Tubulin; microtubule (IC50 = 18-40 nM)

Cevipabulin (0-50 nM, 72 hours) exhibits good activity on cell lines from tumors of the ovary, breast, prostate, and cervical regions (between 18 and 40 nM IC50 values)[1].
Cevipabulin (TTI-237) generates sub-G₁ nuclei at low concentrations (20–40 nM) and strongly inhibits the G₂-M complex at higher concentrations (>50 nM), according to flow cytometry experiments[1].

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TTI-237 causes marked turbidity development with both microtubule protein and purified tubulin. TTI-237 inhibits binding of vinblastine to tubulin. TTI-237 induces multiple spindle poles and multinuclear cells. TTI-237 produces sub-G1 nuclei at a concentration lower than that required for mitotic block. TTI-237 is a poor substrate of P-glycoprotein. [1]

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Good activity (IC50 values between 18 and 40 nM) is demonstrated by cevipabulin (0-50 nM, 72 hours) against ovarian, breast, prostate, and cervical carcinoma cell lines [1]. Cevipabulin (TTI-237) exhibits substantial G2-M blockage at concentrations greater than 50 nM, although low concentrations (20–40 nM) result in sub-G1 nuclei [1].

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At low molar ratios of TTI-237:tubulin heterodimer (about 1:30), TTI-237 enhanced depolymerization kinetics in response to low temperature, but stabilized the aggregates at higher ratios (about 1:4). Similarly, the aggregates induced in microtubule protein by TTI-237 were depolymerized by excess Ca(++) at low TTI-237:tubulin-heterodimer molar ratios, but were stable at higher ratios. TTI-237 inhibited the exchange of [(3)H]GTP at the exchangeable nucleotide site of the tubulin heterodimer, and was similar to vincristine in its effects on the phosphorylation of eight intracellular proteins in HeLa cells. Conclusions: TTI-237 has properties that distinguish it from typical vinca-site and taxoid-site ligands, and therefore it may exemplify a new class of microtubule-active compounds[2].

Cevipabulin (TTI-2370) delivered intravenously and orally to mice (5, 10, 15, and 20 mg/kg every 4 days for 4 cycles) exhibits dose-dependent efficacy against human tumor xenografts and, at doses of 20 and 15, has good anti-tumor activity [1].

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TTI-237 is active by i.v. and p.o. administration against human tumor xenografts.[1]
Because of the successful clinical use of Vinca alkaloids and taxanes in the treatment of cancer, TTI-237 was tested for antitumor efficacy in two mouse xenograft models. In the first, the compound, which has excellent solubility in water, was formulated in 0.9% saline and given i.v. to athymic mice bearing staged tumors of LoVo human colon adenocarcinoma. The compound was given every 4 days for four cycles at doses of 5, 10, 15, and 20 mg/kg/dose. The compound showed dose-dependent effects, with good antitumor activity at 20 and 15 mg/kg (Fig. 6A).[1]
In the second model, U87-MG human glioblastoma, TTI-237 was given both i.v. and p.o. at a single dose of 25 mg/kg to tumor-bearing mice. The compound was about equally effective by the two routes (Fig. 6B).

Tubulin polymerization experiments. [1]
Immediately before use, microtubule protein or purified tubulin was dissolved in ice-cold PEM buffer. When used, GTP was present in PEM buffer at 1 mmol/L. In the case of purified tubulin without added GTP, the PEM buffer contained 10% (w/v) glycerol. The tubulin solution was centrifuged at top speed in an Eppendorf model 5415C microcentrifuge for 10 min at 4°C to remove any particles or aggregates. The supernatant from this centrifugation was dispensed at 100 μL per well to wells of a half-area 96-well plate already containing 10 μL of the compounds of interest. Compounds were diluted in the same buffer used for tubulin solubilization before being added to wells. The final compound concentrations and tubulin concentration are given in figure legends. Each compound was tested in duplicate at each concentration in each experiment. The polymerization plate was prepared at room temperture, and the cold tubulin solution was the final addition. As rapidly as possible after tubulin addition, the plate was put in a SpectraMax Plus plate reader, thermostated at either 24°C or 35°C, and mixed for 15 s using the instrument mix function, and the absorbance of each well at 340 nm was determined every minute for 60 min. An increase in apparent absorbance at 340 nm over the course of the reaction was a measure of the appearance of turbidity believed to be caused by the formation of tubulin polymers of unknown morphology. The absorbance at time 0 for each well was subtracted from each of the subsequent absorbance readings for that well, and then the duplicates were averaged. Every other point is shown in Fig. 2 and Supplementary Figs. S1 to S3 for clarity.

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Competitive binding experiments. [1]
To study possible competition at the Vinca domain and colchicine site, incubations were done under conditions which do not favor polymerization because vinblastine and colchicine bind preferentially to unpolymerized heterodimer. Highly purified tubulin was dissolved in PEM buffer without GTP and used at a final concentration of 1.0 to 1.3 mg/mL (10–13 μmol/L). Aliquots of competitor stock solutions were added to aliquots of the tubulin solution to give final concentrations of 100 μmol/L, and then aliquots of either [3H]vinblastine or [3H]colchicine were added to give final concentrations of 100 nmol/L or 50 nmol/L, respectively. Each reaction was run in quadruplicate. These solutions were incubated at 24°C for 1 h and then applied to MicroSpin G-50 columns which were centrifuged for 2 min at 3,000 rpm in an Eppendorf 5415C microfuge. An aliquot of each column effluent (containing tubulin and bound radioligand) was mixed with scintillation fluid and counted in a liquid scintillation spectrometer. Controls included samples without competitor and samples with unlabeled vincristine, colchicine, or paclitaxel. The ability of the competitor to inhibit the binding of the radioligand was expressed as a percentage of control binding in the absence of any competitor.

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Tubulin polymerization: plate protocol [2]
Immediately before use, microtubule protein or purified tubulin was dissolved in ice-cold PEM buffer. When used, GTP was present in PEM buffer at 1 mM. After holding the tubulin solution on ice for 10–15 min, it was centrifuged at top speed in an Eppendorf model 5415C microcentrifuge for 10 min at 4°C to remove any particles or aggregates. The supernatant from this centrifugation was dispensed to wells of ½-area 96-well plates as described previously, with the following modifications. After recording the increase in absorbance at 340 nm, 30°C, for 60 min, the plate was removed from the SpectraMax Plus plate reader and immediately floated for 30 min in a large tray of water that was pre-cooled to 4°C. Control experiments demonstrated that this was 2–3 times longer than necessary to fully depolymerize control microtubules. During the 4°C incubation no absorbance readings could be taken. At the end of the 4°C incubation, the plate was rapidly dried and returned to the SpectraMax Plus at 30°C for another 60 min of recording. Changes in turbidity which occurred as a result of the low temperature incubation were revealed as sharp discontinuities in the absorbance curves over the 2 h recording. Every sample was run in duplicate in each experiment. Final concentrations of tubulin, compound, and DMSO are given in the legend to Fig. 2. Absorbance measurements were taken every min throughout the assay.

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Tubulin polymerization: spectrophotometer protocol [2]
Reactions were carried out in 1 cm path length quartz cuvettes in a Beckmann Model DU7400 spectrophotometer equipped with a six cuvette automatic sample changer and Peltier temperature controller. Each cuvette contained 270 μl of microtubule protein plus compound; final concentrations of protein, compound, and DMSO are given in the legend to Fig. 2. Polymerization reactions were recorded for 60 min at 24°C, then the temperature was shifted to 12°C and the depolymerization was followed for another 60 min. Absorbance at 340 nm was recorded every min for each sample.

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Tubulin polymerization: depolymerization induced by Ca++ [2]
The spectrophotometer protocol given above was followed, except that after the first 60 min of recording at 25°C, all samples were made 3.6 mM in Ca++ by adding 10 μl of 100 mM CaCl2. The solutions were rapidly mixed, and recording was continued at 25°C for 60 min. Data points were collected at 1 min intervals.

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Binding of [3H]GTP to purified tubulin [2]
Reactions were carried out in standard, flat-bottom, 96-well plates. Highly purified tubulin was used at a final concentration of 11–14 μM heterodimer, and compounds were used at 100 μM final concentrations. In one set of experiments, tubulin was incubated first with compounds in quadruplicate for 30 min at room temperature before addition of [3H]GTP (final concentration of 96.7 μM, specific activity 0.04 Ci/mmol). After a further 30 min incubation, aliquots of each sample were applied to MicroSpin G-50 columns which were centrifuged for 2 min at 3,000 rpm in an Eppendorf 5415C microfuge. An aliquot of each column effluent (containing tubulin and bound [3H]GTP) was mixed with scintillation fluid and counted in a liquid scintillation spectrometer.

Cell cytotoxicity assay[1]
Cell Types: Human cancer cell lines (SK-OV-3, MDA-MB-435, MDA-MB-468, LnCaP and Hela cells).
Tested Concentrations: 0-50 nM
Incubation Duration: 72 hrs (hours)
Experimental Results: IC50 values in SK-OV-3, MDA-MB- are 24±8 nM, 21±4 nM, 18±6 nM, 22±7 nM and 40 nM 435, MDA-MB-468, LnCaP and HeLa cells.

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 Cytotoxicity assay. [1]
Cells were harvested by trypsinization, washed, counted, and distributed to wells of 96-well flat-bottomed microtiter plates at 1,000 cells per well in 200 μL of medium. All plates were incubated at 37°C in humidified 5% CO2 in air for ∼24 h.  On day 2, compounds for test were diluted and added to wells. Compounds were dissolved in DMSO at 10 to 20 mmol/L. For each compound, nine serial 2-fold dilutions were prepared in DMSO. Ten microliters of each dilution was transferred to 100 μL of medium and mixed well, and then 5 μL of this dilution were transferred in triplicate or quadruplicate to wells containing cells. The final high concentration of each compound was typically 5 μmol/L. All cultures, including controls with no compound, contained a final concentration of 0.27% DMSO. After 3 d of culture with test compounds (day 5 overall), the MTS assay (Promega; CellTiter 96 aqueous nonradioactive cell proliferation assay) was done on all wells. The averaged replicates for each compound at each concentration level were plotted against concentration, and the concentration that produced a relative color yield half way between the maximum (no compound) and minimum (all cells killed) was taken as the IC50 value.

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Immunofluorescence microscopy. [1]
HeLa cells were cultured in DMEM containing 10% heat-inactivated fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. For experiments, cells were plated at 2.5 × 104 per 0.5 mL per chamber of Biocoat poly-d-lysine–coated eight-well culture slides. Compounds were added to chambers the next day in a volume of 5 μL DMSO, giving a final DMSO concentration of 1%. Twenty hours later, medium was removed from the wells, the chamber sides were lifted off, and cells were fixed with ice-cold methanol for a minimum of 10 min. The slides were removed from methanol and allowed to air-dry. All remaining steps were done at room temperature. The cells were washed with two changes of PBS (10 min each time, in a Copland jar), then incubated with anti α-tubulin monoclonal antibody (1:500 dilution) in 1% bovine serum albumin (BSA; IgG free and protease free) in PBS for 1 h. Cells were washed twice with PBS for 10 min each time, then incubated with secondary antibody [1:100 dilution of FITC-conjugated goat antimouse IgG, F(ab′)2 specific] in 1% BSA in PBS for 1 h in the dark. After two washes with PBS, 5 min each, cells were stained with Hoechst 33258 at 6 μg/mL in PBS for 10 min, followed by two PBS washes. Finally, cells were mounted in SlowFade Light and examined with an Olympus BX61 microscope using a 40× UPlanApo air objective. Images were acquired with a Cooke SensiCam CCD imaging camera and Sliderule software.

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Flow cytometry. [1]
HeLa cells were plated at 1.25 × 105 in 2 mL/well in 12-well plates and cultured overnight. Then various concentrations of compounds were added as indicated in the figures, and culture was continued for 18 h. Cells were then harvested, taking care to recover all nonadherent as well as adherent cells from each well. This kit contains reagents to dissolve cell membrane lipids with a nonionic detergent, degrade structural proteins with trypsin, remove RNA with RNase, and stabilize the nuclear chromatin with spermine. The cleaned, isolated nuclei were stained with propidium iodide and analyzed by flow cytometry using a FACSCalibur instrument from Becton Dickinson. Nuclei were analyzed instead of cells because this approach gives greater accuracy in DNA content estimates and because we wished to detect nuclei from multinuclear cells, although mitotic cells, which lack a nuclear envelope, may not be accurately measured. Each compound was analyzed in three independent experiments, and data were averaged to make the graphs presented here. Estimates of the fraction of total nuclei in each cell cycle compartment were done by visually setting markers on the population histograms as illustrated in Fig. 5B.

Animal/Disease Models: Athymic nu/nu female mice were implanted subcutaneously (sc) (sc) with 1 × 107 LoVo human colon adenocarcinoma cells in the flank [1].
Doses: 5, 10, 15 and 20 mg/kg
Doses: intravenously (iv) (iv)(iv) every 4 days for a total of 4 cycles.
Experimental Results: The compound demonstrated dose-dependent effects and had good antitumor activity at 20 and 15 mg/kg.

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Animal/Disease Models: Athymic nu/nu female mice were implanted with 1 × 106 U87-MG human glioblastoma cells subcutaneously (sc) (sc) in the flank [1].
Doses: 25 mg/kg.
Doses: Orally or intravenously (iv) (iv)(iv) on days 0, 7, and 14.
Experimental Results: The compound was active against human tumor xenografts administered orally or intravenously (iv) (iv)(iv).

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Tumor xenograft experiments.[1]
Athymic nu/nu female mice were implanted s.c. in the flank with either 1 × 107 LoVo human colon adenocarcinoma cells or 1 × 106 U87-MG human glioblastoma cells. Cells were suspended in culture medium for injection. When tumors attained a mass of between 80 and 120 mg (day 0), animals were randomized into treatment groups each containing 5 to 10 animals. After staging, animals were treated i.v. or p.o. with Cevipabulin (TTI-237)  formulated in 0.9% saline or Klucel according to the schedules given in the figure legend or with vehicle alone. Tumor volumes [(length × width2) / 2] were determined at regular intervals. Results are reported as relative tumor growth (mean tumor volume on day measured divided by the mean tumor volume on day 0) as a function of time after staging. The data were analyzed by a one-sided Student's t test. A P value of ≤0.05 indicated a statistically significant reduction in tumor growth of the treated group compared with that of the vehicle control group.

[1]. TTI-237: a novel microtubule-active compound with in vivo antitumor activity. Cancer Res. 2008 Apr 1;68(7):2292-300.

[2]. The microtubule-active antitumor compound TTI-237 has both DB01229-like and Leurocristine-like properties. Cancer Chemother Pharmacol. 2009 Sep;64(4):681-9.

Cevipabulin has been used in trials studying the treatment and educational/counseling/training of Tumors and Neoplasms.
Cevipabulin is a synthetic, water soluble tubulin-binding agent with potential antineoplastic activity. Cevipabulin appears to bind at the vinca-binding site on tubulin, but seems to act more similar to taxane-site binding agents in that it enhances tubulin polymerization and does not induce tubulin depolymerization. The disruption in microtubule dynamics may eventually inhibit cell division and reduce cellular growth.

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5-Chloro-6-[2,6-difluoro-4-[3-(methylamino)propoxy]phenyl]-N-[(1S)-2,2,2-trifluoro-1-methylethyl]-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine butanedioate (TTI-237) is a microtubule-active compound of novel structure and function. Structurally, it is one of a class of compounds, triazolo[1,5a]pyrimidines, previously not known to bind to tubulin. Functionally, TTI-237 inhibited the binding of [(3)H]vinblastine to tubulin, but it caused a marked increase in turbidity development that more closely resembled the effect observed with docetaxel than that observed with vincristine. The morphologic character of the presumptive polymer is unknown at present. When applied to cultured human tumor cells at concentrations near its IC(50) value for cytotoxicity (34 nmol/L), TTI-237 induced multiple spindle poles and multinuclear cells, as did paclitaxel, but not vincristine or colchicine. Flow cytometry experiments revealed that, at low concentrations (20-40 nmol/L), TTI-237 produced sub-G(1) nuclei and, at concentrations above 50 nmol/L, it caused a strong G(2)-M block. The compound was a weak substrate of multidrug resistance 1 (multidrug resistance transporter or P-glycoprotein). In a cell line expressing a high level of P-glycoprotein, the IC(50) of TTI-237 increased 25-fold whereas those of paclitaxel and vincristine increased 806-fold and 925-fold, respectively. TTI-237 was not recognized by the MRP or MXR transporters. TTI-237 was active in vivo in several nude mouse xenograft models of human cancer, including LoVo human colon carcinoma and U87-MG human glioblastoma, when dosed i.v. or p.o. Thus, TTI-237 has a set of properties that distinguish it from other classes of microtubule-active compounds.[1]

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Purpose: To compare TTI-237 (5-chloro-6-[2,6-difluoro-4-[3-(methylamino)propoxy]phenyl]-N-[(1S)-2,2,2-trifluoro-1-methylethyl]-[1, 2, 4]triazolo[1,5-a]pyrimidin-7-amine butanedioate) with paclitaxel and vincristine in order to better understand the properties of this new anti-microtubule agent. Methods: Tubulin polymerization and depolymerization were followed by turbidimetric assays. Effects of compounds on the binding of [(3)H]guanosine triphosphate ([(3)H]GTP) to tubulin were studied by competition binding assays. Effects of compounds on the phosphorylation of a panel of intracellular proteins were determined by flow cytometry using phosphoprotein-specific antibodies. Results: At low molar ratios of TTI-237:tubulin heterodimer (about 1:30), TTI-237 enhanced depolymerization kinetics in response to low temperature, but stabilized the aggregates at higher ratios (about 1:4). Similarly, the aggregates induced in microtubule protein by TTI-237 were depolymerized by excess Ca(++) at low TTI-237:tubulin-heterodimer molar ratios, but were stable at higher ratios. TTI-237 inhibited the exchange of [(3)H]GTP at the exchangeable nucleotide site of the tubulin heterodimer, and was similar to vincristine in its effects on the phosphorylation of eight intracellular proteins in HeLa cells. Conclusions: TTI-237 has properties that distinguish it from typical vinca-site and taxoid-site ligands, and therefore it may exemplify a new class of microtubule-active compounds.[2]

C22H22CLF5N6O5

580.892301082611

580.126

C, 42.83; H, 4.25; Cl, 5.75; F, 15.40; N, 13.62; O, 18.15

849550-67-0

Cevipabulin;849550-05-6; 852954-81-5 (succinate); 849550-69-2 (fumarate)

71587814

White to off-white solid powder

4.249

4

15

10

39

685

1

C[C@@H](C(F)(F)F)NC1=C(C(=NC2=NC=NN12)Cl)C3=C(C=C(C=C3F)OCCCNC)F.C(=C/C(=O)O)\C(=O)O

TUXZQBYIZLWUKK-AFIAKLHKSA-N

InChI=1S/C18H18ClF5N6O.C4H4O4/c1-9(18(22,23)24)28-16-14(15(19)29-17-26-8-27-30(16)17)13-11(20)6-10(7-12(13)21)31-5-3-4-25-2;5-3(6)1-2-4(7)8/h6-9,25,28H,3-5H2,1-2H3;1-2H,(H,5,6)(H,7,8)/b;2-1+/t9-;/m0./s1

(E)-but-2-enedioic acid;5-chloro-6-[2,6-difluoro-4-[3-(methylamino)propoxy]phenyl]-N-[(2S)-1,1,1-trifluoropropan-2-yl]-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine

Cevipabulin fumarate; Cevipabulin fumarate [USAN]; 849550-69-2; UNII-Q380BYV049; Q380BYV049; Cevipabulin fumarate dihydrate; TTI-237; Cevipabulin fumarate (USAN);

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~86.07 mM)
H2O : ~1.43 mg/mL (~2.46 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.)