VEGFR1 (IC50 = 13 nM); VEGFR2 (IC50 = 4.2 nM); VEGFR3 (IC50 = 46 nM); PDGFRβ (IC50 = 22 nM); Braf (IC50 = 28 nM); VEGFR2 (BRafV600E = 19 nM); Raf-1 (IC50 = 2.5 nM)

Regorafenib potently inhibits VEGFR2 autophosphorylation in NIH-3T3/VEGFR2 cells with an IC50 of 3 nM. Regorafenib has an IC50 of 90 nM and blocks PDGFR-β autophosphorylation in HAoSMCs following PDGF-BB stimulation. With an IC50 of 3 nM, vegf165-stimulated HUVEC proliferation is inhibited by rogorafenib[1]. With a 5 μM IC50, regorafenib inhibits the growth of Hep3B cells in a concentration-dependent manner. The JNK target phospho-c-Jun, but not total c-Jun, is subsequently upregulated by regulatorafenib in Hep3B cells[3].
Regorafenib (0-10 μM, 96 h) exhibits anti-proliferation activity in GIST 882, Thyroid TT, MDA-MB-231, HepG2, A375, and SW620 cells[1].
Regorafenib (BAY 73-4506), a novel oral multikinase inhibitor, potently inhibits these endothelial cell kinases in biochemical and cellular kinase phosphorylation assays. Furthermore, regorafenib inhibits additional angiogenic kinases (VEGFR1/3, platelet-derived growth factor receptor-β and fibroblast growth factor receptor 1) and the mutant oncogenic kinases KIT, RET and B-RAF.
Regorafenib inhibited growth of human Hep3B, PLC/PRF/5, and HepG2 cells in a concentration- and time-dependent manner. Multiple signaling pathways were altered, including MAP kinases phospho-ERK and phospho-JNK and its target phospho-c-Jun. There was evidence for apoptosis by FACS, cleavage of caspases and increased Bax levels; as well as induction of autophagy, as judged by increased Beclin-1 and LC3 (II) levels. Prolonged drug exposure resulted in cell quiescence. Full growth recovery occurred after drug removal, unlike with doxorubicin chemotherapy. Regorafenib is a potent inhibitor of cell growth. Cells surviving Regorafenib treatment remain viable, but quiescent and capable of regrowth following drug removal. The reversibility of tumor cell growth suppression after drug removal may have clinical implications.[3]
Regorafenib (0–3000 nM, 30 min) inhibits FGFR and pERK1/2 as well as the autophosphorylation of VEGFR2, TIE2, and PDGFR-β.
Regorafenib has an IC50 of 5 μM and inhibits Hep3B cell growth in a concentration-dependent manner. Regorafenib then elevates phospho-c-Jun levels in Hep3B cells, a JNK target, but not total c-Jun levels[3].

Regorafenib effectively slows the growth of Colo-205 xenografts at doses between 10 and 100 mg/kg, with a TGI of 75% at day 14 at the 10 mg/kg dose. Regorafenib is highly effective in the MDA-MB-231 model at doses as low as 3 mg/kg, producing a significant TGI of 81%, which rises to 93% at doses of 10 and 30 mg/kg, where tumor stasis is reached[1].
Regorafenib (10 mg/kg, Orally, once or twice daily for 4 days) inhibits tumor growth and tumor vasculature in a rat GS9L glioblastoma model[1].
Regorafenib (0-100 mg/kg, Orally, qd × 9) exhibits antitumorigenic and antiangiogenic effects in the Colo-205, MDA-MB-231 and 786-O model[1].
The antiangiogenic effect of regorafenib was demonstrated in vivo by dynamic contrast-enhanced magnetic resonance imaging. Regorafenib administered once orally at 10 mg/kg significantly decreased the extravasation of Gadomer in the vasculature of rat GS9L glioblastoma tumor xenografts. In a daily (qd)×4 dosing study, the pharmacodynamic effects persisted for 48 hr after the last dosing and correlated with tumor growth inhibition (TGI). A significant reduction in tumor microvessel area was observed in a human colorectal xenograft after qd×5 dosing at 10 and 30 mg/kg. Regorafenib exhibited potent dose-dependent TGI in various preclinical human xenograft models in mice, with tumor shrinkages observed in breast MDA-MB-231 and renal 786-O carcinoma models. Pharmacodynamic analyses of the breast model revealed strong reduction in staining of proliferation marker Ki-67 and phosphorylated extracellular regulated kinases 1/2. These data demonstrate that regorafenib is a well-tolerated, orally active multikinase inhibitor with a distinct target profile that may have therapeutic benefit in human malignancies[1].

Recombinant VEGFR2 (murine aa785–aa1367), VEGFR3 (murine aa818–aa1363), PDGFRβ (aa561–aa1106), Raf-1 (aa305–aa648) and BRafV600E (aa409–aa765) kinase domains are used in in vitro tests. At a constant 1 M Regorafenib concentration, the initial in vitro kinase inhibition profiling is carried out. Select responding kinases, such as VEGFR1 and RET, are used to calculate the 50% inhibitory concentration (IC50) values. Using a recombinant fusion protein of glutathione-S-transferase, the intracellular domain of TIE2, and the peptide biotin-Ahx-EPKDDAYPLYSDFG as substrate, the homogeneous time-resolved fluorescence (HTRF) assay is used to measure TIE2 kinase inhibition.

GIST 882 and TT cells are grown in RPMI medium with L-glutamine for proliferation assays, while MDA-MB-231, HepG2, and A375 cells are grown in DMEM that is always supplemented with 10% hiFBS. Trypsinized cells are plated at a density of 5×104 cells per well in 96-well plates containing complete media containing 10% FBS, and grown overnight at 37 °C. The incubation is continued for another 96 hours with the addition of vehicle or regorafenib, serially diluted in complete growth media to final concentrations between 10 μM and 5 nM, and 0.2% DMSO. Using CellTitre-GloTM, cell proliferation is measured. [1]

---

VEGFR2 phosphorylation was analyzed by enzyme-linked immunosorbent assay (ELISA) and Western blotting[1]
NIH-3T3 cells transfected with human VEGFR2 were plated at 30,000 cells/well in 96-well plates in Dulbecco's Modified Eagle Medium containing 10% FBS; 6 hr after plating, media was changed to 0.1% BSA/DMEM and incubation continued for 24 hr. Cells were treated with vehicle or various concentrations of Regorafenib in 0.1% BSA/DMEM/0.1% dimethylsulfoxide (DMSO) for 1 hr at 37°C, prior to stimulation with recombinant VEGF165 at 30 ng/mL final concentration for 5 min. Cells were washed with cold phosphate-buffered saline (PBS) and lysed in 100 μL of lysis buffer (50 mM HEPES, pH 7.2, 1% Triton X-100, 1 mM Na3VO4, 150 mM NaCl, 10% glycerol, 1.5 mM ethylene glycol tetraacetic acid and complete protease inhibitor cocktail).

---

Regorafenib treatment [3]
Each cell line was seeded at 0.3×105 cells/2ml of DMEM containing 10% FBS in 35 mm tissue culture dishes. The cells were incubated for 24 h to allow attachment, and then the medium was replaced by fresh culture medium containing Regorafenib at increasing concentrations (1 μM, 2.5 μM, 5 μM, 7.5 μM and 10 μM). In these experimental conditions, the cells were allowed to grow for 72 or 96 h. Time-course experiments on Hep3B cells were performed with 7.5 μM of Regorafenib at short (15, 60, 180 min.), middle (24, 48, 72 and 96 h) or long times (up to seven days). When the cells were treated for long times the drug was replaced with a fresh one. Each experiment included a control with the equivalent concentration of DMSO (solvent control) as the one used for adding Regorafenib. Each experiment was performed in triplicate and repeated 3 times. Subsequent analyses were performed at specific Regorafenib concentrations and incubation times.

---

Recovery/Reversibility [3]
To study the recovery in cell proliferation after drug withdrawal, Hep3B cells were treated with Regorafenib 5 or 7.5 μM for 3-7 days, then the medium was removed and replaced with fresh medium without drug. The rate of cell recovery was evaluated by MTT test at different subsequent time points. Doxorubicin treatment at 0.01or 0.05 or 0.1 μM was used as positive control to study the apoptotic process.

---

FACS analysis for apoptosis [3]
The FITC-annexin V kit was used to detect apoptosis as specified by the supplier. Briefly, 1×106 cells treated with various Regorafenib concentrations for 48 h were harvested and washed with PBS. Cells were resuspended in binding buffer and then incubated for 5 min at room temperature in the dark after 5 μl AnnexinV-FITC and 10μl 7-amino actinomycin D (7AAD) intercalates into DNA. Intact cells were discriminated from apoptotic cells.

\n \nDynamic contrast-enhanced magnetic resonance imaging [1]
\nFor DCE-MRI experiments, Fischer 344 rats were inoculated with 3×106 GS9L cells intramuscularly into the left thigh. Treatment was initiated when the tumor reached between 300 and 700 mm3. MRI was performed using a Siemens 1.5T Avanto MRI system equipped with a dedicated animal receiver coil. Regorafenib was administered orally, either as single administration (daily [qd]×1) or qd×4 at a dose of 10 mg/kg body weight. DCE-MRI examinations were performed using the contrast agent Gadomer-17 before therapy and 4, 8, 24, 48 hr and 4 days after the last regorafenib administration. For MRI, animals were anesthetized using 1.5% Isoflurane in O2/N2O. Gadomer-17 was injected intravenously at a dose of 50 μmol Gd/kg body weight into the tail vein at a rate of 0.5 mL/s using an automated injection device. For DCE-MRI data acquisition, a 2D turbo flash saturation recovery pulse sequence was used with the settings: echo time (TE): 1.63 ms, repetition time (TR): 350 ms, inversion time (TI): 180 ms, flip angle (FL): 10°, four averages, 5 mm slice thickness at 0.8 × 0.8 mm2 in plane resolution. The acquisition time for 1 image was 1.4 s, and 254 images were acquired over ∼6 min. Before contrast agent injection, six images were acquired as baseline. For data evaluation, a region of interest was defined covering the complete tumor on one acquired slice. Signal intensity in the region of interest over time was analyzed. Area under the curve of the initial 360 seconds after Gadomer-17 injection (IAUC360) of MRI signal intensity over time graphs in tumor were normalized to muscle as nonaffected reference tissue in each animal and used for data evaluation. Tumor volume was determined at various time points: at staging (predose), at qd×4 of oral dosing (day 4 post-treatment) and at days 6 and 8 after staging using MRI pulse sequence set at: 3D gradient recalled echo, TE: 9 ms, TR: 16 ms, FL: 40°, one average, 60 slices at 0.7 mm slice thickness and 0.35 × 0.35 mm2 in plane resolution. Volume was calculated by slice per slice tumor area evaluation. Statistical analysis was performed using unpaired two-sided Student's t test.\n

---

\n\nExamination of microvessel area, Ki-67 and MAPK in tumor xenograft models [1]
\nAnimals with tumors of ∼200 mg were treated orally with regorafenib at 10 and 30 mg/kg on a qd×5 schedule. Subsequently, tumors were harvested, paraffin-embedded, and analyzed by immunohistochemistry (IHC). Tumor endothelial cells were detected using an antibody against CD-31 (#M-20, 1:750). Inhibition of cell signaling was assessed using an antibody against pERK1/2 (#9101L, 1:100), and tumor cell proliferation was analyzed using an antibody against Ki-67, as previously described. \n
\nFor tumor MVA determinations, CD-31 stained slides were coded before analysis. Tissue sections were viewed using a 10× objective magnification (0.644 mm2 per field). Four fields per section were randomly analyzed, excluding peripheral surrounding connective and central necrotic tissues. CD-31-positive areas were quantified using the software Image-Pro Plus version 3.0 and SIS image analysis. The data are presented as MVA, %. Data were analyzed statistically with one-way analysis of variance on ranks using Kruskal–Wallis, and the Dunnett method was used for comparison with the vehicle group; p < 0.05 was considered significant.\n

---

\nTumor xenograft experiments [1]
\nFemale athymic NCr nu/nu mice, kept in accordance with Federal guidelines, were subcutaneously inoculated with 5×106 Colo-205 or MDA-MB-231 cells or implanted with 1 mm3 786-O tumor fragments. When tumors reached a volume of ∼100 mm3, regorafenib or vehicle control was administered orally qd×21 in the 786-O model, and qd×9 in the Colo-205 and MDA-MB-231 models, respectively, at doses of 100, 30, 10, and 3 mg/kg. Paclitaxel was administered intravenously at 10 mg/kg in ethanol/Cremophor EL®/saline (12.5%/12.5%/75%) every 2 days × 5. Tumor size (volume) was estimated twice weekly (l×w2)/2, and the percentage of tumor growth inhibition (TGI) was obtained from terminal tumor weights (1-T/C×100). Mice were weighed every other day starting from the first day of treatment. The general health status of the mice was monitored daily.\n

---

\n

PEG400/125 mM aqueous methanesulfonic acid (80/20) or polypropylene glycol/PEG400/Pluronic F68 (42.5/42.5/15 + 20% Aqua); 3, 10, 30, 200 mg/kg; oral

Female athymic NCr nu/nu mice with Colo-205, MDA-MB-231 or 786-O

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.

---

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).

---

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.

---

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]. Targeted therapy for metastatic renal cell carcinoma: current treatment and future directions. Ther Adv Med Oncol, 2010, 2(1), 39-49.

[3]. Fluoro-Sorafenib (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).

---

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.

C₂₁H₁₆CL₂F₄N₄O₃

519.28

518.053

835621-07-3

Regorafenib;755037-03-7;Regorafenib monohydrate;1019206-88-2

11167602

White to off-white solid

6.968

3

8

5

33

686

0

Cl.O=C(NC1C(F)=CC(OC2C=C(C(NC)=O)N=CC=2)=CC=1)NC1C=C(C(F)(F)F)C(Cl)=CC=1

ACSWJKPZXNIVMY-UHFFFAOYSA-N

InChI=1S/C21H15ClF4N4O3.ClH/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);1H

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

BAY-734506 HCl; BAY 734506; BAY734506; Regorafenib HCl. Brand name: Stivarga

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 2 mg/mL (3.85 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: 30% PEG400+0.5% Tween80+5% Propylene glycol : 30mg/mL

---

 (Please use freshly prepared in vivo formulations for optimal results.)