EGFRL858R (IC50 = 0.4 nM); EGFR (IC50 = 0.5 nM); EGFRL858R/T790M (IC50 = 10 nM); HER2 (IC50 = 14 nM); HER3

Heregulin-stimulated HER3 phosphorylation can be prevented by afatinib oxalate at a concentration of 100 nM [1]. Effectively suppressing the anchorage-independent proliferation of NIH-3T3 cells ectopically expressing EGFR mutations as well as the cell proliferation of H1666, H3255, and NCI 1975 cells is achieved with the use of Afatinib oxalate (0-10000 nM) [1]. SLMT-1, EC-1, HKESC-1, and HKESC-2 cells show growth suppression in response to Afatinib oxalate (48–72 hours) [2]. In ESCC cell lines, afatinib oxalate (0–1 μM, 24-48 hours) suppresses the AKT and MAPK pathways as well as EGFR and AKT phosphorylation [2]. HKESC-2 and EC-1 experience G0/G1 cell cycle arrest when exposed to afatinib oxalate (0–1 μM) for 16–48 hours [2]. In HKESC-2 and EC-1, afatinib oxalate (0–1 μM, 24-48 hours) efficiently causes apoptosis [2].

EGFR, HER2, HER3, and AKT phosphorylation were all significantly downregulated and tumor regression was observed when oral afatinib oxalate (0–20 mg/kg) was administered daily for 25 days [1]. Strongly inhibiting the growth of HKESC-2 tumors was afatinib oxalate (15 mg/kg), taken orally for 5 days, followed by 2 days off, for a duration of 2 weeks [2].

EGFR kinase: 10 μL of inhibitor in 50% Me2SO, 20 μL of substrate solution (200 mM HEPES pH 7.4, 50 mM Mg-acetate, 2.5 mg/mL poly (EY), 5 μg/mL bio-pEY), and 20 µL enzyme preparation were included in each 100 µL enzyme reaction. The addition of 50 µL of a 100 µM ATP solution prepared in 10 mM MgCl2 initiates the enzymatic reaction. After 30 minutes of assaying at room temperature, 50 µL of stop solution (250 mM EDTA in 20 mM HEPES pH 7.4) is added to end the assay. 100 µL are added to a microtiterplate coated with streptavidin, and after 60 minutes of room temperature incubation, the plate is cleaned with 200 µL of wash solution (50 mM Tris, 0.05% Tween20). The wells are filled with a 100 µL aliquot of PY20H Anti-Ptyr:HRP, a 250 ng/mL HRPO-labeled anti-PY antibody. Following a 60-minute incubation period, the plate is three times cleaned using a 200 µL wash solution. Following that, 100µL of TMB Peroxidase Solution (A:B=1:1) is used to develop the samples. After ten minutes, the reaction is stopped. After the plate is placed in an ELISA reader, the extinction at OD450nm is calculated. The enzyme HER2-IC: The assay of enzyme activity is conducted in 50% Me2SO with or without serial inhibitor dilutions. Similar components as described for the EGFR kinase assay are included in each 100 µL reaction, along with the addition of 1000 µM Na3VO4. The addition of 50µL of a 500 µM ATP solution prepared in 10 mM magnesium acetate initiates the enzymatic reaction. The enzyme is diluted to the point where the amount of enzyme and the amount of time it takes for phosphate to be incorporated into bio-pEY are linear. The mixture of 20 mM HEPES pH 7.4, 130 mM NaCl, 0.05% Triton X-100, 1 mM DTT, and 10% glycerol is used to dilute the enzyme preparation. After 30 minutes of assaying at room temperature, 50 µL of stop solution is added to end the procedure. Src kinase assays: 10 µL of inhibitor in 50% Me2SO, 20 µL of enzyme preparation, and 20 µL of substrate solution enhanced with 1000 µM Na3VO4 were included in each 100 µL reaction. The addition of 50 µL of a 1000 µM ATP solution prepared in 10 mM Mg-acetate initiates the enzymatic reaction. Assay for BIRK kinase: 50 µL of a 2 mM ATP solution prepared in 8 mM MnCl2 and 20 mM Mg-acetate is added to 250 mM Tris pH 7.4, 10 mM DTT, 2.5 mg/mL poly(EY), and 5 mg/mL bio-pEY as the substrate solution to initiate the enzymatic reaction. HGFR kinase and VEGF2 assays: The assay is completed by adding 10 µL of 5% H3PO4 after it has been running at room temperature for 20 minutes. The precipitate is then collected using a 96 well filter mate universal harvester and trapped onto GF/B filters. The filter plate is thoroughly cleaned, dried for one hour at 50°C, sealed, and the radioactivity is measured using scintillation counting with either a TopCountTM or a Microbeta b counterTM.

Cell proliferation assay [1]
Cell Types: NIH-3T3 cells, H1666, H3255 and NCI 1975 Cell
Tested Concentrations: 0, 1, 10, 100, 1000, 10000 nM
Incubation Duration:
Experimental Results: Effective inhibition of NIH- anchorage-dependent proliferation 3T3 Cells ectopically express EGFR mutants. It inhibits the anchorage-independent cell proliferation of multiple lung cancer cell lines (H1666, H3255 and NCI 1975 cells) with IC50 values of 60 nM, 0.7 nM and 99 nM respectively.

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Cell viability assay[2]
Cell Types: HKESC-1, HKESC-2, SLMT-1 and EC-1 Cell Line
Tested Concentrations:
Incubation Duration: 48 and 72 hrs (hours)
Experimental Results: More than 95% growth inhibition was observed. Respective IC50 concentrations at 48 hrs (hours) (HKESC-1=0.078 μM, HKESC-2=0.115 μM, KYSE510=3.182 μM, SLMT-1=4.625 μM and EC-1=1.489 μM) and 72 hrs (hours) (HKESC-1=0.002) μM, HKESC-2=0.002 μM, KYSE510=1.090 μM, SLMT-1=1.161 μM and EC-1=0.109 μM) are all in the lower micromolar range.

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Western Blot Analysis[2]
Cell Types: HKESC-2 cells and EC-1 cells
Tested Concentrations: 0, 0.01 a

Animal/Disease Models: Athymic NMRI-nu/nu female mice (21–31 g, 5 to 6 weeks old, transgenic mouse lung cancer model and xenograft model) [1]
Doses: 15 mg/kg, 20 mg/kg given Medication: Orally administered daily for 25 days
Experimental Results: In a standard xenograft model of the epidermoid cancer cell line A431, tumors Dramatically regressed with a cumulative treatment/control tumor volume ratio (T/C ratio) of 2%, and EGFR and AKT downregulates phosphorylation. Induced regression of large tumors in this HER2-driven model and effectively controlled xenograft tumor formation in the NCIH1975 cell line expressing EGFR L858R/T790M, with a T/C value of 12% at a dose of 20 mg/kg. After 4 weeks of treatment, the tumor was diminished by more than 50%. Downregulates EGFR, HER2 and HER3 phosphorylation.

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Animal/Disease Models: Sixweeks old female athymic nude mice (nu/nu) (16-20 g)[2]
Doses: 15 mg/kg
Route of Administration: po (oral gavage), according to a schedule of 5 days plus 2 days of rest , lasted for two weeks.
Experimental Results: Str

[1]. BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models. Oncogene. 2008 Aug 7;27(34):4702-11.

[2]. Preclinical evaluation of afatinib (BIBW2992) in esophageal squamous cell carcinoma (ESCC). Am J Cancer Res. 2015 Nov 15;5(12):3588-99.

[3]. Afatinib circumvents multidrug resistance via dually inhibiting ATP binding cassette subfamily G member 2 in vitro and in vivo. Oncotarget. 2014 Dec 15;5(23):11971-85.

[4]. Antitumor activity of pan-HER inhibitors in HER2-positive gastric cancer. Cancer Sci. 2018 Apr;109(4):1166-1176.

Afatinib is a quinazoline compound with a 3-chloro-4-fluoroaniline group at position 4, a 4-dimethylamino-trans-but-2-enamid group at position 6, and a (S)-tetrahydrofuran-3-oxy group at position 7. It (in the form of dimaleate) is used as first-line treatment for patients with metastatic non-small cell lung cancer. It is a tyrosine kinase inhibitor and an antitumor drug. It belongs to the quinazoline, furan, organofluorine, enamide, aromatic ether, tertiary amine, monochlorobenzene, and secondary amide classes. Afatinib is a 4-aniline quinazoline tyrosine kinase inhibitor, available in the form of dimaleate, marketed under the brand name Gilotrif and manufactured by Boehringer Ingelheim. Afatinib tablets, taken orally, can be used as a first-line (initial) treatment for patients with metastatic non-small cell lung cancer (NSCLC) diagnosed by FDA-approved testing methods with common epidermal growth factor receptor (EGFR) mutations. Gelotinib (afatinib) is Boehringer Ingelheim's first FDA-approved oncology treatment. Afatinib is a kinase inhibitor. Its mechanism of action is as a protein kinase inhibitor. Afatinib is a tyrosine kinase receptor inhibitor used to treat certain types of metastatic non-small cell lung cancer. Afatinib treatment can cause a transient increase in serum transaminase levels, and there are reports of clinically significant acute liver injury, and in rare cases, even death. Afatinib is an orally bioavailable aniline-quinazoline derivative and an inhibitor of epidermal growth factor receptor (ErbB; EGFR) family receptor tyrosine kinases (RTKs), exhibiting antitumor activity. Following administration, afatinib selectively and irreversibly binds to and inhibits epidermal growth factor receptors 1 (ErbB1; EGFR), 2 (ErbB2; HER2), and 4 (ErbB4; HER4), as well as certain EGFR mutants, including those caused by EGFR exon 19 deletion mutations or exon 21 (L858R) mutations. This may result in suppression of tumor growth and angiogenesis in tumor cells that overexpress these RTKs. Furthermore, afatinib also inhibits EGFR T790M gatekeeper mutations resistant to first-generation EGFR inhibitors. EGFR, HER2, and HER4 are RTKs belonging to the EGFR superfamily; they play important roles in tumor cell proliferation and tumor angiogenesis and are overexpressed in various cancer cell types. A quinazoline and butenamide derivative, as a tyrosine kinase inhibitor of the epidermal growth factor receptor (ERBB receptor), is used to treat metastatic non-small cell lung cancer.

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Drug Indications
Afatinib is a kinase inhibitor indicated for first-line monotherapy in the following cases: (a) patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) who have not received prior treatment with an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) or (a) patients with a non-resistant EGFR mutation detected in the tumor by an FDA-approved assay, and (b) adult patients with locally advanced or metastatic squamous cell carcinoma NSCLC whose disease has progressed after platinum-based chemotherapy. Recently, as of January 2018, the U.S. FDA approved Boehringer Ingelheim's supplemental New Drug Application (NDA) for Gilotrif (afatinib) for first-line treatment of patients with metastatic non-small cell lung cancer (NSCLC) whose tumors are found to have non-resistant epidermal growth factor receptor (EGFR) mutations by FDA-approved assays. The new label includes data for three additional EGFR mutations: L861Q, G719X, and S768I.

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Pharmacodynamics
Abnormal ErbB signaling triggered by receptor mutations, amplification, and/or receptor ligand overexpression leads to a malignant phenotype. EGFR mutations define a unique molecular subtype of lung cancer. In non-clinical disease models of ErbB pathway dysregulation, afatinib, as a monotherapy, effectively blocks ErbB receptor signaling, thereby inhibiting tumor growth or leading to tumor regression. In both nonclinical and clinical settings, non-small cell lung cancer (NSCLC) tumors carrying common activating EGFR mutations (Del 19, L858R) and some less common EGFR mutations (located in exon 18 (G719X) and exon 21 (L861Q)) are particularly sensitive to afatinib treatment. Limited nonclinical and/or clinical activity has been observed in NSCLC tumors carrying exon 20 insertion mutations. Acquired secondary T790M mutations are the main mechanism of acquired resistance to afatinib, and the gene dose of the T790M mutation allele is correlated with the degree of resistance in vitro. Approximately 50% of tumors in patients who progress on afatinib treatment have T790M mutations; for these patients, EGFR-TKIs targeting T790M may be considered as a second-line treatment option. Preclinical studies have suggested other potential mechanisms of afatinib resistance, and MET gene amplification has also been observed clinically. Meanwhile, an open-label, single-arm study evaluated the effects of multiple doses of afatinib (50 mg once daily) on cardiac electrophysiology and QTc interval in patients with relapsed or refractory solid tumors. Ultimately, no significant change in mean QTc interval (i.e., >20 ms) was detected in this study.

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Absorption
The time to peak plasma concentration (Tmax) after oral administration is 2 to 5 hours. Within the 20 to 50 mg dose range, the increase in maximum concentration (Cmax) and area under the concentration-time curve (AUC0-∞) is slightly greater than that of dose proportion. The geometric mean relative bioavailability of the 20 mg tablet is 92% compared to the oral solution. Furthermore, compared to fasting administration, systemic exposure to afatinib is reduced by 50% (Cmax) and 39% (AUC0-∞) when co-administered with a high-fat meal, respectively. Based on population pharmacokinetic data from clinical trials across various tumor types, AUCss decreases by a mean of 26% when food is consumed within 3 hours before or 1 hour after administration of afatinib.

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Elimination Pathway
In the human body, afatinib is primarily excreted via feces. Following oral administration of 15 mg afatinib solution, 85.4% of the dose is recovered in feces and 4.3% in urine. Of the recovered dose, the parent compound afatinib accounts for 88%.
Volume of Distribution
The volume of distribution of afatinib recorded in healthy male volunteers is 4500 liters. Such a high plasma volume of distribution suggests that its tissue distribution may be high.
Clearance
The geometric mean of the apparent systemic clearance of afatinib recorded in healthy male volunteers is as high as 1530 mL/min.
Metabolism/Metabolites
Enzymatic metabolism has negligible effect on afatinib in vivo. Protein covalent adducts are the main circulating metabolites of afatinib.
Biological Half-Life
The effective half-life of afatinib is approximately 37 hours. Therefore, steady-state plasma concentrations of afatinib are reached within 8 days after multiple doses, resulting in a 2.77-fold (AUC0-∞) and 2.11-fold (Cmax) increase in cumulative drug dose. In patients treated with afatinib for more than 6 months, the estimated terminal half-life is 344 hours.

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Protein Binding
In vitro, afatinib binds to approximately 95% of human plasma proteins. Afatinib binds to proteins via both non-covalent (traditional protein binding) and covalent binding.

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Mechanism of Action
Afatinib is a potent, selective, and irreversible ErbB family blocker. Afatinib covalently binds to all homodimers and heterodimers of ErbB family members EGFR (ErbB1), HER2 (ErbB2), ErbB3, and ErbB4, irreversibly blocking their signal transduction. Specifically, afatinib covalently binds to the kinase domains of EGFR (ErbB1), HER2 (ErbB2), and HER4 (ErbB4), irreversibly inhibiting autophosphorylation of tyrosine kinases, thereby downregulating the ErbB signaling pathway. Certain mutations in EGFR, including non-resistant mutations in its kinase domain, can lead to increased receptor autophosphorylation, thereby activating the receptor, sometimes even in the absence of ligand binding, and promoting the proliferation of non-small cell lung cancer (NSCLC) cells. Non-resistant mutations are mutations occurring in the exons constituting the EGFR kinase domain that lead to increased receptor activation, and their efficacy can be predicted by: 1) clinically significant tumor shrinkage at the recommended dose of afatinib; and/or 2) the maintenance of afatinib at the recommended dose, according to validated methods, to inhibit cell proliferation or EGFR tyrosine kinase phosphorylation. The most common of these mutations are exon 21 L858R substitution and exon 19 deletion. Furthermore, at the concentrations achieved in patients, afatinib inhibits autophosphorylation and/or in vitro proliferation in cell lines expressing wild-type EGFR, as well as cell lines expressing specific EGFR exon 19 deletion mutations, exon 21 L858R mutations, or other less common non-resistant mutations. Additionally, afatinib inhibits the in vitro proliferation of HER2-overexpressing cell lines.

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Genetic alterations in the epidermal growth factor receptor (EGFR) kinase domain in non-small cell lung cancer (NSCLC) patients are associated with sensitivity to small molecule tyrosine kinase inhibitors. Although first-generation reversible ATP-competitive inhibitors have shown encouraging clinical efficacy in lung adenocarcinoma tumors carrying these EGFR mutations, almost all patients eventually develop resistance to these inhibitors. Resistance to first-generation EGFR inhibitors is typically associated with acquired T790M point mutations in the EGFR kinase domain or activation of the HER3 downstream signaling pathway. Overcoming these resistance mechanisms, as well as primary resistance to reversible EGFR inhibitors driven by partial EGFR mutations, is crucial for developing effective targeted therapies. In this article, we found that BIBW2992 is an aniline quinazoline compound designed to irreversibly bind to EGFR and HER2, effectively inhibiting the kinase activity of wild-type and activated EGFR and HER2 mutants, including erlotinib-resistant subtypes. Consistent with this activity, BIBW2992 inhibited cancer cell transformation, suppressed cancer cell line survival, and induced tumor regression in xenograft and transgenic lung cancer models, with activity superior to erlotinib. These findings encourage further testing of BIBW2992 in lung cancer patients carrying EGFR or HER2 oncogenes. [1]

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Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer death in the United States. Treatment of locally advanced disease is accompanied by significant acute side effects and can lead to chronic disability, while the prognosis of recurrent or metastatic disease is extremely poor. This highlights the need for better treatment options. Epidermal growth factor receptor (EGFR) is overexpressed in 90% of HNSCC patients, making it an attractive therapeutic target in this patient population. Afatinib is a potent, irreversible pan-ErbB inhibitor. Preliminary studies in HNSCC have shown encouraging activity. This article reviews current data evaluating small molecule inhibitors of the ErbB family for the treatment of HNSCC, focusing on the second-generation irreversible pan-ErbB inhibitor afatinib. The article also describes the pharmacological properties, pharmacokinetics, and toxicity profiles of afatinib, as well as details of published and ongoing clinical trials evaluating its efficacy in patients with head and neck squamous cell carcinoma (HNSCC). Expert opinion: Phase II clinical trials in HNSCC have shown that daily oral afatinib is well tolerated. The most common toxicities were rash and diarrhea. Afatinib has clinical activity as monotherapy in some patients with refractory and/or metastatic HNSCC. Ongoing Phase III clinical trials promise to better elucidate the role of this compound in the treatment of HNSCC. [2]

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Hepatotoxicity/Hepatotoxicity: Elevated serum transaminase levels are common during afatinib treatment, occurring in 20% to 50% of patients, but only 1% to 2% of patients have transaminase levels exceeding 5 times the upper limit of normal. Liver failure has been reported in 0.2% of patients, leading to several deaths. Hepatotoxicity appears to be a class effect of EGFR2 protein kinase inhibitors, although liver injury caused by gefitinib appears to be more common and severe than that caused by afatinib and erlotinib. Specific details of afatinib-related liver injury, such as latency, serum enzyme profiles, clinical features, and course, have not been published. Other EGFR inhibitors, such as erlotinib and gefitinib, typically cause liver injury within days or weeks of starting treatment, manifested as elevated hepatocyte enzymes, with a moderate to severe course. Immune hypersensitivity and autoimmune features are uncommon. Patients with a history of cirrhosis or liver impairment due to hepatic tumor burden have a higher probability of developing clinically significant liver injury and liver failure. Likelihood Score: D (Possibly a cause of clinically significant liver injury). Elevated serum transaminase levels are common during afatinib treatment, occurring in 20% to 50% of patients, but only 1% to 2% of patients have transaminase levels exceeding five times the upper limit of normal. Liver failure has been reported in 0.2% of patients, leading to several deaths. Hepatotoxicity appears to be a class effect of EGFR2 protein kinase inhibitors, although liver injury caused by gefitinib appears to be more common and severe than that caused by afatinib and erlotinib. Specific details regarding afatinib-related liver injury, such as latency, serum enzyme profiles, clinical features, and disease course, have not been published. Other EGFR inhibitors, such as erlotinib and gefitinib, typically cause liver injury within days or weeks of treatment initiation, characterized by elevated hepatocyte enzymes and rapid, moderate to severe progression. Immune hypersensitivity and autoimmune features are uncommon. Patients with a history of cirrhosis or liver dysfunction due to hepatic tumor burden have a higher risk of clinically significant liver injury and liver failure. Probability score: D (Possibly the cause of clinically obvious liver damage).

C26H27CLFN5O7

575.97328877449

665.153

1398312-64-5

Afatinib;850140-72-6;Afatinib dimaleate;850140-73-7;(E/Z)-Afatinib;439081-18-2;Afatinib-d6;1313874-96-2

66547001

Typically exists as solid at room temperature

6

16

10

46

773

1

CN(C)C/C=C/C(=O)NC1=C(C=C2C(=C1)C(=NC=N2)NC3=CC(=C(C=C3)F)Cl)O[C@H]4CCOC4.C(=O)(C(=O)O)O.C(=O)(C(=O)O)O

NHTYOYNDYHAMAE-IACUOYJGSA-N

InChI=1S/C24H25ClFN5O3.2C2H2O4/c1-31(2)8-3-4-23(32)30-21-11-17-20(12-22(21)34-16-7-9-33-13-16)27-14-28-24(17)29-15-5-6-19(26)18(25)10-15;2*3-1(4)2(5)6/h3-6,10-12,14,16H,7-9,13H2,1-2H3,(H,30,32)(H,27,28,29);2*(H,3,4)(H,5,6)/b4-3+;;/t16-;;/m0../s1

(E)-N-[4-(3-chloro-4-fluoroanilino)-7-[(3S)-oxolan-3-yl]oxyquinazolin-6-yl]-4-(dimethylamino)but-2-enamide;oxalic acid

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.)