Akt; ERK; Human mitochondrial caseinolytic protease P (ClpP) was identified as a direct binding protein. ONC201 bound ClpP with approximately 10-fold lower affinity compared to TR compounds in competition assays. [3]

ONC201/TIC10 is a small-molecule inducer of the TRAIL gene under current investigation as a novel anticancer agent. In this study, we identify critical molecular determinants of ONC201 sensitivity offering potential utility as pharmacodynamic or predictive response markers. By screening a library of kinase siRNAs in combination with a subcytotoxic dose of ONC201, we identified several kinases that ablated tumor cell sensitivity, including the MAPK pathway-inducer KSR1. Unexpectedly, KSR1 silencing did not affect MAPK signaling in the presence or absence of ONC201, but instead reduced expression of the antiapoptotic proteins FLIP, Mcl-1, Bcl-2, cIAP1, cIAP2, and survivin. In parallel to this work, we also conducted a synergy screen in which ONC201 was combined with approved small-molecule anticancer drugs. In multiple cancer cell populations, ONC201 synergized with diverse drug classes, including the multikinase inhibitor sorafenib. Notably, combining ONC201 and sorafenib led to synergistic induction of TRAIL and its receptor DR5 along with a potent induction of cell death [1].

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- ONC201 inhibited cell proliferation in triple-negative breast cancer cells SUM159 and MDA-MB-231 with IC50 values of 2.96 μM (SUM159) and 4.20 μM (MDA-MB-231) after 72 h treatment (MTS assay). [3]
- ONC201 induced the integrated stress response (ISR) markers ATF4 and CHOP in SUM159 and MDA-MB-231 cells in a dose- and time-dependent manner. The concentration required to increase ATF4 closely reflected the IC50 for growth inhibition. CHOP induction peaked earlier than ATF4. [3]
- ONC201 (10 μM, 24 h) increased CHOP protein level in wild-type SUM159 cells, but this effect was abolished in ClpP knockdown cells. [3]
- ONC201 (10 μM, 24 h) reduced mitochondrial proteins TFAM and TUFM in SUM159 cells; this reduction was prevented by ClpP knockdown. [3]
- ONC201 significantly inhibited total protein synthesis (>50%) after 24 h treatment in SUM159 cells as measured by 35S-methionine/cysteine incorporation; this inhibition was strongly attenuated by ClpP knockdown. [3]
- ONC201 (up to 30 μM) did not significantly increase caspase 3/7 activity (using fluorogenic ACE-DEVD-AMC substrate) nor PARP cleavage in SUM159 cells, indicating cytostatic rather than apoptotic effects under the tested conditions. Staurosporine (10 nM) served as positive control for caspase activation. [3]
- ONC201 (300 nM, 3 μM, 30 μM for 24 h) did not induce significant caspase activity in SUM159 cells. [3]
- Cell counting experiments confirmed that ONC201 treatment did not reduce total cell number below initial seeding value even after 72 h incubation, consistent with cytostatic effects. [3]

In a mouse xenograft model of hepatocellular carcinoma, we demonstrated that ONC201 and sorafenib cooperatively and safely triggered tumor regressions. Overall, our results established a set of determinants for ONC201 sensitivity that may predict therapeutic response, particularly in settings of sorafenib cotreatment to enhance anticancer responses [1].

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TIC10 and TRAIL treatment causes tumor regression in the HCT116 p53−/− xenograft to a comparable extent when both are administered as multiple doses. TIC10 also induces regression of MDA-MB-231 human triple-negative breast cancer xenografts, whereas TRAIL-treated tumors progressed. In DLD-1 colon cancer xenografts, TIC10 induces tumor stasis one week after treatment, whereas TRAIL-treated tumors advance after a single dose. The SW480 xenograft also exhibits a sustained regression after receiving a single dose of TIC10, and this effect is seen whether the drug is administered orally or intraperitoneally. This suggests that TIC10 has a favorable oral bioavailability. TIC10 causes tumor-specific cell death by TRAIL-mediated direct and bystander effects. TIC10 is an effective antitumor agent against orthotopic human glioblastoma multiforme tumors. [2]

siRNA kinase library screen[1]
HCT116 p53−/− cells were transfected at 20,000 cells/well in 96-well plates. Cells were transfected with the Stealth RNAi human kinase library using RNAiMax in Optimem. Scramble and AllStars Hs Cell Death Control siRNA were used as negative and positive controls, respectively. Transfection was carried out overnight and the following day complete media containing ONC201 or DMSO was added to the plates following removal of the transfection media. CellTiterGlo analysis was performed according to the manufacturer’s instructions at indicated time points.

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ChIP assays [2]
ChIP assays were carried out as previously described for the TRAIL promoter with a ChIP-grade antibody for Foxo3a or an equivalent concentration of rabbit immunoglobulin G as a nonspecific control.

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Enzyme Assay: - Affinity chromatography competition assay: HeLa cell lysates were mixed with vehicle (2% DMSO) or different concentrations of ONC201 (concentrations not specified in text for this assay) for 15 min, then combined with TR-80 agarose beads and rotated for 1 h at room temperature. Beads were washed, boiled in SDS sample buffer, resolved by SDS-PAGE, and immunoblotted with ClpP antibodies. ONC201 reduced ClpP binding to TR-81 beads in a dose-dependent manner when applied to cell lysates or live cells. [3]
- Recombinant human ClpP peptidase activity assay: Purified recombinant human ClpP (1 μg/mL) was incubated in assay buffer (50 mM Tris, 10 mM MgCl2, 100 mM KCl, 1 mM DTT, 4 mM ATP, 0.02% Triton X-100, 5% glycerol, pH 8.0) with 10 μM fluorogenic substrate Ac-WLA-AMC. Two protocols were used: Protocol #1 – reaction initiated instantly by direct mixing of enzyme, substrate, and compound; Protocol #2 – enzyme and compound preincubated for 60 min at 37°C before substrate addition. Fluorescence (excitation 350 nm, emission 460 nm) was monitored. ONC201 increased ClpP peptidase activity in a dose- and time-dependent manner. [3]
- ClpP protease activity assay (casein degradation): Recombinant ClpP was preincubated for 1 h at 37°C with DMSO or ONC201 in assay buffer, then incubated for an additional hour with 5 μM α-casein. Reaction products were resolved by 12% SDS-PAGE and silver stained. ONC201 increased casein proteolysis with a half-maximal dose of approximately 1.25 μM. [3]

Small molecule synergy screen[1]
Procedures were performed with a Biomek 2000 robot using pin tools for drug treatment. Cells were seeded at 5×104 cells/mL in 96-well black-wall plates and treatment was performed 12 hours later. Combinatorial activity was initially assessed by calculating the difference between the observed activity with the combination and the sum of the monoagent activities.

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Cell-based assays[1]
Cell TiterGlo, Western blot analysis, and cell cycle flow cytometry analysis were performed as previously described. Cell surface TRAIL and DR5 were assessed following fixation with 4% paraformaldehyde in PBS for 20 minutes, rinsed in PBS, incubated overnight at 1:200 with anti-TRAIL or anti-DR5 antibodies, rinsed in PBS, incubated with fluorophore-conjugated secondary antibodies at 1:250 for 30 minutes, and analyzed by flow cytometry. pERK was assessed with an antibody for p-T202/Y204 (Cell Signaling) and pAkt was assessed with antibody for pT308.

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Cells were treated with 10 μM ONC201 or DMSO for 24 h.
Colony formation assays [2]
The indicated cell lines were plated at 500 cells per well and allowed to adhere, and then treated the next day in fresh complete medium. At 3 days after treatment, the medium was replaced with drug-free medium and cells were propagated for 10 days, with fresh medium given once every 3 days. At the end of the 10-day period, cells were washed in PBS, fixed with methanol, stained with Coomassie blue, rinsed, and dried for quantification.

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Western blot analysis [2]
Western blot analysis was conducted as previously described with NuPAGE 4 to 12% bis-tris gel and visualized with SuperSignal West Femto and x-ray film. Densitometry was performed with NIH ImageJ. Nuclear and cytoplasmic extracts were prepared with a cytoplasmic lysis buffer (10 mM Hepes, 10 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol) followed by a nuclear lysis buffer (20 mM Hepes, 420 mM NaCl, 1.5 mM MgCl2, 250 μM EDTA, 25% glycerol). For all lysis buffers, fresh protease inhibitor and 1 mM sodium orthovanadate were added immediately before use.

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- Cell viability (resazurin assay): SUM159 cells (1000 cells/well) were plated in 96-well plates, allowed to adhere overnight, then treated with ONC201 at indicated concentrations for 72 h. Resazurin (0.6 mM, 20 μL) was added and incubated for 30 min at 37°C. Relative fluorescence of resorufin (540-20 excitation, 590-20 emission) was measured. IC50 was calculated using GraphPad Prism 7. [3]
- Total cell counting (Hoechst stain): SUM159 cells (1000 cells/well) were plated in 96-well plates, treated with ONC201, and at indicated time points (0, 24, 48, 72 h), media was aspirated and Hoechst stain (1 μg/mL) added for 30 min at 37°C. Total cell number was quantified using Celigo Imaging Cytometer. [3]
- Crystal violet colony formation assay: SUM159 cells (1000 cells/well) were plated in 6-well plates, treated with ONC201 for 48 h, then media replaced with fresh media with or without drug. Cells were incubated until one well reached 100% confluence, stained with 0.5% crystal violet in 20% methanol for 10 min at RT, rinsed, and dried. Staining was quantified by dissolving in Sorenson's buffer (0.1 M sodium citrate, 50% ethanol, pH 4.2) and measuring absorbance at 570 nm. [3]
- Immunoblotting: SUM159 or MDA-MB-231 cells were treated with compounds, rinsed with cold PBS, lysed in RIPA buffer (no SDS) supplemented with phosphatase and protease inhibitors. Lysates were clarified, resolved by SDS-PAGE, transferred to membranes, and probed with primary antibodies (ATF4, CHOP, β-actin, TFAM, TUFM, ClpP) diluted 1:1000, followed by secondary antibodies (1:10,000). Blots were developed with ECL reagents and imaged. [3]
- siRNA reverse transfection: siRNA stocks (20 μM) were resuspended. Dharmafect I diluted 1:266 in OptiMEM, mixed with siRNA (final 125 nM), incubated 30 min at RT, added to plates. SUM159 cells were trypsinized, resuspended in DMEM:F12 with 5% FBS, 5 μg/mL insulin, 1 μg/mL hydrocortisone at 1×10^5 cells/mL (6-well) or 1×10^4 cells/mL (96-well), added to siRNA-containing wells, and incubated for 24 h. ClpP knockdown was verified by immunoblotting. [3]
- Caspase 3/7 activity assay: SUM159 cells (8×10^5 cells per 6 cm plate) were treated for 24 h with ONC201 (300 nM, 3 μM, 30 μM), DMSO (0.1%), or staurosporine (10 nM). Cells were scraped into lysis buffer (50 mM HEPES pH 7.4, 5 mM CHAPS, 5 mM DTT), and caspase activity was measured using fluorogenic peptide substrate (7-amido-4-methylcoumarin). [3]
- Nascent protein synthesis assay (35S metabolic labeling): SUM159 cells were incubated in methionine- and cysteine-free medium with ONC201 for 15 min, then 35S-labeled methionine/cysteine (125 μCi) was added for 30 min. Cells were washed, lysed in RIPA buffer, protein concentration determined by Bradford assay. TCA (20% final) was added, precipitated proteins captured on glass microfiber filters, washed with 20% TCA and 100% ethanol, air-dried, and radioactivity quantified by scintillation counting. Results normalized to protein concentration. [3]

6-week-old athymic nude mice were obtained from Charles River Laboratories. 107 HepG2 cells in 200 µL (1:1, PBS: Matrigel) were injected into each rear flank. Measurable tumors were assessed 1 week later. Treatment was then initiated as indicated. Sorafenib and ONC201 were administered as 100 µL solutions by oral gavage. ONC201 was administered 12 hours following sorafenib treatment. Tumor volume was assessed with digital calipers and calculated as a spheroid. Tumor dimensions and body weights were assessed twice a week. IHC (Vector Labs) and TUNEL (Millipore) analyses were performed as previously described [1].

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For subcutaneous xenografts, 4- to 6-week-old female athymic nu/nu mice (Charles River Laboratories) were inoculated with 1 × 106 cells (2.5 × 106 for T98G) of the indicated cell lines in each rear flank as a 200-μl suspension of 1:1 Matrigel (BD)/PBS. All subcutaneous tumors were allowed to establish for 1 to 4 weeks after injection until reaching a volume of ~125 mm3 before treatment initiation.[2]

Absorption
The maximum concentration (Cmax) of dodavelnipron was 2.8 mcg/mL (42%), and the total systemic exposure (AUC) was 23 h × mcg/mL (48%). Within the dose range of 125 to 625 mg, the Cmax and AUC of dodavelnipron increased dose-proportionately. The median time to reach maximum plasma concentration (Tmax) of dodavelnipron was 1.4 h (0.5 h, 5.6 h). Co-administration with a high-fat meal (800 to 1000 calories, 50% fat) decreased the Cmax of dodavelnipron by 40%, while the AUC remained unchanged.
Elimination Routes
Following a single dose of radiolabeled dodavelnipron, approximately 70% of the dose was excreted in the urine and 20% in the feces. No significant unmetabolized dodavelnipron was detected in the urine or feces.
Volume of Distribution
The apparent (oral) volume of distribution of dodavepron is 450 L (40%). The median in vitro plasma concentration is 0.67. Dodavepron can cross the blood-brain barrier.
Clearance
The apparent clearance is approximately 27 L/hr (48%).
Protein Binding
The plasma protein binding of dodavepron is 95% to 97%, and is concentration-independent in vitro.
Metabolites/Metabolites
Dodavepron is primarily metabolized by CYP3A4, with smaller contributions from CYP2B6, CYP2C8, CYP2C9, CYP2D6, and CYP3A5. Its metabolic pathways and metabolites are not fully elucidated.
Biological Half-Life
The mean terminal half-life of dodavepron is 11 hours (30%).

Efficacy and Safety: This study comprehensively evaluated the efficacy of treatment in 50 adult and pediatric patients with recurrent H3 K27M mutant diffuse midline gliomas from five open-label, non-randomized clinical trials conducted in the United States (ONC006 [NCT02525692], ONC013 [NCT03295396], ONC014 [NCT03416530], ONC016 [NCT05392374], and ONC018 [NCT03134131]). The efficacy evaluation population included patients with H3 K27M mutant diffuse midline gliomas who received monotherapy with dovetroxone and were diagnosed with measurable disease progression according to the Neuro-oncology Response Assessment - High-Grade Gliomas (RANO-HGG) criteria. All patients had received radiotherapy for at least 90 days, had adequate clearance from prior anticancer therapy, a Karnofsky Performance Status (KPS)/Lansky Functional Status (LPS) score ≥60, and had stable or gradually decreasing glucocorticoid dosage. Patients with diffuse entropional gliomas, primary spinal cord tumors, atypical histological types, or cerebrospinal fluid dissemination were excluded. The primary efficacy endpoint was objective response rate (ORR), assessed by an independent center-blinded review (BICR) according to RANO 2.0 criteria, with duration of response (DOR) as a secondary efficacy endpoint. The ORR was 22% (95% CI: 12, 36), and the median DOR was 10.3 months (95% CI: 7.3, 15.2). Among the 11 patients who achieved an objective response, 73% had a DOR ≥6 months, and 27% had a DOR ≥12 months. Prescription information for dodavirone included warnings and precautions regarding allergies, QTc interval prolongation, and embryo-fetal toxicity.

[1]. Cancer Res.2015 Apr 15;75(8):1668-74;
[2]. Sci Transl Med.2013 Feb 6;5(171):171ra17.

[3]. ACS Chem Biol. 2019 May 17;14(5):1020-1029.

ONC201/TIC10 is a small molecule inducer of the TRAIL gene, and its potential as a novel anticancer drug is currently under investigation. This study aimed to identify key molecular determinants of ONC201 sensitivity, which are expected to serve as biomarkers for pharmacodynamics or predictive efficacy. By screening a kinase siRNA library and combining it with subcytotoxic doses of ONC201, we identified several kinases capable of eliminating tumor cell sensitivity, including the MAPK pathway inducer KSR1. Surprisingly, silencing KSR1, regardless of the presence of ONC201, did not affect the MAPK signaling pathway; instead, it reduced the expression of anti-apoptotic proteins FLIP, Mcl-1, Bcl-2, cIAP1, cIAP2, and survivin. Simultaneously, we conducted a synergistic screening, combining ONC201 with approved small molecule anticancer drugs. In various cancer cell populations, ONC201 exhibited synergistic effects with multiple drug classes, including the multi-kinase inhibitor sorafenib. Notably, the combination of ONC201 and sorafenib synergistically induces the expression of TRAIL and its receptor DR5 and potently induces cell death. In a mouse xenograft model of hepatocellular carcinoma, we demonstrated that ONC201 and sorafenib synergistically and safely induce tumor regression. Overall, our findings identify a number of determinants of ONC201 sensitivity that may predict treatment response, especially in combination with sorafenib to enhance the anticancer response. [1] Recombinant tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL) is an antitumor protein currently undergoing clinical trials as a potential anticancer therapy, but its pharmacodynamic properties have some limitations, such as short serum half-life, poor stability, high cost, and uneven biodistribution (especially in the brain). To overcome these limitations, we discovered a highly effective, orally available, and stable small molecule compound—TRAIL-inducing compound 10 (TIC10). TIC10 can induce TRAIL expression in a p53-independent manner and cross the blood-brain barrier. TIC10 can induce sustained upregulation of TRAIL in tumor cells and normal cells, which may be one of the reasons for its significant antitumor activity. TIC10 can inhibit the activity of kinase Akt and extracellular signal-regulated kinase (ERK), thereby promoting the translocation of Foxo3a to the nucleus and binding to the TRAIL promoter in the nucleus, thereby upregulating gene transcription. TIC10 is an effective antitumor therapeutic drug that acts on tumor cells and their microenvironment to increase the concentration of the endogenous tumor suppressor TRAIL. [2]

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Pharmacodynamics: Dodavirpron is an antitumor drug: it has shown antitumor activity in cell-based assays and in vivo models of H3 K27M mutant diffuse glioma. Dodavirpron causes concentration-dependent QTc interval prolongation. At 1.2 times the maximum recommended dose, the estimated mean change in QTcF was 11.8 ms (90% CI: 9.8, 13.7). The exposure-response relationship and pharmacodynamic response timeline of dodavirpron are not fully elucidated, therefore its safety and efficacy remain unclear. Mechanism of Action: Dodavirpron is a protease activator of mitochondrial casein hydrolase P (ClpP, a mitochondrial serine protease). Diffuse midline gliomas carrying the H3 K27M mutation are associated with the loss of H3 K27 trimethylation. In vitro experiments have shown that dodavirpron can induce apoptosis and alter mitochondrial metabolism, thereby restoring histone H3 K27 trimethylation levels in an H3 K27M mutant diffuse glioma model. Dodavirpron can activate ATF4 and induce endoplasmic reticulum (ER) stress or integrated stress response (ISR). Furthermore, dodavirpron can inhibit dopamine D2 receptors, which are frequently overexpressed in various cancers, including glioblastoma.

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- ONC201 (also known as TIC10) is a first-in-class imipridone molecule originally identified from an NCI chemical library screen for its ability to induce TRAIL gene transcription in HCT116 human colon cancer cells. [3]
- ONC201 has shown growth inhibitory effects in multiple cancer cell lines and antitumor activity in animal models of glioblastoma, colorectal cancer, non-Hodgkins lymphoma, and pancreatic cancer (reviewed in reference 2). [3]
- As of 2019, ONC201 was in 15 clinical trials (ClinicalTrials.gov identifier: NCT02863991). In 2018, ONC201 was granted Fast Track Designation for the treatment of adult recurrent H3 K27M mutant high-grade gliomas. [3]
- Previous proposed mechanisms (TRAIL induction, Akt/ERK inhibition, dopamine receptor targeting) have been controversial; this study identifies mitochondrial ClpP as a direct target. [3]
- ONC201 was reported to kill breast cancer cells by targeting mitochondria and inducing an integrated stress response (ISR) with ATF4 and CHOP induction, and was ineffective in Rho0 cells (cells with impaired mitochondrial function). [3]

C24H28CL2N4O

459.411323547363

458.164

C, 62.75; H, 6.14; Cl, 15.43; N, 12.20; O, 3.48

1638178-82-1

41276-02-2 (isomer);1616632-77-9;1638178-82-1 (HCl);1777785-71-3 (HBr); 2007141-57-1 (2HBr);

121596510

Solid powder

2

3

4

31

693

0

Cl.Cl.O=C1C2CN(CC3C=CC=CC=3)CCC=2N2CCN=C2N1CC1C=CC=CC=1C

HKBXPCQCBQFDML-UHFFFAOYSA-N

InChI=1S/C24H26N4O.2ClH/c1-18-7-5-6-10-20(18)16-28-23(29)21-17-26(15-19-8-3-2-4-9-19)13-11-22(21)27-14-12-25-24(27)28/h2-10H,11-17H2,1H32*1H

7-benzyl-4-(2-methylbenzyl)-1,2,6,7,8,9-hexahydroimidazo[1,2-a]pyrido[3,4-e]pyrimidin-5(4H)-one dihydrochloride

ONC-201 Dihydrochloride, ONC-201 HCl, ONC201; ONC 201 ONC-201; NSC350625; NSC-350625; NSC 350625; ONC-201 Dihydrochloride; 1638178-82-1; ONC201 HCl; 53VG71J90J; 7-Benzyl-4-(2-methylbenzyl)-2,4,6,7,8,9-hexahydroimidazo[1,2-a]pyrido[3,4-e]pyrimidin-5(1H)-one dihydrochloride; 11-benzyl-7-[(2-methylphenyl)methyl]-2,5,7,11-tetrazatricyclo[7.4.0.02,6]trideca-1(9),5-dien-8-one;dihydrochloride; Imidazo(1,2-a)pyrido(3,4-E)pyrimidin-5(1H)-one, 2,4,6,7,8,9-hexahydro-4-((2-methylphenyl)methyl)-7-(phenylmethyl)-, hydrochloride (1:2); Imidazo[1,2-a]pyrido[3,4-e]pyrimidin-5(1H)-one, 2,4,6,7,8,9-hexahydro-4-[(2-methylphenyl)methyl]-7-(phenylmethyl)-, hydrochloride (1:2); imipridone; TIC10; TIC 10; Dordaviprone; TIC-10 TRAIL inducing compound 10.

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; > 10 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.)