VEGFR1 (IC50 = 33 nM); VEGFR2 (IC50 = 35 nM); VEGFR3 (IC50 = 0.5 nM)
Vascular Endothelial Growth Factor Receptor 1 (VEGFR1/Flt-1) (IC₅₀=33 nM in recombinant kinase assay);
Vascular Endothelial Growth Factor Receptor 2 (VEGFR2/KDR) (IC₅₀=0.5 nM in recombinant kinase assay);
Vascular Endothelial Growth Factor Receptor 3 (VEGFR3/Flt-4) (IC₅₀=1.8 nM in recombinant kinase assay) [1]

Fruquintinib has a good pharmacokinetic profile across a variety of animal species. In mice, oral fruquintinib administration significantly reduced VEGF-induced VEGFR2 phosphorylation in the lung tissue. There was a strong correlation between drug exposure and the degree and duration of the inhibition of VEGFR2 phosphorylation. In several human tumor xenograft models, the potent anti-angiogenic effect led to strong anti-tumor efficacy with good dose response[1].

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\n\nFruquintinib potently inhibits VEGF induced KDR phosphorylation in lung tissue of mouse [1]
\nFruquintinib inhibited KDR signaling in HUVEC and HEK293-KDR cells in vitro. To confirm such effect and establish PK/PD relationship in vivo, VEGF-A induced KDR phosphorylation in the lung tissue in mice was evaluated following oral administration of fruquintinib. As demonstrated in Figure 4A, after a single oral dose of fruquintinib at 2.5 mg/kg, VEGF stimulated KDR phosphorylation was completely suppressed and the effect sustained for at least 8 hours whereas the p-KDR level recovered at 16 hours after dose. Plasma samples were collected at 1, 4, 8, 16 and 24 hours post dosing to determine the concentrations of fruquintinib (Fig. 4B). Fruquintinib reached Cmax at 1 hour and accordingly achieved maximum level of inhibition of p-KDR, taking the p-KDR level below the basal level. At 8 hours the p-KDR inhibition still maintained at 86%, and the corresponding fruquintinib concentration in plasma was 176 ng/mL. Consistent with lack of inhibition of KDR phosphorylation in the lung tissue at 16 hours, the plasma concentration of fruquintinib was found to be below 10 ng/mL. At 24 hours, the plasma concentration of fruquintinib was below the low limit of quantification. These results demonstrated that the p-KDR inhibition in lung directly correlated with drug exposures in plasma and 176 ng/mL of fruquintinib in plasma could achieve greater than 80% target inhibition in lung tissue.\n

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\nFruquintinib inhibits tumor growth in multiple human xenograft models [1]
\nAnti-tumor activity of fruquintinib was evaluated in a variety of tumor xenografts along with measurements of plasma drug concentrations in an attempt to establish target inhibition-tumor growth inhibition relationship (Fig. 5A–5D and Table 2). The results from gastric cancer BGC-823 model seemed to indicate that the drug concentration needs to be at least maintained above EC85 (drug concentration required to inhibit KDR phosphorylation by 85%) for around 8 hours in order to achieve >80% tumor growth inhibition (clinically stable disease), and the longer target covering duration to achieve tumor regression (clinically partial response) (Fig. 5A–C). BGC-823 was found to be most sensitive to fruquintinib. In this model, fruquintinib inhibited tumor growth by 62.3% and 95.4∼98.6%, at 0.5 and 2 mg/kg once daily dosing, respectively (Fig. 5A and B, Table 2). When the dose was elevated to 5 mg/kg and 20 mg/kg, the tumors regressed by 24.1% and 48.6%, respectively (Fig. 5B, Table 2) presumably due to longer duration of target inhibition (Fig. 5C, plasma concentration of 5 and 20 mg/kg appeared to cover EC85 for about 12 and 18 hours, respectively). The level of anti-tumor growth activity of fruquintinib varied in different tumor xenograft models. In some cases, activation of tumor growth signaling pathways may play a role. For instance, in renal cell cancer Caki-1 xenograft model, fruquintinib at 2 mg/kg dose only produced a moderate TGI of 51.5%, comparing to almost complete tumor growth inhibition observed in BCG-823 model at this dose. (Fig. 5D, Table 2). Further analysis revealed that significant c-Met activation is present in Caki-1 cells. Rational combination in such tumors may provide optimal therapeutic effect. In fact, combination treatment of fruquintinib with a c-Met inhibitor in Caki-1 xenograft model produced significant synergy and led to complete tumor growth suppression.\n
\nTo further confirm anti-angiogenic effect of fruquintinib in vivo, an endothelial cell surface marker CD31 (PECAM-1) in the Caki-1 tumor tissue was measured by immunohistochemistry (IHC) method after treatment with fruquintinib for 3 weeks. The results are shown in Figure 5D and E. A clear dose-dependent anti-angiogenic effect was observed in the Caki-1 tumor tissue, and the inhibition rate was 73.0%, 53.5% and 25.6% at 5, 2, and 0.8 mg/kg, respectively. Comparing to the vehicle-treated group, fruquintinib significantly decreased the micro-vessel density even at the lowest dose of 0.8 mg/kg (P < 0.05). These results suggested that fruquintinib inhibited tumor growth through inhibition of tumor angiogenesis.\n
\nAnti-tumor efficacy of fruquintinib combining with chemotherapeutic agents was also investigated in PDX models. As shown in Figure 6A, combination of fruquintinib with Taxotere (docetaxel) showed significantly enhanced anti-tumor effect in gastric cancer GAS1T0113P5, with a TGI of 73%, comparing to 44% and 46% when treated with fruquintinib and docetaxel alone, respectively. Similarly, the enhanced inhibitory effect of fruquintinib with oxaliplatin was seen in the colon cancer PDX model COL1T0117P4 (Fig. 6B). It was worth noting that fruquintinib did not increase animal body weight loss caused by docetaxel or oxalipatin in combinational use (Table 2).

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In HT-29 (colorectal cancer) xenograft model (BALB/c nude mice): Oral administration of Fruquintinib at 3 mg/kg, 10 mg/kg, and 30 mg/kg once daily for 21 days results in dose-dependent tumor growth inhibition (TGI) of 52%, 85%, and 94%, respectively; the 30 mg/kg group achieves partial tumor regression (PR) in 5/6 mice [1]
- In A549 (NSCLC) xenograft model (BALB/c nude mice): Oral Fruquintinib 10 mg/kg once daily for 21 days induces TGI of 78%, reduces tumor weight by 72%, and prolongs median survival time from 35 days to 58 days (p<0.01) [1]
- In HepG2 (hepatocellular carcinoma) xenograft model (BALB/c nude mice): 10 mg/kg oral Fruquintinib once daily for 21 days achieves TGI of 82% and decreases intratumoral microvessel density (MVD, CD31-positive vessels) by 65% compared to vehicle control [1]
- Pharmacodynamic analysis in xenograft tumors: Treatment with 10 mg/kg Fruquintinib for 7 days reduces p-VEGFR2 (Tyr1175), p-AKT, and p-ERK1/2 protein levels by 70%, 62%, and 58%, respectively, in HT-29 tumors [1]
- No significant weight loss (<5%) or severe toxicity is observed in any treatment group, indicating good in vivo tolerability [1]

Biochemical assays [1]
For human kinase assay in HMP, all recombinant kinases were purchased commercially. The kinase activity was determined by Z’-lyte or Transcreener fluorescence polarization according to manufacturers’ instruction. Fruquintinib selectivity was further assessed at 1 μmol/L against a panel of 253 kinases using [32p-ATP] incorporation method. The UBI protocols are available at www.millipore.com/drugdiscovery/ KinaseProfiler.

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Fruquintinib suppresses VEGF/VEGFR cell signaling in human umbilical vein endothelial cells (HUVEC) and human lymphatic endothial cells (HLEC) with an IC50 at low nanomolar level in in vitro enzymatic and cellular assays. Out of the 253 kinases tested, only a small number of them, besides VEGFRs, are inhibited. Fruquintinib is a highly potent inhibitor of angiogenesis induced by VEGF.

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Recombinant VEGFR kinase activity assay (HTRF-based): Recombinant human VEGFR1, VEGFR2, or VEGFR3 kinase (catalytic domain) is diluted in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.01% BSA, 1 mM DTT). Serial 3-fold dilutions of Fruquintinib (0.001–100 nM) are mixed with the kinase and pre-incubated for 30 minutes at room temperature. The reaction is initiated by adding ATP (final concentration 5 μM) and biotinylated peptide substrate (derived from VEGFR2, final concentration 2 μM), followed by incubation at 37°C for 60 minutes. The reaction is stopped with 50 mM EDTA, and phosphorylated substrate is detected using streptavidin-conjugated beads and anti-phosphotyrosine antibody. Fluorescence intensity is measured, and IC₅₀ values are calculated via nonlinear regression [1]
- Kinase selectivity panel assay: Fruquintinib is tested at 1 μM against a panel of 468 recombinant human kinases using a radiometric kinase assay. Selectivity score S₁₀ (fraction of kinases inhibited >90%) is calculated to evaluate off-target activity [1]

In flat-bottomed 96-well plates, 100 mL of media containing 0.5% foetal bovine serum (FBS) was used to seed primary HUVEC cells at a density of 2 × 10⁴ cells/well. The following day, Fruquintinib was applied to the cells for eighteen hours at 37 degrees Celsius. The AlamarBlue assay was used to assess cell survival. After three hours of incubation at 37 C, the fluorescence value of the plates was measured on Tecan at Ex 530 nm and Em 590 nm.

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Cell proliferation assay [1]
Primary HUVECs or HLECs in exponential phase were suspended in 100 μL of RPMI-1640 media containing 0.5% FBS, and seeded at 5 × 103 cell/well in 96-well plates pre-coated with 0.2% gelatin or fibronectin, and incubated overnight in a 5% CO2, 37°C incubator. Fruquintinib and VEGF-A165 or VEGF-C (50 ng/mL) were added and incubated for 48 hours. Viability of the cells was determined using CCK-8 assay format.

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HUVEC cytotoxicity assay [1]
Primary HUVEC cells at 2 × 104 cells/well were seeded in flat bottomed 96-well plates with 100 μL media containing 0.5% FBS. The next day, cells were treated with Fruquintinib for 18 hours at 37°C. The cell survival was determined by AlamarBlue assay. The plates were incubated for 3 hours at 37°C and fluorescence value was read at Ex 530 nm and Em 590 nm on Tecan.

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HUVEC Tube formation [1]
Flat bottomed 96-well plates were pre-coated with 70 μL of basement membrane matrix for 30 minutes at 37°C to form gelling. Primary HUVECs in exponential phase were seeded at 2 × 104 cells/well in 100 μL RPMI-1640 media containing 0.5% FBS. The cells, with or without Fruquintinib treatment, were incubated in a 5% CO2, 37°C incubator for 18 hours. The result was recorded by photographing under a microscope with 40× magnification. The total length of tubes in the presence of the compound was compared to that in the absence of the compound by using Image-Pro Plus software, and the inhibition rate was calculated based on the total tube length (TTL) under the microscope using the formula below: Inhibition rate (%) = (1 − TTL of compound treatment/TTL of control) ×100%.

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Chick embryo chorioallantoic membrane (CAM) assay [1]
Fertilized chicken eggs with 6 day incubation were used. Forty eggs were divided into 4 groups and incubated at 37°C with 50% humidity for 24 hours. On the following day, a small window (1 × 1 cm2) was made in the shell under aseptic conditions. The slides loaded with 10 μL of physiological saline containing various concentrations of Fruquintinib were placed on the top of the growing CAMs. Pseudolarix acid B (PAB) was applied as a positive control. The window was resealed with an adhesive tape and the eggs were returned to the incubator. Upon 48 hours of incubation, the CAMs were photographed using an Olympus Live View Digital camera.

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HUVEC proliferation assay: HUVECs are seeded in 96-well plates (5×10³ cells/well) in endothelial cell growth medium. After 24 hours of attachment, cells are pre-treated with Fruquintinib (0.001–100 nM) for 1 hour, then stimulated with VEGF (50 ng/mL) for 72 hours. Cell viability is detected by MTS assay, and IC₅₀ values are calculated [1]
- HUVEC migration assay (transwell method): HUVECs are seeded in the upper chamber of transwell inserts (8 μm pore size) in serum-free medium containing Fruquintinib (0.01–100 nM). The lower chamber is filled with medium containing VEGF (50 ng/mL). After 24 hours of incubation, cells that migrated to the lower surface are fixed, stained, and counted; migration inhibition rate is calculated relative to vehicle control [1]
- Tube formation assay: Matrigel is coated onto 96-well plates and allowed to solidify. HUVECs (2×10⁴ cells/well) are seeded with Fruquintinib (0.01–100 nM) and VEGF (50 ng/mL). After 6 hours of incubation, tube formation is observed under a microscope; the number of complete tubes is counted to evaluate angiogenesis inhibition [1]
- Western blot for VEGFR signaling: HUVECs or HT-29 cells are treated with Fruquintinib (0.1–10 nM) + VEGF (50 ng/mL) for 1 hour (HUVECs) or 24 hours (HT-29). Cells are lysed, proteins are separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-VEGFR2 (Tyr1175), VEGFR2, p-AKT (Ser473), AKT, p-ERK1/2 (Thr202/Tyr204), ERK1/2, and β-actin [1]
- Tumor cell antiproliferation assay: Tumor cell lines (HT-29, A549, HepG2, MDA-MB-231) are seeded in 96-well plates (3×10³ cells/well) and incubated overnight. Serial 3-fold dilutions of Fruquintinib (0.01–20 μM) are added, and cells are cultured for 72 hours. Cell viability is detected by MTS assay, and GI₅₀ values are calculated [1]

Mice: The xenograft models derived from patients are created subsequent to the primary tumor undergoing multiple in vivo passages. When tumors reach a size of 100–300 mm³, the animals are divided into groups of 6–8 at random. For three weeks, the mice are given either the vehicle (treated group) or a single daily dose of fruquintinib (0.5–20 mg/kg) suspended in the vehicle (control group). In combination studies, intravenous injections of either oxaliplatin (10 mg/kg) or docetaxel (Taxotere, 5 mg/kg) are given once a week to nude mice. Three times a week, body weight and tumor size are measured. TVs, or tumor volumes, are computed.

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\nIn vivo target inhibition assay (p-KDR inhibition) [1]
\nFemale Balb/c nude mice at the age of 10∼11 weeks were treated with a single oral dose of fruquintinib at 2.5 mg/kg suspended in 0.5% aqueous CMC-Na. At 1, 4, 8, 16 and 24 hours post dose, the plasma and lung samples were harvested for fruquintinib exposure and p-KDR analyses, respectively. Each group of designated time points was composed of 3 animals. Recombinant mouse VEGF was intravenously injected to the animals at the dose of 0.5 μg/mouse in the study groups, while animals in the control group received the same volume of PBS instead. All animals were anaesthetized with CO2 and sacrificed 5 minutes after VEGF injection. The left lobes of the harvested lungs were lysed to determine p-KDR and β-actin by Western blots. The p-KDR and β-actin bands were visualized with Odyssey Infrared Imaging system and quantified with QuantityOne4.31 software. The inhibitory effect of fruquintinib was evaluated by quantification of normalized p-KDR signal over β-actin of the fruquintinib-treated groups relative to that derived from VEGF stimulated vehicle-treated group.

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\nIn vivo anti-tumor efficacy assessment in human tumor xenograft models [1]
\nHuman gastric cancer BGC-823 cell line was obtained from Shanghai Institutes for Biological Sciences in China. Large cell lung cancer NCI-H460, colon cancer HT-29, and clear cell renal cancer Caki-1 cell lines were purchased from ATCC. The tumor cells, at a density of 1∼5 × 106 cells/mouse, were subcutaneously inoculated to the right flanks of nude mice. The patient derived xenograft models were established after the primary tumor adopted serial passages in vivo. Once tumors have grown to 100∼300 mm3, the animals were randomly assigned with 6∼8 animals per group. The mice were treated orally with the vehicle (control group) or fruquintinib at a dose range of 0.5∼20 mg/kg suspended in the vehicle (treated group) once daily for 3 weeks. In combination studies, docetaxel (Taxotere, 5 mg/kg) or oxaliplatin (10 mg/kg) was administered to nude mouse via intravenous injection, once a week. Tumor size and body weights were measured 3 times a week. Tumor volumes (TV) were calculated using the formula: TV = (length × [width]2)/2. The percentage of tumor growth inhibition (%TGI = 100 × [1− (TV final - TVinitial for drug treated group)/(TVfinal – TVinitial for the control group)]) was used for evaluation of anti-tumor efficacy.

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\nIn vivo anti-angiogenesis analysis [1]
\nAt the end of Caki-1 anti-tumor efficacy study, the subcutaneous tumors from control- and fruquintinib-treated groups were collected and fixed in Zinc-formalin, and paraffin embedded sections were prepared. Immunohistochemistry staining of CD31 was analyzed. In each section, 5 non-necrosis areas were randomly chosen and CD31 positive area was determined by NIKON BR-3.0 software. The anti-angiogenic effect of fruquintinib was evaluated by CD31 inhibition calculated with the formula: Inhibition% = 100 × [1− (CD31 positive area/tumor area for compound treated group)/(CD31 positive area/tumor area for the control group)].

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\nHT-29 colorectal cancer xenograft model: BALB/c nude mice (6–8 weeks old) are subcutaneously implanted with 5×10⁶ HT-29 cells (suspended in 50% Matrigel/PBS) into the right flank. When tumors reach 100–150 mm³, mice are randomized into vehicle control and treatment groups (n=6/group). Fruquintinib is formulated in 0.5% carboxymethylcellulose sodium (CMC-Na) + 0.1% Tween 80 and administered orally at 3 mg/kg, 10 mg/kg, or 30 mg/kg once daily for 21 days. Tumor size is measured every 3 days with calipers, and tumor volume is calculated as length×width²×0.5 [1]
\n- A549 NSCLC xenograft model: BALB/c nude mice are subcutaneously implanted with 5×10⁶ A549 cells. When tumors reach 100–150 mm³, mice are treated with Fruquintinib 10 mg/kg oral once daily for 21 days. Survival is monitored for 60 days; tumor weight is measured at study end [1]
\n- HepG2 hepatocellular carcinoma xenograft model: BALB/c nude mice are implanted with 5×10⁶ HepG2 cells. After tumor establishment, mice are treated with Fruquintinib 10 mg/kg oral once daily for 21 days. Tumors are harvested for CD31 immunohistochemistry (to assess MVD) and western blot analysis of signaling proteins [1]
\n- Pharmacodynamic sampling: Mice bearing HT-29 xenografts are treated with Fruquintinib 10 mg/kg oral once daily for 7 days. Tumors are harvested, frozen in liquid nitrogen, and analyzed by western blot for p-VEGFR2, p-AKT, and p-ERK1/2 levels [1]

Absorption, Distribution and Excretion
At the recommended dose, the steady-state geometric mean maximum concentration (Cmax) of fruquintinib is 300 ng/mL (28%), and the area under the concentration-time curve (AUC0-24h) is 5880 ng∙h/mL (29%). Within the dose range of 1 to 6 mg (0.2 to 1.2 times the recommended dose), the Cmax and AUC0-24h of fruquintinib are dose-dependent. Steady-state of fruquintinib is reached after 14 days, with a mean AUC0-24h cumulative effect of 4 times. The median time (min, maxima) for fruquintinib to reach Cmax is approximately 2 hours (0, 26 hours). No clinically significant differences in the pharmacokinetics of fruquintinib were observed after administration of a high-fat meal (800 to 1000 calories, 50% fat).
After oral administration of 5 mg of radiolabeled fruquintinib, approximately 60% of the dose was recovered in the urine (0.5% unchanged) and 30% in the feces (5% unchanged).
The mean (standard deviation) apparent volume of distribution of fruquintinib was approximately 46 (13) L.
The apparent clearance (standard deviation) of fruquintinib was 14.8 (4.4) mL/min.

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Metabolism/Metabolites
Fruquintinib is primarily eliminated via CYP450 and non-CYP450 metabolism (i.e., sulfation and glucuronidation). CYP3A, and to a lesser extent CYP2C8, CYP2C9, and CYP2C19, are the CYP450 enzymes involved in the metabolism of fruquintinib.

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Biological Half-Life
The mean (SD) elimination half-life of fruquintinib was approximately 42 (11) hours.

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Oral bioavailability: 68% in rats (10 mg/kg orally) and 75% in dogs (5 mg/kg orally) [1]
-Plasma pharmacokinetics: In rats, oral administration of 1–30 mg/kg resulted in dose-proportional Cmax (0.7–21.5 μg/mL) and AUC₀–24h (4.8–156.2 μg·h/mL); terminal half-life (t₁/₂) = 9.6 hours [1]
-In dogs, oral administration of 5 mg/kg resulted in Cmax = 5.8 μg/mL, AUC₀–24h = 46.3 μg·h/mL, and t₁/₂ = 11.3 hours [1]
-Tissue distribution: In rats, fruquintinib was widely distributed in tissues, with the highest concentrations in the liver, kidneys, and tumors; 4 hours after administration Hourly tumor/plasma concentration ratio = 4.2 [1]
- Metabolism: Primarily metabolized in human liver microsomes via cytochrome P450 3A4 (CYP3A4); three major metabolites (M1, M2, M3) have been identified, with 20-50 times lower inhibitory efficacy against VEGFR2 than the parent drug [1]
- Excretion: In rats, the cumulative excretion rate over 72 hours was 65% (feces) and 18% (urine); 42% of fecal excretion was the parent drug [1]
- Plasma protein binding: 95-97% in human, rat, and canine plasma (equilibrium dialysis, 0.1-10 μg/mL) [1]

Hepatotoxicity
In pre-marketing clinical trials of fruquintinib, abnormal liver function was common during its use, similar to other multi-kinase inhibitors. In two large placebo-controlled trials, 39% and 44% of patients in the fruquintinib group, respectively, experienced elevated serum ALT or AST, compared to 27% and 30% in the placebo group, respectively. In the treatment group, 6% and 4% of patients, respectively, had transaminase levels exceeding five times the upper limit of normal, compared to 3% and 2% in the placebo group, respectively. Only one patient in the treatment group experienced elevated serum transaminase levels with jaundice; this patient developed ALT 270 U/L, AST 131 U/L, and total bilirubin 4.8 mg/dL 58 days after discontinuation of treatment. The delayed onset of symptoms was considered unrelated to treatment. Among the 1101 patients in the two controlled trials, no life-threatening liver injury events or liver-related deaths occurred. Therefore, no clinically significant liver injury was observed during fruquintinib treatment, but elevations in ALT, AST, and bilirubin were common during treatment, although these elevations were not always caused by fruquintinib in these patients with advanced colorectal cancer. In addition, several other VEGF receptor inhibitors have been associated with cases of acute liver injury with symptomatic jaundice (pazopanib, regorafenib, sorafenib). Since the approval and widespread use of fruquintinib, no published cases of clinically significant liver injury with jaundice have been reported. Probability score: E (Unproven but suspected rare cause of clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation There is currently no information regarding the clinical use of fruquintinib during lactation. Because fruquintinib binds to plasma proteins at a rate of up to 95%, its concentration in breast milk is likely to be low. However, the manufacturer recommends discontinuing breastfeeding during treatment with fruquintinib and for 2 weeks after the last dose.
◉ Effects on breastfed infants
As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding
The plasma protein binding rate of fruquintinib is approximately 95%.

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Acute toxicity (mice): A single oral dose of 500 mg/kg fruquintinib did not result in death or serious toxicity; 2 out of 6 mice experienced mild, transient diarrhea [1]
- Subchronic toxicity (rats, 28 days): No significant changes in body weight, food intake, or hematological/biochemical indicators (ALT, AST, BUN, creatinine) were observed at oral doses up to 30 mg/kg/day; no histopathological abnormalities were found in major organs (liver, kidney, heart, lung) [1]
- Genotoxicity: The Ames test and chromosome aberration test results were negative [1]
- No significant inhibition of CYP450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) was observed at concentrations up to 10 μM [1]

[1]. Discovery of fruquintinib, a potent and highly selective small molecule inhibitor of VEGFR 1, 2, 3 tyrosine kinases for cancer therapy. Cancer Biol Ther. 2014;15(12):1635-45.

Fruquintinib belongs to the quinazoline class of compounds. Its structure is quinazoline, with [2-methyl-3-(methylcarbamoyl)-1-benzofuran-6-yl]oxy, methoxy, and methoxy substitutions at positions 4, 6, and 7, respectively. It is a highly potent and selective inhibitor of VEGFR 1, 2, and 3 (IC50 values of 33, 0.35, and 35 nM, respectively) used to treat metastatic colorectal cancer. Fruquintinib possesses various pharmacological activities, including EC 2.7.10.1 (receptor protein tyrosine kinase) inhibitor, antitumor drug, and vascular endothelial growth factor receptor antagonist. It belongs to the quinazoline class, aromatic ether class, 1-benzofuran class, and secondary amide class. Fruquintinib is a novel small-molecule anti-VEGFR drug that targets VEGFR-1, -2, and -3, inhibiting angiogenesis. Tumor angiogenesis is one of the key biological processes that increase the oxygen and nutrient supply to cancer cells, and the VEGF/VEGFR pathway is one of the key pathways in this phenomenon. In fact, oncogene activation, loss of tumor suppressor function, and hypoxia, often caused by cancer cells, are all known to upregulate VEGF expression. Currently, there are two main approaches to combating tumor angiogenesis: one is to neutralize VEGF/VEGFR activity with monoclonal antibodies, and the other is to block VEGFR kinase activity with small molecule inhibitors. A typical example of the first approach is the VEGF-A trap antibody [bevacizumab]. Although bevacizumab can effectively inhibit the target, its clinical application is limited by drawbacks such as requiring intravenous injection, immunogenicity, and the potential to induce autoimmune diseases. For small molecule drugs, most early VEGFR inhibitors, such as sunitinib, sorafenib, regorafenib, and pazopanib, have poor selectivity, thus increasing the risk of off-target toxicity. Therefore, the emergence of fruquintinib, a new generation of VEGFR inhibitors with high kinase selectivity, demonstrates the feasibility of the small molecule inhibitor strategy. On November 8, 2023, the U.S. Food and Drug Administration (FDA) approved fruquintinib (brand name: Fruzaqla) for the treatment of adult patients with metastatic colorectal cancer (mCRC) who have previously received fluorouracil, oxaliplatin, and irinotecan chemotherapy, anti-vascular endothelial growth factor (VEGF) therapy, and (if RAS wild-type and medically appropriate) anti-epidermal growth factor receptor (EGFR) therapy. This approval is based on favorable results from the FRESCO and FRESCO-2 trials, both of which observed an increase in overall survival. Fruquintinib is an oral, small-molecule vascular endothelial growth factor receptor (VEGFR) inhibitor with potential anti-angiogenic and anti-tumor activity. After oral administration, fruquintinib inhibits VEGF-induced phosphorylation of VEGFR 1, 2, and 3, thereby inhibiting endothelial cell migration, proliferation, and survival, inhibiting microangiogenesis, and inhibiting tumor cell proliferation and tumor cell death. VEGFR expression may be upregulated in various tumor cell types. Drug Indications Fruquintinib is indicated for the treatment of adult patients with metastatic colorectal cancer (mCRC) who have previously received fluorouracil, oxaliplatin, and irinotecan chemotherapy, anti-VEGF therapy, and (if RAS wild-type and medically feasible) anti-EGFR therapy. Colorectal Cancer Treatment Mechanism of Action Fruquintinib is a small molecule kinase inhibitor that inhibits vascular endothelial growth factor receptors (VEGFR)-1, -2, and -3, with IC50 values of 33, 35, and 0.5 nM, respectively. Pharmacodynamics In vitro studies have shown that fruquintinib inhibits VEGF-mediated endothelial cell proliferation. In vitro studies have shown that fruquintinib promotes vascular endothelial cell formation, and in vivo studies have confirmed that fruquintinib inhibits tumor growth in a mouse model of colon cancer xenograft. Both in vitro and in vivo studies have demonstrated that fruquintinib inhibits VEGF-induced VEGFR-2 phosphorylation. The exposure-response relationship and pharmacodynamic response timeline of fruquintinib are unclear. At the approved recommended dose, no mean increase in the QTc interval exceeding 20 ms was observed. The VEGF/VEGFR signaling axis has been identified as an important target for developing novel cancer therapies. One of the challenges facing small-molecule VEGFR inhibitors is achieving sustained target inhibition at tolerable doses, something previously only achievable with long-acting biologics. This requires high activity (low effective drug concentration) and sufficient drug exposure at tolerable doses to maintain drug concentrations above the effective drug concentration during dosing, thereby achieving target inhibition. Fruquintinib (HMPL-013) is a small-molecule inhibitor with potent and highly selective activity against the VEGFR family and is currently in Phase II clinical trials. Phase I pharmacokinetic data analysis showed that fruquintinib achieved complete inhibition of VEGFR2 (drug concentration maintained above the concentration required for >85% inhibition of VEGFR2 phosphorylation in mice) at the maximum tolerated dose of once-daily oral administration, and this effect lasted for 24 hours. This article will present preclinical data of fruquintinib, including kinase activity and selectivity, cellular VEGFR inhibition and VEGFR-driven functional activity, inhibition of VEGFR phosphorylation in mouse lung tissue, and inhibition of tumor growth in various tumor xenograft models and patient-derived xenograft models. In addition, this article will provide pharmacokinetic and target inhibition data to elucidate the correlation between target inhibition and tumor growth inhibition. [1]

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Fruquintinib’s potent in vitro activity against VEGFR was confirmed in vivo after oral administration. Studies have shown that fruquintinib inhibits VEGF-stimulated VEGFR2 phosphorylation (p-KDR) in mouse lung tissue in an exposure-dependent manner. Following a single oral administration of 2.5 mg/kg fruquintinib in mice, p-KDR was almost completely (>85%) inhibited, and this inhibition was maintained in lung tissue for at least 8 hours. At this point, the corresponding plasma concentration of fruquintinib was 176 ng/mL (85% effective target inhibition concentration, EC85). In antitumor efficacy studies, a once-daily oral dose of approximately 2 mg/kg achieved statistically significant tumor growth inhibition in various human tumor xenograft mouse models, indicating that maintaining complete inhibition of the VEGFR2 pathway for approximately 8 hours or longer can produce significant antitumor efficacy. However, for optimal antitumor activity, it is best to maintain complete target inhibition for 24 hours/day, similar to an antibody. EC85 (176 ng/mL for fruquintinib) can be used to assess the duration of target inhibition at the recommended dose in clinical trials. Based on pharmacokinetic data from a Phase I clinical trial, the plasma concentration of the maximum tolerated dose of fruquintinib maintained at EC85 for 24 hours with a once-daily dosing regimen, suggesting that fruquintinib may provide sustained target inhibition for cancer patients. In summary, fruquintinib is a potent, highly selective, and orally effective inhibitor of VEGFR1, 2, and 3 tyrosine kinases. In vitro, fruquintinib inhibits VEGF-induced VEGFR2 phosphorylation, endothelial cell proliferation, and tubular formation, and also inhibits VEGFR2 phosphorylation in lung tissue. Fruquintinib exhibits significant tumor growth inhibitory activity in mouse human tumor xenograft models. In various PDX models, fruquintinib, when used in combination with chemotherapeutic agents, enhances tumor growth inhibition. Fruquintinib possesses equally potent inhibitory activity against both VEGFR2 and VEGFR3, potentially offering significant anti-tumor growth and metastasis efficacy. These excellent properties, coupled with its favorable pharmacokinetic and toxicity profiles, make fruquintinib a highly promising candidate for clinical investigation. [1]

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Fruquintinib is a potent, highly selective small molecule VEGFR1,2,3 tyrosine kinase inhibitor for the treatment of advanced solid tumors.[1]
- Its mechanism of action involves competitive binding to the catalytic domains of VEGFR1,2,3 to ATP, inhibiting their tyrosine kinase activity and blocking VEGF-mediated downstream signaling pathways (PI3K/AKT and MAPK/ERK), thereby inhibiting angiogenesis (the formation of new blood vessels) and suppressing tumor growth and metastasis.[1]
- Its high selectivity for the VEGFR family minimizes off-target effects, distinguishing it from multi-target kinase inhibitors with broader specificity.[1]
- Preclinical data in xenograft models of colorectal cancer, non-small cell lung cancer, and hepatocellular carcinoma showed significant antitumor efficacy and good tolerability, supporting its clinical development.[1]
- This product is an oral tablet, and the predicted human therapeutic dose range based on preclinical pharmacokinetic/pharmacodynamic calculations is 3–10 mg/day. [1]
- VEGFR2 is the main target mediating its antitumor activity because it plays a central role in VEGF-induced endothelial cell proliferation, migration, and angiogenesis, processes that are crucial for tumor progression. [1]

C21H19N3O5

393.39

393.132

C, 64.12; H, 4.87; N, 10.68; O, 20.34

1194506-26-7

44480399

White to off-white solid powder

1.3±0.1 g/cm3

600.5±55.0 °C at 760 mmHg

317.0±31.5 °C

0.0±1.7 mmHg at 25°C

1.639

3.08

1

7

5

29

579

0

O1C(C([H])([H])[H])=C(C(N([H])C([H])([H])[H])=O)C2C([H])=C([H])C(=C([H])C1=2)OC1C2=C([H])C(=C(C([H])=C2N=C([H])N=1)OC([H])([H])[H])OC([H])([H])[H]

BALLNEJQLSTPIO-UHFFFAOYSA-N

InChI=1S/C21H19N3O5/c1-11-19(20(25)22-2)13-6-5-12(7-16(13)28-11)29-21-14-8-17(26-3)18(27-4)9-15(14)23-10-24-21/h5-10H,1-4H3,(H,22,25)

6-(6,7-dimethoxyquinazolin-4-yl)oxy-N,2-dimethyl-1-benzofuran-3-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)

Solubility in Formulation 1: ≥ 0.59 mg/mL (1.50 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 5.9 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 0.59 mg/mL (1.50 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 5.9 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 0.59 mg/mL (1.50 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 5.9 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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