EGFR/HER

In nude mice bearing N87 human gastric cancer xenografts, Poziotinib (0.5 mg/kg p.o.) alone significantly inhibits the growth of tumors, and coadministraion of Poziotinib and 5-FU causes more effective tumor inhibition. In addition, HM781-36B/Poziotinib shows excellent antitumor activity in a variety of EGFR- and HER-2-dependent tumor xenograft models, including erlotinib-sensitive HCC827 NSCLC cells, erlotinib-resistant NCI-H1975 NSCLC cells, HER-2 overexpressing Calu-3 NSCLC cells, NCI-N87 gastric cancer cells, SK-Ov3 ovarian cancer cells and EGFR-overexpressing A431 epidermoid carcinoma cancer cells.

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Then, we assessed the in vivo efficacy of synergism between Poziotinib/HM781-36B and 5-FU using a nude mouse bearing N87 human gastric cancer xenograft model. The growth of tumors in mice treated with HM781-36B alone or in combination with 5-FU was significantly inhibited compared with control mice, and tumor volume in mice receiving coadministraion of HM781-36B and 5-FU was smaller than tumor volume in mice receiving HM781-36B only (Fig. 5C). In addition, we found that the administration of HM781-36B with other chemotherapeutic agents (paclitaxel, oxaliplatin, docetaxel or SN-38) also exerted synergistic activity against HER2 amplified cells (SNU216 and N87; Fig. 5D). Taken together, these results suggest that HM781-36B induces a synergistic effect when administered with chemotherapeutic agents (5-FU, cisplatin, paclitaxel, oxaliplatin, docetaxel or SN-38) in HER2 amplified cancer cells (SNU216, N87), and that these effects are particularly strong in both HER2 amplified and HER2 non-amplified cells when it is administered with 5-FU or cisplatin.[1]

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HM781-36B/Poziotinib shows excellent anti-tumor activity in EGFR-dependent xenograft models [2]
The in vivo activity of Poziotinib/HM781-36B was assessed in xenograft mice models with various EGFR-dependent cancer cell lines through a direct comparison with BIBW2992, an irreversible EGFR/HER-2 inhibitor and erlotinib, an EGFR selective inhibitor.29, 43 To evaluate the in vivo activity of HM781-36B, we generated a standard xenograft model with NSCLC cell line HCC827 having the erlotinib-sensitive EGFR DelE746_A750 mutation. Daily oral treatments of HM781-36B at 0.3 mg/kg/day or 1 mg/kg/day for 10 days resulted in a dramatic reduction of tumor size with an 83% maximum inhibition rate (mIR, IR = [1 − (relative tumor growth in treated group/relative tumor growth in control group)] × 100) at 0.3 mg/kg/day without body-weight loss. As shown in Figure 3a, the antitumor efficacy upon treatment with HM781-36B (1 mg/kg/day, mIR 89%, p < 0.01; Kruskal–Wallis test) was comparable to that with 10 mg/kg/day of BIBW2992 (mIR 93%, p < 0.001; Kruskal–Wallis test) and 100 mg/kg/day of erlotinib (mIR 91%, p < 0.001; Kruskal–Wallis test). We also evaluated the effect of HM781-36B on HCC827 tumor endothelium by immunohistochemistry and observed the significant reduction in the expression level of pEGFR, pAKT and pERK upon treatment with HM781-36B (0.3 mg/kg/day) for 10 days (Fig. 3b). In this dose schedule with statistically significant inhibition of tumor growth, the anti-tumor efficacy of Poziotinib/HM781-36B was studied in mice models xenografted with NCI-H1975 NSCLC cell line harboring erlotinib-resistant EGFRL858R/T790M and with Calu-3 NSCLC cell line harboring a high level of HER-2 expression (Figs. 3c and 3d, respectively). HM781-36B showed the strong in vivo efficacy, with 80.8% mIR at 5 mg/kg/day for 10 days (p < 0.001; Kruskal–Wallis test) in the NCI-H1975 xenograft model and with 67.4% mIR at 1 mg/kg/day for 10 days (p < 0.05; Kruskal–Wallis test) in the Calu-3 xenograft model. In fact, the comparable in vivo efficacy was achieved with BIBW2992 at much higher doses; 40 mg/kg/day and 30 mg/kg/day in the NCI-H1975 and the Calu-3 xenograft model, respectively (p < 0.05; Kruskal–Wallis test). [2]
HM781-36B/Poziotinib was also effective in different kinds of cancers with a high level of HER-2 expression, including NCI-N87, a human gastric cancer cell line, and SK-Ov3, a human ovarian cancer cell line (Figs. 3e and 3f). HM781-36B effectively induced tumor regression with 84.4% mIR for 10 days at 1 mg/kg/day (p < 0.001; Kruskal–Wallis test) in the NCI-N87 model and 92.3% mIR at 1 mg/kg/day for 10 days (p < 0.05; Kruskal–Wallis test) in the SK-Ov3 model. Again, the comparable in vivo efficacy was achieved with BIBW2992 at much higher doses; 89.6% mIR at 30 mg/kg/day (p < 0.01; Kruskal–Wallis test) in the NCI-N87 model and 97.1% mIR at 50 mg/kg/day (p < 0.05; Kruskal–Wallis test) in the SK-Ov3 model. We also confirmed the excellent in vivo efficacy in the mice xenograft model of A431 epidermoid carcinoma cell line with a high level of wild-type EGFR expression through the observation of a dramatic reduction in tumor size with an 80.7% mIR upon treatment of HM781-36B at 0.3 mg/kg/day (p < 0.001; Kruskal–Wallis test) without body-weight loss (Fig. 3g).

Enzyme activity assay [1]
To determine the IC50 values of gefitinib, BIBW-2992 and Poziotinib/HM781-36B for kinase inhibition, enzymes of EGFR, HER2, and HER4 were expressed as recombinant proteins in Sf9 insect cells. Enzyme selectivity screening was then performed using a tyrosine kinase assay kit. Briefly, the reactions were performed in 96 well polystyrene round-bottomed plates containing kinase buffer composed of 100 mM HEPES (pH 7.4), 25 mM MgCl2, 10 mM MnCl2 and 250 μM Na3VO4. The reactions were initiated by the addition of 100 ng/assay enzyme, 100 μM ATP, and 10 ng/ml poly(Glu, Tyr). After 1 h of incubation at room temperature, the reactions were terminated by adding 6 mM EDTA solution and then anti-phosphotyrosine antibody, PTK Green Tracer, and FP dilution buffer mixtures. The fluorescence polarization values were then measured after 30 min at room temperature using a Victor3 microplate reader. Finally, the IC50 values were calculated using the following equation: Y = bottom + (top–bottom)/(1 + ).

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In vitro kinase assay [2]
EGFRWT, EGFRT790M, EGFRT790M/L858R, HER-2 and HER-4 were expressed by baculovirus in Sf21 insect cells and used for kinase assays and IC50 determinations, which were performed as previously described.38 To determine the selectivity of HM781-36B against various kinases, the SelectScreen™ Kinase Profiling service was used.

Cell growth inhibition assay [1]
Viable cell growth was determined by an MTT reduction assay. Briefly, all cell lines were seeded at a density of 3 × 103 per well in 96-well culture plates and then incubated at 37 °C for 24 h. The cells were then treated with 0.001, 0.01, 0.1, or 10 μmol/L of Poziotinib/HM781-36B, Gefitinib, Lapatinib, BIBW-2992 and CI-1033 and 0.01, 0.1, 1, 10, or 100 μg/ml of Trastuzumab. Three days later, 50 μg of tetrazolium-dye(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide, MTT) were added to each well and the samples were then incubated for 4 h to reduce the dye. Next, the samples were treated with dimethylsulfoxide, after which the absorbance of the converted dye in the living cells was measured at a wavelength of 540 nm. Six replicate wells were used for each analysis, and at least three independent experiments were conducted. Data points shown represent the mean while bars represent the SE. [1]
To evaluate the effects of Poziotinib/HM781-36B administered in conjunction with chemotherapeutic agents (5-FU, cisplatin, paclitaxel, oxaliplatin, docetaxel or SN-38), cells were treated with serial dilutions of each drug individually and with both drugs simultaneously at a fixed ratio of doses that corresponded to the individual IC50. Specifically, HER2 amplified gastric cancer cell lines (SNU216 and N87) were exposed to various concentrations of HM781-36B (0.00025, 0.0005, 0.001, 0.002, 0.004 μmol/L) and chemotherapeutic agents (5-FU, cisplatin, paclitaxel, oxaliplatin, docetaxel or SN-38) at a ratio of 1:100, while other gastric cancer cell lines (SNU1, 5, 16, 484, 601, 620, 638, 668) were exposed to various concentrations of HM781-36B (0.05, 0.25, 0.5, 2.5, 5 μmol/L) and 5-FU or cisplatin at a ratio of 1:1. After 72 h of exposure, cell viability was measured using the MTT assay. The methods described by Chou and Talalay were then used to determine if a synergistic effect existed. The analysis of the median effect was conducted using the Calcusyn software to determine a combination index value (CI > 1: antagonistic effect, CI = 1: additive effect, CI < 1: synergistic effect).

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Cell cycle analysis [1]
After incubation with Poziotinib/HM781-36B under various concentrations (0.001, 0.01, 0.1 μmol/L) for 48 h, the cells were centrifuged at 3000 rpm for 5 min, after which they were fixed in 70% alcohol and stored at −20 °C. The samples were then dissolved in 10 μL RNAse (100 μg/mL) and subsequently incubated at 37 °C for 10 min. Next, the samples were treated with propidium iodide, after which the DNA contents of the cells (10,000 cells per experimental group) were determined using a FACS Calibur flow cytometer equipped with a ModFit LT program, as previously described.

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Annexin V binding assay for apoptosis [1]
After the cells were exposed to HM781-36B for 48 h, the degree of apoptosis was assessed by the Annexin V binding assay using the protocols of the manufacturer. The harvested cell suspension was then incubated with Annexin V for 15 min at room temperature in the dark and then analyzed by flow cytometry, as described previously.

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Cell growth inhibition assay [2]
Calu-3, NCI-H1975, NCI-H358, NCI-H1781, HCC827, A549, A431, SK-Br3, BT-474, MDA-175, NCI-N87, Hs-27 and Balb/c3t3 (clone A31) cells were purchased from the American Type Culture Collection. Calu-3 and A549 cells were maintained in Minimum Essential Medium (MEM) and F-12K culture medium, respectively. NCI-H1975, NCI-H358, NCI-H1781, HCC827, SK-Br3 and NCI-N87 cell lines were cultured in RPMI medium containing 1 mM sodium pyruvate. A431 and Hs-27 cell lines were maintained in high-glucose Dulbecco's modified Eagle's medium (DMEM), and Balb/c3t3 cell line was maintained in low-glucose DMEM. BT-474 and MDA-175 cells were incubated in Hybri-Care medium and L-15 medium, respectively. All culture media were supplemented with 1% penicillin–streptomycin and 10% fetal bovine serum (FBS) except for Balb/c3t3 cells (10% fetal calf serum and 1% penicillin–streptomycin). Cells were incubated in a humidified atmosphere under 5% CO2 except for MDA-175 cells (CO2 free) at 37°C. Cell growth inhibition assays and GI50 and GI90 determinations were performed as previously described.

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Western blot analysis [2]
Cells were treated with erlotinib or Poziotinib/HM781-36B in the presence of media supplemented with 10% FBS for 24 hr. After harvesting the cells, total cell lysate was immunoblotted. Primary antibodies against p-EGFR (pY1086 in H1975 and pY1148 in HCC827), p-HER-2 (pY877) and p-AKT (pS473) and EGFR, HER-2, AKT, p-ERK1 and ERK1/2 were used.

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Prolongation of phosphorylation inhibition [2]
Cells were plated at a density of 5 × 105/well in 6-well plates under normal culture conditions (10% FBS and 1% penicillin-streptomycin). After 24 hr, the media were changed to 0.1% FBS media and cells were incubated for 16 hr. Cells were then treated with 1 μM erlotinib, BIBW2992 or HM781-36B for 4 hr. Each set was washed four times with warmed compound-free medium and incubated for 0, 8 and 24 hr. Each set was stimulated with EGF (100 ng/ml) for 5 min. The phosphorylation rates of EGFR or HER-2 were measured by ELISA using Human EGFR and HER-2 immunoassay kit.

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Irreversible binding studies with Cy3-HM781-36B [2]
Enzymes (1 μg of EGFRWT, EGFRT790M or PDGFRα) were incubated with 0.1% DMSO or 1 μM of Cy3-HM781-36B in the presence or absence of 5 μM of unlabeled Poziotinib/HM781-36B for 15 min at 0°C. Subsequent to incubation, samples were boiled for 5 min in SDS buffer, and proteins were separated by SDS-PAGE (10% acrylamide).

Xenograft mouse model [1]
To determine the in vivo activity of Poziotinib/HM781-36B, 4–6 week-old female BALB/c athymic nude mice were used. The mice were permitted to acclimatize to local conditions for 1 week before being injected with cancer cells. Mice were injected s.c. with N87 cells in 100 μL of PBS (1 × 108 cells per 100 μL PBS). When the tumor reached a volume of 400 mm3, mice were randomized into treatment group (n = 7 per group) to receive vehicle control, HM781-36B suspended in 23% PEG/TW in water, 5-FU or a combination of HM781-36B and 5-FU. HM781-36B was administered via oral gavage once daily at a concentration of 0.5 mg/kg for 3 weeks. A dose of 50 mg/kg of 5-FU was given intraperitoneally once weekly for 3 weeks. The tumor volume was measured every other day using a caliper, and it was calculated according to following formula: [(width)2 × (height)]/2. After the final treatment on day 22, all mice were euthanized.

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Xenograft mouse model [2]
The SK-Ov3 human ovarian cancer cell line was obtained from ATCC (Manassas/VA). SK-Ov3 cells were maintained in McCoy's 5A containing 1% penicillin-streptomycin and 10% FBS in a humidified atmosphere containing 5% CO2 at 37°C. In xenograft models, a total of 30 mg of mouse tumor fragments were implanted subcutaneously into the right flank of NU/NU Balb/C mice (female, body-weight range: 20 ± 5 g). Treatment was initiated approximately 7 days after implantation. Animals were randomized into treatment groups (n = 8) with similar mean tumor volumes. Distilled water containing 20% PEG400 and 3% Tween80 was used as vehicle in the in vivo studies. Different doses of Poziotinib/HM781-36B, BIBW2992, erlotinib or vehicle alone were orally administered once daily for 10 days. Tumor volumes (mg) and body weights (g) were recorded twice a week from all groups using a Vernier caliper and balance.
For immunohistochemistry staining of HCC827 model, two tumor-bearing mice in the group of control and Poziotinib/HM781-36B (0.3 mg/kg) group were killed 6 hr after the last dose and tumor sections were then stained with antibodies recognizing phospho-EGFR (Tyr1173), phospho-AKT (Ser473) and phospho-ERK1/2 (Tyr202/Tyr204) by HM781-36B. The samples were observed by using an optical microscope.

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Suspended in 23% PEG/TW in water; 0.5 mg/kg; p.o

BALB/c athymic nude mice bearing N87 xenografts

[1]. Cancer Lett.2011 Mar 28;302(2):155-65;
[2]. Int J Cancer.2012 May 15;130(10):2445-54.

Poziotinib Hydrochloride is the hydrochloride salt form of poziotinib, an orally bioavailable, quinazoline-based, irreversible pan-epidermal growth factor receptor (EGFR or HER) inhibitor, with potential antineoplastic activity. Upon oral administration, poziotinib inhibits EGFR (HER1 or ErbB1), HER2 and HER4, thereby inhibiting proliferation of tumor cells in which these receptors are overexpressed and/or mutated. EGFRs, cell surface receptor tyrosine kinases upregulated or mutated in a variety of cancer cell types, play key roles in cellular proliferation and survival.

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Trastuzumab, a HER2 directed treatment has shown clinical benefit in HER2 amplified gastric cancer. This study demonstrated the potent antitumor activity of HM781-36B, a quinazoline-based irreversible pan-HER inhibitor, in HER2 amplified gastric cancer cells (SNU216 and N87) in vitro and in vivo. HM781-36B inhibited phosphorylation of HER family and downstream signaling molecules, and induced apoptosis and G1 arrest. Furthermore, HM781-36B exerted synergistic effects with chemotherapeutic agents in both HER2 amplified and HER2 non-amplified gastric cancer cells. Therefore, HM781-36B may be useful for the treatment of HER2 amplified gastric cancer alone or in combination with chemotherapeutic agents.[1]

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The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases has been implicated in a variety of cancers. In particular, activating mutations such as the L858R point mutation in exon 21 and the small in-frame deletions in exon 19 of the EGFR tyrosine kinase domain are correlated with sensitivity to EGFR tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC) patients. Clinical treatment of patients is limited by the development of drug resistance resulting mainly from a gatekeeper mutation (T790M). In this study, we evaluated the therapeutic potential of a novel, irreversible pan-HER inhibitor, HM781-36B. The results from this study show that HM781-36B is a potent inhibitor of EGFR in vitro, including the EGFR-acquired resistance mutation (T790M), as well as HER-2 and HER-4, compared with other EGFR tyrosine kinases inhibitors (erlotinib, lapatinib and BIBW2992). HM781-36B treatment of EGFR DelE746_A750-harboring erlotinib-sensitive HCC827 and EGFR L858R/T790M-harboring erlotinib-resistant NCI-H1975 NSCLC cells results in the inhibition of EGFR phosphorylation and the subsequent deactivation of downstream signaling proteins. Additionally, HM781-36B shows an excellent efficacy in a variety of EGFR- and HER-2-dependent tumor xenograft models, including erlotinib-sensitive HCC827 NSCLC cells, erlotinib-resistant NCI-H1975 NSCLC cells, HER-2 overexpressing Calu-3 NSCLC cells, NCI-N87 gastric cancer cells, SK-Ov3 ovarian cancer cells and EGFR-overexpressing A431 epidermoid carcinoma cancer cells. On the basis of these preclinical results, HM781-36B is the most potent pan-HER inhibitor, which will be advantageous for the treatment of patients with NSCLC including clinical limitation caused by acquired mutation (EGFR T790M), breast cancer and gastric cancer.[2]

C23H22CL3FN4O3

527.8

526.074

C, 52.34; H, 4.20; Cl, 20.15; F, 3.60; N, 10.62; O, 9.09

1429757-68-5

54767257

Typically exists as solid at room temperature

2

7

6

34

684

0

C=CC(=O)N1CCC(OC2=C(OC)C=C3N=CN=C(NC4=C(F)C(Cl)=C(Cl)C=C4)C3=C2)CC1.Cl

OMYSOLOMWJFVNK-UHFFFAOYSA-N

InChI=1S/C23H21Cl2FN4O3.ClH/c1-3-20(31)30-8-6-13(7-9-30)33-19-10-14-17(11-18(19)32-2)27-12-28-23(14)29-16-5-4-15(24)21(25)22(16)26;/h3-5,10-13H,1,6-9H2,2H3,(H,27,28,29);1H

1-[4-[4-(3,4-dichloro-2-fluoroanilino)-7-methoxyquinazolin-6-yl]oxypiperidin-1-yl]prop-2-en-1-one;hydrochloride

Poziotinib hydrochloride; 1429757-68-5; HM-781-36B hydrochloride; X4Z7U6JL1C; Poziotinib hydrochloride [USAN]; UNII-X4Z7U6JL1C; HM781-36B HYDROCHLORIDE; 2-Propen-1-one, 1-(4-((4-((3,4-dichloro-2-fluorophenyl)amino)-7-methoxy-6-quinazolinyl)oxy)-1-piperidinyl)-, hydrochloride (1:1);

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)