JNK/SAPK; CEP-1347 is a multi-target kinase inhibitor whose primary well-characterized target is the mixed lineage kinase family .

em>In vitro activity: In vitro, CEP-1347 efficiently induced differentiation and inhibited the self-renewal and tumor-initiating capacities of human cancer stem cells from glioblastoma as well as from pancreatic and ovarian cancers at clinically-relevant concentrations, without impairing the viability of normal fibroblasts and neural stem cells.
Kinase Assay: CEP-1347 (also known as KT7515), a derivative of the natural product K-252a found in broths of Narcodiopsis bacterium, is a novel and potent JNK inhibitor with IC50 of 30 nM on JNK1.
Cell Assay: Knockdown of survivin overexpressed in ovarian Chemoresistance of cancer stem cells (CSCs) resulted in increased sensitivity to paclitaxel. Treatment at clinically relevant concentrations with CEP-1347, a mixed lineage kinase inhibitor with a known safety profile in humans, reduced survivin expression in ovarian CSCs and sensitized them to paclitaxel.

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CEP-1347 reduces survivin expression and sensitizes ovarian CSCs to paclitaxel. Given that survivin expression is involved in paclitaxel resistance of ovarian CSCs, we next wished to identify drugs with which to therapeutically target survivin expression in order to overcome paclitaxel resistance associated with survivin overexpression. Since a parallel search for signaling molecules differentially expressed in ovarian CSCs and their differentiated counterparts revealed that the expression levels of MLK3 closely paralleled those of survivin in both A2780 CSLC and TOV21G CSLC cell lines (Figure 1), we surmised that MLK3 might have a role in survivin expression and therefore utilized CEP-1347, a small-molecule MLK inhibitor with a known safety profile in humans. The results indicated that CEP-1347 efficiently inhibited survivin expression in both ovarian CSC lines (Figure 3). Importantly, CEP-1347 reduced survivin expression in a concentration-dependent manner at 300 nM or below, i.e. at concentrations that have been shown to be clinically relevant in human studies (Figure 3A). Time-course analysis demonstrated that survivin expression in cells treated with CEP-1347 at 200 nM decreased over the course of 3 days (Figure 3B).[1]

Having demonstrated the ability of CEP-1347 to inhibit survivin expression, we next asked whether CEP-1347 sensitizes ovarian CSCs to paclitaxel. To examine this, the ovarian CSC lines were treated with paclitaxel in the presence and absence of CEP-1347. Treatment with CEP-1347 alone at 200 nM modestly induced cell death in both ovarian CSC lines, which may be in line with our earlier observation that knockdown of survivin alone was sufficient to reduce their viability (Figure 4A). Treatment of the cells with paclitaxel alone at 2 nM also caused marginal levels of cell death (Figure 4A) as shown earlier (Figure 2B). However, strikingly, treatment of the ovarian CSC lines with the combination of CEP-1347 and paclitaxel resulted in a significant increase in the proportion of dead cells (Figure 4A), in support of the idea that CEP-1347 sensitized the cells to paclitaxel. Further analysis showed that this cell death was accompanied by caspase activation, as indicated by the increased expression of apoptosis-specific cleaved caspase 3 (Figure 4B), suggesting that the caspase-dependent apoptotic program may be involved in cell death induced by CEP-1347 in the presence and absence of paclitaxel.[1]

Finally, in order to exclude the possibility that CEP-1347 was simply advancing the time-kinetics of cell death that would otherwise eventually occur, we determined whether concurrent treatment with CEP-1347 and paclitaxel for a defined period leads not only to short-term increase in cell death, but also to sustained inhibition of cellular growth, which may be of significance also from a therapeutic point of view. When ovarian CSCs treated with a combination of drugs for 3 days were then allowed to grow in the absence of the drugs, growth was inhibited more efficiently by the combination of CEP-1347 and paclitaxel than either treatment alone (Figure 5). These results suggest that the combinatorial use of CEP-1347 and paclitaxel may be beneficial in achieving net reduction in the population of ovarian CSCs[1] .

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CEP-1347 promotes the differentiation of CSCs into non-CSCs in vitro [2]
To identify novel CSC-targeting agents by drug repositioning, we screened a panel of candidate existing drugs that were presumed to have CSC-inhibitory activity based on their mechanisms of action, by examining their effect on the expression of stem cell marker expression in CSCs. Among such candidate drugs was CEP-1347, an inhibitor of MLKs that act upstream of c-jun N-terminal kinase (JNK) in the JNK signaling cascade. Since CEP-1347 as such can inhibit the JNK signaling pathway, which has been shown to be essential for the maintenance of a variety of CSCs, we examined its effect on human CSCs known to be dependent on JNK. [2]
First, we treated CSCs in vitro with CEP-1347 at 300 nM or below, concentrations that has been shown to be clinically-relevant in human studies and confirmed not to be toxic to normal human lung fibroblasts or rat neural stem cells and also minimally toxic to CSCs (Supplementary Figure 1), to analyze the impact of the drug on the stem cell properties of CSCs. Culture of three different glioma stem cells (GS-Y01, GS-Y03, and GS-NCC01) in the presence of CEP-1347 resulted in substantial declines in the proportion of cells expressing a stem cell marker CD133 on the cell surface, which was similarly observed when other CSCs such as pancreatic (PANC-1 CSLC) and ovarian (A2780 CSLC and TOV21G CSLC) CSCs were tested (Figure 1A). To determine whether the loss of the cell surface expression of CD133 caused by CEP-1347 treatment represented loss of stem cell properties by CSCs, we examined the expression levels of other stem cell markers and also those of differentiation markers at the same time. The results indicated that CEP-1347 treatment reduced the expression levels of stem cell markers such as Sox2 and Bmi1 in all CSCs examined while inducing the expression of respective differentiation markers such as glial fibrillary acidic protein (GFAP) in glioma stem cells and E-cadherin in ovarian CSCs (Figure 1B). Increase in E-cadherin expression, however, was not observed in PANC-1 CSLC cells at least when the cells were treated with CEP-1347 for up to 6 days. To continuously monitor, therefore, the impact of CEP-1347 on E-cadherin expression in PANC-1 CSLC cells beyond 6 days, we cultured the cells in the absence of CEP-1347 thereafter because long-term exposure to CEP-1347 substantially reduced their viability. Strikingly, we found that E-cadherin expression progressively increased accompanied by constant decrease in Sox2 expression even in the absence of CEP-1347 (Figure 1C). The results, while providing evidence that CEP-1347 promotes differentiation also in PANC-1 CSLC cells, suggested that transient exposure to CEP-1347 may be sufficient to commit CSCs to differentiation into non-CSCs.

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CEP-1347 inhibits the self-renewal and tumor-initiating capacities of CSCs in vitro [2]
Having demonstrated that CEP-1347 is a potent driver of CSC differentiation, we next determined the effect of CEP-1347 treatment on the self-renewal and tumor-initiating capacities of CSCs by sphere formation and xenograft assays, respectively. When CSCs were cultured in the sphere-forming condition in the absence of CEP-1347 after being treated in its presence for 6 days, the number of spheres formed was significantly reduced by the preceding CEP-1347 treatment, suggesting that the CEP-1347 pretreatment had impaired their self-renewal capacity by the time they were subjected to the sphere formation assay (Figure 2). Similarly, when CSCs treated for 6 days with CEP-1347 were implanted into mice, the cells failed to form tumors with the exception of one case, whereas control-treated CSCs invariably gave rise to tumors that grew progressively (Figure 3). Notably, in a mouse implanted with CEP-1347-treated PANC-1 CSLC cells, a large (>1,000 mm3) tumor was formed, which nonetheless regressed spontaneously (Figure 3B, Left). This observation suggested the possibility that CEP-1347 impaired the ability of CSCs to perpetuate tumor growth without interfering with their engraftment in mice (see Discussion). Together, the results indicated that CEP-1347 effectively inhibits the self-renewal and tumor-initiating capacities of CSCs in vitro.

In vivo, a 10-day systemic administration of CEP-1347 at a dose that was less than 1/10 the mouse equivalent of the dose safely given to humans for 2 years was sufficient to effectively reduce tumor-initiating cancer stem cells within established tumors in mice. Furthermore, the same treatment protocol significantly extended the survival of mice receiving orthotopic implantation of glioma stem cells. Together, the findings suggest that CEP-1347 is a promising candidate for cancer stem cell-targeting therapy and that further clinical and preclinical studies are warranted to evaluate its efficacy in cancer treatment.

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Systemically administered CEP-1347 inhibits CSCs in vivo [2]
Encouraged by the potent CSC-inhibitory activity of CEP-1347 demonstrated in vitro, we went on to determine in vivo whether systemically administered CEP-1347 can target and inhibit CSCs in situ, i.e., in tumors in animal models of human cancer. Previous clinical studies in humans demonstrated that CEP-1347 up to 100 mg/day (50 mg, twice a day orally) was well tolerated and sufficient to raise the plasma concentration of CEP-1347 to submillimolar levels. Since a dose of 100 mg/day in adult humans translates to ∼20 mg/kg/day in mice based on the Km values of human and mouse, we chose a starting dose of 1.5 mg/kg/day of CEP-1347, which is less than 1/10 the above dose and has indeed been administered successfully to mice for 1 week via the intraperitoneal route. After confirming that intraperitoneal administration of 1.5 mg/kg/day CEP-1347 for 10 consecutive days does not impair the general health status of mice as monitored by their body weight (Figure 4A), we conducted a serial transplantation assay to evaluate whether systemic CEP-1347 reduces the tumor-initiating CSC population within xenograft tumors in tumor-bearing mice. To this end, we treated mice bearing subcutaneous tumors formed by the implantation of glioma stem cells according to the above treatment schedule (intraperitoneal injection of 1.5 mg/kg CEP-1347 once a day for 10 days). On the next day of the final drug administration, the subcutaneous tumors were excised and, after dissociation, tumor cells were transplanted orthotopically into the brains of new mice, which were then observed thereafter without any additional treatment. The results of the serial transplantation assay indicated that transplantation of tumor cells (5 × 104 and 1 × 104) derived from control-treated primary tumors invariably leads to brain tumor formation and subsequent mortality, though the survival time varied depending on the number of transplanted tumor cells, i.e., reflecting the number of CSCs transplanted (Figure 4B). The results also indicated that transplantation of tumor cells (5 × 104) derived from CEP-1347-treated primary tumors eventually resulted in brain tumor formation in all recipient mice. However, survival was significantly extended compared with that of the corresponding control, consistent with the idea that CEP-1347 treatment reduced tumor initiating CSCs within the primary tumors (Figure 4B and 4C). Indeed, this idea was corroborated by the observation that mice transplanted with 1 × 104 tumor cells derived from CEP-1347-treated primary tumors survived significantly longer than the corresponding control mice, with one of them even surviving longer than 240 days without any signs of brain tumor formation (Figure 4B and 4D). Notably, the identical CEP-1347 treatment schedule apparently failed to inhibit the growth of the primary tumors, which is considered to be driven mainly by the proliferation of non-CSCs (Figure 4E). Collectively, these results suggested that CEP-1347 selectively inhibits tumor-initiating cells in vivo, preferentially targeting CSCs over non-CSCs.

Given that the low dose of CEP-1347 was sufficient to reduce glioma stem cells in subcutaneous tumors significantly, we next sought to determine whether the same treatment schedule was effective in inhibiting tumor-initiation by glioma stem cells in their orthotopic location, i.e., in the brain parenchyma. To this end, we stereotactically implanted glioma stem cells into mouse brain and initiated CEP-1347 treatment (intraperitoneal injection of 1.5 mg/kg CEP-1347 once a day for 10 consecutive days) on the next day of implantation. Consistent with the results of the serial transplantation assay, the identical CEP-1347 treatment schedule was similarly effective in this brain tumor xenograft model, significantly extending the survival of mice compared to control treatment (Figure 5). Thus, the results suggested that CEP-1347 can therapeutically target glioma stem cells in the brain, in line with its reported ability to penetrate the blood-brain barrier.

In cell-free assays, the mechanism of action is determined by directly measuring the inhibition of recombinant MLK3 kinase activity. Purified MLK3 is incubated with an ATP-containing buffer and a peptide substrate in the presence of serially diluted CEP-1347. The compound acts as a competitive inhibitor at the ATP-binding site, preventing substrate phosphorylation. Quantification of the remaining kinase activity, typically via the IC₅₀ value, showed that CEP-1347 inhibited recombinant MLK3 with an IC₅₀ of approximately 23 nM . This biochemical approach confirmed that the MLK family is a direct molecular target of CEP-1347, providing the basis for its subsequent in-depth investigation as an inhibitor of the JNK signaling pathway .

Cell viability assay. [1]
Cell viability assay was conducted as described previously. Briefly, cell viability was determined by tetrazolium salt reduction method using WST-8 according to the manufacture's instruction. Cells (500/well) plated in 96-well collagen I-coated plates were treated or not on the next day with 2 nM paclitaxel in the absence or presence of 200 nM CEP-1347 for 3 days, followed by culture in the absence of any drugs for another 4 days. WST-8 reagent was then added and the cells were incubated for 1-3 h at 37°C. Absorbance at 450 nm was measured using a microplate reader (Model 680; Bio-Rad). Relative cell viability was calculated as a percentage of absorbance of treated samples relative to that of controls. Cell viability assays were performed in triplicate.

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Cell death assay. [1]
Cell death assay was performed as previously described. Briefly, cells were incubated in situ with PI (1 μg/ml) and Hoechst 33342 (10 μg/ml) for 5 min at 37°C in a CO2 incubator to stain dead cells and cell nuclei, respectively. Then the numbers of PI-and Hoechst-positive cells were scored under a fluorescence microscope, and the percentage of PI-positive cells (dead cells) relative to Hoechst-positive cells (total cells) was determined. Cell death assays were performed in triplicate.

Mice (n = 6 for each group) implanted intracranially with GS-Y03 cells (1 × 104) underwent a daily intraperitoneal injection of the control vehicle or CEP-1347 (1.5 mg/kg/day) for 10 consecutive days, which started on the next day of intracranial implantation.
Mice with an orthotopic brain tumor model

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For serial transplantation, primary tumors treated as described in the figure legend were excised, and, after a wash in chilled sterile PBS, were transferred into DMEM/F12, minced with scissors, and incubated in TrypLE™ Express for 30 min at 37°C. After being rinsed with DMEM/F12, the cells were resuspended in DMEM/F12 and filtered through a 70-μm strainer. The single cell suspension was then intracranially injected after cell number and viability were determined. For systemic administration of CEP-1347, the CEP-1347 stock solution (1 mM in DMSO) was diluted in PBS to prepare 200 μL solutions for each injection. The CEP-1347 solutions were injected intraperitoneally into nude mice. All control- and CEP-1347-treated mice received an equal volume of DMSO per body weight (3.6 mL/kg). [2]

CEP-1347 is an orally active small molecule with a molecular weight of 615.19 g/mol, XLogP of 7.52, and topological polar surface area of 145.32 Ų . In HIV-infected patients receiving CEP-1347 at 50 mg twice daily, the compound demonstrated significant pharmacokinetic interactions with atazanavir (ATV), increasing ATV accumulation ratio by 15% (p = 0.007) and prolonging ATV half-life from 12.7 to 15.9 hours (p = 0.002), while no significant impact on ritonavir pharmacokinetics was observed . In a short-term study of 30 Parkinson's disease patients, CEP-1347 had no acute effect on levodopa pharmacokinetics, making it well suited for longer-term studies .

CEP-1347 was evaluated in a large-scale Phase 2/3 clinical trial for early Parkinson's disease (NCT00040404), where it demonstrated a favorable safety and tolerability profile, although it failed to show clinical efficacy . At a dose of 50 mg twice daily in HIV-infected patients, CEP-1347 was well tolerated with no significant adverse effects reported in the drug interaction study . In preclinical studies, systemic administration of CEP-1347 at a dose less than 1/10 the mouse equivalent of the dose safely given to humans for 2 years was sufficient to reduce tumor-initiating cancer stem cells in vivo without impairing the viability of normal fibroblasts and neural stem cells . A known safety profile in humans has been established through prior clinical investigations . No severe adverse events or black box warnings have been reported for this investigational compound.

[1]. Anticancer Res.2018 Aug;38(8):4535-4542;
[2]. Oncotarget.2017 Oct 24;8(55):94872-94882.

CEP-1347 is a semi-synthetic compound that has been shown to protect a variety of nerve cells from various types of damage, thereby preventing programmed cell death (apoptosis), which may help improve the survival rate of dopaminergic neurons before and after transplantation. Drug Indications Its application/treatment in asthma and Parkinson's disease is under investigation. Mechanism of Action CEP-1347 is an orally active molecule and a selective and potent inhibitor of the stress-activated protein kinase pathway. This pathway is an intracellular signaling pathway and an important component of the stress response leading to neuronal death. In vitro cell culture systems and in vivo models of Parkinson's disease in mice and non-human primates have shown that CEP-1347 can protect dopaminergic neurons in the substantia nigra (a brain region affected by Parkinson's disease). Background: Chemotherapy resistance in cancer stem cells (CSCs) is considered a major cause of recurrence after ovarian cancer treatment, negatively impacting patient prognosis. Materials and Methods: We used CSCs derived from two different ovarian cancer cell lines to look for molecules associated with chemotherapy resistance in ovarian CSCs and drugs targeting these molecules. Results: Knockdown of survivin overexpression in ovarian CSCs increased their sensitivity to paclitaxel. Treatment with a clinically relevant concentration of the mixed lineage kinase inhibitor CEP-1347 (which has a known safety profile in humans) reduced survivin expression in ovarian cancer stem cells and made them sensitive to paclitaxel. Conclusion: Survivin overexpression plays a key role in chemotherapy resistance in ovarian cancer stem cells. Using CEP-1347, which targets survivin expression in ovarian cancer stem cells, as a sensitizer for conventional ovarian cancer chemotherapy may be a reasonable and feasible treatment strategy for ovarian cancer. [1] In summary, the evidence we present supports the view that survivin overexpression may be one of the main mechanisms of enhanced chemotherapy resistance in ovarian cancer stem cells. This study confirms that CEP-1347 can effectively inhibit survivin expression and enhance the chemosensitivity of ovarian cancer stem cells at clinically relevant concentrations. Therefore, its application may be a reasonable and promising strategy to improve the efficacy of traditional chemotherapy and thus improve the survival rate of ovarian cancer patients. CEP-1347 is a mixed-lineage kinase inhibitor that has been tested in large-scale phase II/III clinical trials for early-stage Parkinson's disease, showing good safety and tolerability, but its efficacy has not yet been confirmed. This study identifies CEP-1347 as a potential anti-cancer stem cell drug through drug repositioning. In vitro experiments show that at clinically relevant concentrations, CEP-1347 can effectively induce differentiation of human cancer stem cells such as glioblastoma, pancreatic cancer, and ovarian cancer, and inhibit their self-renewal and tumor initiation capabilities, without affecting the activity of normal fibroblasts and neural stem cells. In vivo experiments show that a 10-day systemic administration of CEP-1347 to mice, at a dose less than one-tenth of the safe human dose for two years, can effectively reduce tumor-initiating cancer stem cells within existing tumors in mice. In addition, the same treatment regimen significantly prolonged the survival of mice that received orthotopic transplantation of glioma stem cells. In summary, our results suggest that CEP-1347 is a promising candidate for cancer stem cell-targeted therapy, and further clinical and preclinical studies are needed to evaluate its efficacy in cancer treatment. [2] In conclusion, this study shows that CEP-1347 can promote the differentiation of cancer stem cells (CSCs) at clinically relevant concentrations in vitro, thereby effectively inhibiting tumor formation of CSCs in vivo. Although the exact mechanism by which CEP-1347 inhibits CSCs and the role of MLK in this process remains to be elucidated, our results, combined with the drug’s good safety record in humans, suggest that adding CEP-1347 to current cancer treatment regimens is a reasonable and feasible strategy to prevent recurrence and/or metastasis in surviving CSCs after treatment, especially those that rely on JNK to maintain their stem cell status. [2]

615.76

C33H33N3O5S2

615.186

C, 64.37; H, 5.40; N, 6.82; O, 12.99; S, 10.41

156177-65-0

9917013

White to off-white solid powder

6.49

2

7

8

43

1150

3

CCSCC1=CC2=C(C=C1)N3[C@H]4C[C@@]([C@](O4)(N5C6=C(C=C(C=C6)CSCC)C7=C8CNC(=O)C8=C2C3=C75)C)(C(=O)OC)O

SCMLRESZJCKCTC-KMYQRJGFSA-N

InChI=1S/C33H33N3O5S2/c1-5-42-15-17-7-9-22-19(11-17)26-27-21(14-34-30(27)37)25-20-12-18(16-43-6-2)8-10-23(20)36-29(25)28(26)35(22)24-13-33(39,31(38)40-4)32(36,3)41-24/h7-12,24,39H,5-6,13-16H2,1-4H3,(H,34,37)/t24-,32+,33+/m1/s1

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.)