In GIST 882, thyroid TT, MDA-MB-231, HepG2, A375, and SW620 cells, regorafenib mesylate (0–10 μM, 96 h) has anti-proliferative action [1]. In addition to inhibiting FGFR and pERK1/2, regorafenib mesylate (0-3000 nM, 30 minutes) also prevents VEGFR2, TIE2, and PDGFR-β from autophosphorylating[1]. Hep3B cell growth is inhibited by regorafenib mesylate in a concentration-dependent manner, with an IC50 of 5 μM. The JNK target phosphorylated c-Jun is then upregulated by regorafenib in Hep3B cells, but not total c-Jun [2].

Regorafenib mesylate (10 mg/kg, PO, as a single dosage or daily for 4 days) reduces tumor vasculature and tumor development in the rat GS9L glioblastoma model [1]. Regorafenib mesylate (0-100 mg/kg, oral, qd × 9) demonstrates anti-tumor and anti-angiogenic actions in Colo-205, MDA-MB-231 and 786-O models [1].

Cell Proliferation Assay[1]
Cell Types: GIST 882, Thyroid TT, MDA-MB-231, HepG2, A375 and SW620 Cell
Tested Concentrations: 10 μM and 5 nM
Incubation Duration: 96 hrs (hours)
Experimental Results: In GIST 882, Thyroid TT, MDA- For MB-231, HepG2, A375 and SW620 cells, the IC50 values are 45 ± 20, 34 ± 8, 401 ± 88, 560 ± 200, 900, 967 ± 287 nM. respectively.

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Western Blot Analysis[1]
Cell Types: NIH-3T3/VEGFR2 cells, (CHO)-TIE2 cells, HAoSMCs cells, MCF-7 cells
Tested Concentrations: 0, 10, 30, 100, 300, 1000, 3000 nM
Incubation Duration: 30 Minute
Experimental Results: Inhibits autophosphorylation of VEGFR2, TIE2, and PDGFR-β with IC50 values of 3, 31, and 90 nM, respectively, inhibits FGFR signaling in FGF10-stimulated MCF-7 breast cancer (BC) cells, and shows inhibition Phosphorylates FGFR substrate 2 (pFRS2) and the downstream signaling kinase pERK1/2.

Animal/Disease Models: Rat GS9L glioblastoma xenograft[1]
Doses: 10 mg/kg
Route of Administration: po (po (oral gavage)) single dose or one time/day for 4 days
Experimental Results: Inhibition of rat GS9L glioblastoma Tumor vasculature and tumor growth in models.

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Animal/Disease Models: Female athymic NCr nu/nu (nude) mice, various xenograft models, including those derived from CRC (Colo-205), BC (MDA-MB-231) and RCC (786-O) tumors [1]
Doses: 0, 3, 10, 30, 100 mg/kg
Route of Administration: po (po (oral gavage)) qd × 9
Experimental Results: Effectively inhibited the growth of Colo-205, MDA-MB-231 and 786-O models. Dramatically reduces tumor MVA, effectively inhibits the RAF/MEK/ERK signaling cascade reaction, and Dramatically inhibits tumor cell proliferation.

Absorption, Distribution and Excretion
Cmax = 2.5 μg/mL; Tmax = 4 hours; AUC = 70.4 μgh/mL; Steady-state Cmax = 3.9 μg/mL; Steady-state AUC = 58.3 μgh/mL; The mean relative bioavailability of tablets is 69% to 83% compared to oral solutions. Within 12 days after administration of 120 mg of the radiolabeled oral solution, approximately 71% of the radiolabeled dose is excreted in feces (47% as the parent compound and 24% as metabolites), and 19% is excreted in urine (17% as glucuronides). Regorafenib circulates enterohepaticly, and multiple plasma concentration peaks were observed within a 24-hour dosing interval. Metabolisms/Metabolites Regorafenib is metabolized by CYP3A4 and UGT1A9. The major circulating metabolites of regorafenib, as determined by steady-state assays in human plasma, are M-2 (N-oxide) and M-5 (N-oxide and N-demethyl), both of which exhibit similar in vitro pharmacological activities and steady-state concentrations to regorafenib. M-2 and M-5 have high protein binding rates (99.8% and 99.95%, respectively). Regorafenib is a P-glycoprotein inhibitor, while its active metabolites M-2 (N-oxide) and M-5 (N-oxide and N-demethyl) are substrates of P-glycoproteins.

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Biological half-life
Regorafenib, oral administration 160 mg = 28 hours (14-58 hours); M-2 metabolite, oral administration 160 mg = 25 hours (14-32 hours); M-5 metabolite, oral administration 160 mg = 51 hours (32-72 hours).

Hepatotoxicity
Elevated serum transaminase levels are common in large clinical trials of regorafenib, occurring in 39% to 45% of patients, with 3% to 6% of patients having transaminase levels exceeding five times the upper limit of normal (ULN). Furthermore, several cases of clinically significant liver injury during regorafenib treatment have been reported; these injuries are usually severe and occasionally fatal, with an estimated incidence of approximately 0.3%. Therefore, routine monitoring of liver enzymes is recommended. Regorafenib-induced liver injury can present in various patterns or phenotypes. Some patients develop acute liver necrosis within days of starting regorafenib, with elevated serum transaminase and lactate dehydrogenase levels, accompanied by mild jaundice, but with prolonged INR and signs of liver failure. This injury can be severe but is usually self-limiting, with rapid and complete recovery. Other patients present with a clinical pattern similar to acute viral hepatitis, with hepatocellular (or mixed) elevations in serum enzymes and jaundice, which can persist for a long time and, in some cases, even lead to death. Autoimmune and allergic reactions are uncommon. Additionally, rare cases of regorafenib-related liver injury present as sinusoidal obstruction syndrome or pseudocirrhosis, with prominent liver nodules and ascites, but these eventually improve or resolve. Finally, regorafenib, like other multi-kinase inhibitors (sunitinib, imatinib, sorafenib), is associated with hyperammonemia-induced coma, typically occurring within days or weeks of starting treatment and rapidly reversible upon discontinuation.
Probability Score: B (Very likely to cause clinically significant liver injury).

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Pregnancy and Lactation Effects
◉ Overview of Use During Lactation
There is currently no information on the clinical use of regorafenib during lactation. Due to the high plasma protein binding rate of regorafenib (99.5%), its concentration in breast milk may be low. However, one of its metabolites has a long half-life of up to 70 hours and may accumulate in the infant. The manufacturer recommends discontinuing breastfeeding during regorafenib treatment and for two weeks after the last dose.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
No published information found as of the revision date.

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Protein binding
Regorafenib binds very well to human plasma proteins (99.5%).

[1]. Regorafenib (BAY 73-4506): a new oral multikinase inhibitor of angiogenic, stromal and oncogenic receptor tyrosine kinases with potent preclinical antitumor activity. Int J Cancer, 2011, 129(1), 245-255.

[2]. Fluoro-Bay 43-9006 (Regorafenib) effects on hepatoma cells: growth inhibition, quiescence, and recovery. J Cell Physiol, 2013, 228(2), 292-297.

Regorafenib is a pyridinecarboxamide compound formed by the condensation of 4-[4-({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-3-fluorophenoxy]pyridine-2-carboxylic acid with methylamine. It is used to treat patients with metastatic colorectal cancer who have previously received chemotherapy, anti-EGFR, or anti-VEGF therapy. It has antitumor, tyrosine kinase inhibitory, and hepatotoxic effects. It is an aromatic ether, pyridinecarboxamide, monochlorobenzene, (trifluoromethyl)benzene, monofluorobenzene, and phenylurea compound. Regorafenib is an oral multi-kinase inhibitor used to treat metastatic colorectal cancer, advanced gastrointestinal stromal tumors, and hepatocellular carcinoma. Regorafenib was approved by the FDA on September 27, 2012. In April 2017, the indication for regorafenib was expanded to include the treatment of hepatocellular carcinoma. Anhydrous regorafenib is a kinase inhibitor. Its mechanisms of action include acting as a kinase inhibitor, a cytochrome P450 2C9 inhibitor, a breast cancer resistance protein inhibitor, a UGT1A9 inhibitor, and a UGT1A1 inhibitor. Regorafenib is an oral multi-kinase inhibitor used to treat refractory metastatic colorectal cancer, hepatocellular carcinoma, and gastrointestinal stromal tumors. Elevated serum transaminases are common during regorafenib treatment, and rare but sometimes severe and even fatal cases of clinically significant liver injury have been reported. Anhydrous regorafenib is the anhydrous form of regorafenib, a small molecule drug with high oral bioavailability and potential anti-angiogenic and antitumor activities. Regorafenib binds to and inhibits the activity of vascular endothelial growth factor receptors (VEGFR) 2 and 3, as well as Ret, Kit, PDGFR, and Raf kinases, thereby inhibiting tumor angiogenesis and tumor cell proliferation. VEGFR is a receptor tyrosine kinase that plays an important role in tumor angiogenesis; receptor tyrosine kinases RET, KIT, and PDGFR, as well as serine/threonine-specific Raf kinases, are involved in tumor cell signaling.
Regorafenib is the hydrated form of regorafenib, a small molecule drug with high oral bioavailability and potential anti-angiogenic and antitumor activity. Regorafenib binds to and inhibits the activity of vascular endothelial growth factor receptors (VEGFR) 2 and 3, as well as Ret, Kit, PDGFR, and Raf kinases, thereby inhibiting tumor angiogenesis and tumor cell proliferation. VEGFR is a receptor tyrosine kinase that plays an important role in tumor angiogenesis; receptor tyrosine kinases RET, KIT, and PDGFR, as well as serine/threonine-specific Raf kinases, are involved in tumor cell signaling.
See also: Regorafenib monohydrate (active ingredient).

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Drug Indications
Regorafenib is indicated for the treatment of patients with metastatic colorectal cancer (CRC) who have previously received fluorouracil, oxaliplatin, and irinotecan chemotherapy, anti-VEGF therapy, and (if KRAS wild-type) anti-EGFR therapy. Regorafenib is also indicated for the treatment of patients with locally advanced, unresectable, or metastatic gastrointestinal stromal tumors (GIST) who have previously received imatinib mesylate and sunitinib malate. Regorafenib is also indicated for the treatment of patients with hepatocellular carcinoma (HCC) who have previously received sorafenib.

FDA Label
Stivarga is indicated for monotherapy in the following adult patients: patients with metastatic colorectal cancer (CRC) who have previously received or are not eligible for existing therapies (including fluorouracil chemotherapy, anti-VEGF therapy, and anti-EGFR therapy); patients with unresectable or metastatic gastrointestinal stromal tumors (GIST) whose disease has progressed or who are intolerant to prior treatment with imatinib and sunitinib; and patients with hepatocellular carcinoma (HCC) who have previously received sorafenib.
Treatment of all malignancies (excluding hematopoietic and lymphoid tissue tumors)
Mechanism of Action

Regorafenib is a small molecule inhibitor that inhibits a variety of membrane-bound and intracellular kinases involved in normal cellular function and pathological processes such as tumorigenesis, tumor angiogenesis, and maintenance of the tumor microenvironment. In in vitro biochemical or cell experiments, regorafenib and its main human active metabolites M-2 and M-5 inhibited the activities of RET, VEGFR1, VEGFR2, VEGFR3, KIT, PDGFR-α, PDGFR-β, FGFR1, FGFR2, TIE2, DDR2, TrkA, Eph2A, RAF-1, BRAF, BRAFV600E, SAPK2, PTK5, and Abl at clinically applicable concentrations. In in vivo models, regorafenib exhibited anti-angiogenic activity in rat tumor models and demonstrated inhibitory tumor growth and anti-metastatic activity in various mouse xenograft models, including some human colorectal cancers.

C21H15CLF4N4O3.CH4O3S

578.9211

578.065

835621-08-4

Regorafenib;755037-03-7;Regorafenib monohydrate;1019206-88-2;Regorafenib-d3;1255386-16-3

11167602

Typically exists as solid at room temperature

6.81

3

8

5

33

686

0

C1(C(NC)=O)=NC=CC(OC2=CC=C(NC(=O)NC3=CC=C(Cl)C(C(F)(F)F)=C3)C(F)=C2)=C1.S(C)(=O)(O)=O

FNHKPVJBJVTLMP-UHFFFAOYSA-N

InChI=1S/C21H15ClF4N4O3/c1-27-19(31)18-10-13(6-7-28-18)33-12-3-5-17(16(23)9-12)30-20(32)29-11-2-4-15(22)14(8-11)21(24,25)26/h2-10H,1H3,(H,27,31)(H2,29,30,32)

4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino]-3-fluorophenoxy]-N-methylpyridine-2-carboxamide

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.)