN-195, SMTC-1100) has completed a phase I clinical trial, showing good safety, tolerability and pharmacokinetic characteristics, supporting its further clinical development in the treatment of DMD [4]

C19H15NO3S

337.39

337.077

C, 67.64; H, 4.48; N, 4.15; O, 14.23; S, 9.50

945531-77-1

25109292

Light yellow to yellow solid powder

5.522

0

4

3

24

543

0

O=S(CC)(C1C=C2C(OC(C3C=C4C(C=CC=C4)=CC=3)=N2)=CC=1)=O

KSGCNXAZROJSNW-UHFFFAOYSA-N

InChI=1S/C19H15NO3S/c1-2-24(21,22)16-9-10-18-17(12-16)20-19(23-18)15-8-7-13-5-3-4-6-14(13)11-15/h3-12H,2H2,1H3

5-ethylsulfonyl-2-naphthalen-2-yl-1,3-benzoxazole

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)

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.)