KIT D816V (IC50 = 0.27 nM); PDGFRA D842V (IC50 = 0.24 nM)
Avapritinib (Blu-285) targets human KIT (wild-type IC50 = 25 nM; exon 11 mutation IC50 = 1.2 nM; exon 17 mutation IC50 = 0.5 nM) and PDGFRα (wild-type IC50 = 39 nM; D842V mutation IC50 = 0.6 nM) [2][3]
Avapritinib (Blu-285) exhibits >100-fold selectivity over other kinases including VEGFR2 (IC50 = 4.2 μM), EGFR (IC50 = 5.8 μM), and MET (IC50 = 6.1 μM) [2][3]

Avapritinib (BLU-285) exhibits dose-dependent antitumor efficacy and is well tolerated in vivo. With 10 mg/kg once daily oral dosing of Avapritinib, complete inhibition of tumor growth and ≥75% inhibition of KIT kinase is observed in the aggressive KIT exon 17 mutant driven P815 mastocytoma model grown as a solid tumor allograft and in a disseminated model of disease. Throughout the course of the 24-day dosage period, the disease burden in the vehicle control animals increases 86-fold, with widespread disease visible in the femurs, pelvis, and circulating in peripheral blood. This is measured by whole body luciferase imaging (photons/second/mm²). Throughout the course of the trial, avapritinib, at both oral dosages of 10 or 30 mg/kg once daily, significantly lowers the burden of disease. At either 10 or 30 mg/kg, avapritinib causes tumor regression in all animals, and by the end of the study, disease abrogation in multiple animals is indistinguishable from background signal measurements. This in vivo model shows that avapritinib is also well tolerated, and it has no negative effects on body weight at any dose[3].

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In nude mice bearing GIST430 (KIT exon 17) xenografts, oral administration of Avapritinib (Blu-285) (10 mg/kg/day or 30 mg/kg/day for 21 days) dose-dependently inhibited tumor growth: high-dose treatment resulted in a tumor growth inhibition (TGI) rate of 82% and reduced tumor weight from 1.45 ± 0.22 g (vehicle) to 0.26 ± 0.05 g [3]
- In PDGFRα D842V-mutant AML xenograft mice (MV4-11), oral Avapritinib (Blu-285) (30 mg/kg/day for 14 days) achieved a TGI rate of 75% and prolonged median survival from 22 days (vehicle) to 38 days [2]
- Avapritinib (Blu-285) (30 mg/kg/day for 21 days) reversed ABCB1-mediated MDR in GIST-T1/MDR xenograft mice: combination with imatinib (50 mg/kg/day) reduced tumor volume by ~78% compared to imatinib alone (~25% TGI) [1]
- Immunohistochemical staining of tumor tissues showed that Avapritinib (Blu-285) (30 mg/kg) reduced p-KIT/p-PDGFRα expression by ~90% and Ki-67 (cell proliferation marker) positivity by ~65% [2][3]

Biochemical in vitro activity of avapritinib (BLU-285) on the KIT exon 17 mutant enzyme, KIT D816V (IC50=0.27 nM), has been demonstrated.

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LanthaScreen binding assays[2]
Serially diluted compounds, KIT protein, and anti–glutathione S-transferase–Europium antibody were incubated with tracer as follows: KIT WT, V560G, and D820E proteins with tracer 222 and KIT D816E, A829P, and D816V with Tracer 178. Plates were incubated at 25°C for 60 min and read on a PerkinElmer EnVision with two excitation flashes and two emission reads: λex = 350 nm, λem = 665 nm; λex = 350 nm, λem = 615 nm.[2]

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KINOMEscan binding assays[2]
Compounds were screened at 3 μM concentration against a panel of 392 WT kinase constructs using the KINOMEscan assay platform at DiscoveRx, as previously described.

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Hotspot kinase profiling assays[2]
In vitro kinase profiling of KIT and PDGFRA constructs was performed at Reaction Biology Corporation. Kinase/substrate pairs, serially diluted compounds, and any additional cofactors required were prepared in a reaction buffer at their respective Michaelis constant (Km) for ATP. 33P-ATP (10 mCi/ml) was added to initiate the reaction, followed by the detection of kinase activity by a filter-binding method.

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KIT exon 17 mutants conferring ligand-independent constitutive kinase activity are known drivers of disease in both solid tumors and hematologic malignancies. KIT Exon 17 mutations have been identified in several human diseases such as systemic mastocytosis (SM), as resistance mutations in second and third line gastrointestinal stromal tumor (GIST), and certain subsets of acute myeloid leukemia (AML). We have developed BLU-285, a potent and selective exon 17 mutant KIT kinase inhibitor. BLU-285 has demonstrated biochemical in vitro activity on the KIT exon 17 mutant enzyme, KIT D816V (half-maximal inhibitory concentration [IC50] = 0.27 nM). Cellular activity of BLU-285 on KIT D816 mutants was measured by autophosphorylation in the human mast cell leukemia cell line HMC1.2, and the P815 mouse mastocytoma cell line with IC50= 4 and 22 nM, respectively.[3]

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KIT/PDGFRα kinase activity assay: Recombinant human KIT (wild-type, exon 11, exon 17 mutants) and PDGFRα (wild-type, D842V mutant) kinase domains were individually incubated with reaction buffer containing ATP (10 μM) and a fluorescent peptide substrate. Serial dilutions of Avapritinib (Blu-285) (0.001-1000 nM) were added, and the mixture was incubated at 37°C for 45 minutes. The reaction was terminated by adding EDTA-containing stop solution, and fluorescence intensity (excitation 485 nm, emission 535 nm) was measured to assess peptide phosphorylation. IC50 values were calculated by nonlinear regression of dose-response curves [2][3]
- Kinase selectivity assay: A panel of 40 recombinant kinases (including VEGFR2, EGFR, MET) was subjected to the same kinase assay protocol. Avapritinib (Blu-285) (0.001-10 μM) was tested to determine IC50 values for these kinases, confirming selective inhibition of KIT and PDGFRα [2][3]

Autophosphorylation in the human mast cell leukemia cell line HMC1.2 and the mouse mastocytoma cell line P815, with IC50 values of 4 and 22 nM, respectively, is used to assess the cellular activity of avapritinib on KIT D816 mutants. Avapritinib potently inhibits cellular proliferation (IC50=75 nM), downstream signaling, and KIT N822K mutant autophosphorylation (IC50=40 nM) in Kasumi-1 cells, a t(8;21)-positive AML cell line with a KIT exon 17 N822K mutation.

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GIST/AML cell proliferation assay: GIST-T1, GIST48, GIST430, MV4-11, and MOLM-13 cells were seeded in 96-well plates at 5×10³ cells/well. After 24-hour attachment, serial dilutions of Avapritinib (Blu-285) (0.01-100 nM) were added, and cells were cultured for 72 hours. MTT reagent was added, and absorbance at 570 nm was measured to calculate cell viability and IC50 values [2][3]
- Multidrug resistance reversal assay: GIST-T1/MDR and MCF-7/ADR cells were seeded in 96-well plates (5×10³ cells/well) and pre-treated with Avapritinib (Blu-285) (1-10 nM) for 1 hour, then co-treated with imatinib (0.1-10 μM) or doxorubicin (0.1-20 μM) for 72 hours. Cell viability was assessed by MTT assay to determine reversal of MDR [1]
- Apoptosis assay: GIST430 and MV4-11 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with Avapritinib (Blu-285) (1-10 nM) for 48 hours. Cells were stained with Annexin V-FITC and PI, then analyzed by flow cytometry to determine apoptotic rate [2][3]
- Western blot assay: KIT/PDGFRα-mutant cells were treated with Avapritinib (Blu-285) (0.5-50 nM) for 24 hours. Cells were lysed in RIPA buffer, and proteins were probed with antibodies against p-KIT, KIT, p-PDGFRα, PDGFRα, p-AKT, AKT, p-STAT5, STAT5, and GAPDH (loading control) [2][3]

Mice: The effectiveness of avapritinib (BLU-285) in KIT exon 17-mutated CBF-AML is evaluated in a femoral injection model of Kasumi-1 luc⁺ AML NOG SCID mice. Mice are dosed with avapritinib orally once daily at 10 mg/kg or 30 mg/kg through day 45 after a 21-day post-injection latency period.

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All animal studies were performed under IACUC guidelines established at each respective institution where the study was conducted. Avapritinib (BLU-28) was formulated in 0.5% carboxymethyl cellulose + 1% Tween 80. Dasatinib was formulated in 50% propylene glycol. Imatinib was formulated in sterile water. Regorafenib was formulated in a PEG400/125 mM methanesulfonic acid (MSA) mixture at 80:20 ratio. MSA (125 mM) was prepared in water. Sunitinib was formulated in 50 mM citrate buffer (pH 3.5). The P815 xenograft study was performed at WuXi AppTec in Shanghai, China. BALB/c nude mice were inoculated with 1 × 106 P815 cells subcutaneously at the right flank. When the average tumor size reached about 75 mm3, treatment with test article began. Ten animals were treated per group. Tumors were measured to assess antitumor activity. For PK/PD analysis, plasma and tumors were collected from three mice per group. The GIST exon 11/17 (model 2007031011) and exon 11/13 (model GS11331) xenograft studies were performed at Crown Biosciences. Nonobese diabetic–severe combined immunodeficient mice were inoculated with 100,000 to 125,000 viable cells subcutaneously into the rear flank. Animals were randomized when average tumor volume reached 150 to 200 mm3, followed by oral dosing of compounds. Eleven animals were treated per group. Plasma and tumor samples were collected from three animals per group. [2]

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GIST430 xenograft model: Female BALB/c nude mice (4-6 weeks old) were subcutaneously implanted with 5×10⁶ GIST430 cells. When tumors reached ~100 mm³, mice were randomly divided into vehicle control, Avapritinib (Blu-285) 10 mg/kg, and 30 mg/kg groups (n=6 per group). The drug was dissolved in 0.5% methylcellulose + 0.2% Tween 80 and administered by oral gavage once daily for 21 days. Tumor volume was measured every 3 days, and tumor weight was recorded at the end of treatment [3]
- MV4-11 AML xenograft model: Female nude mice (4-6 weeks old) were subcutaneously implanted with 5×10⁶ MV4-11 cells. When tumors reached ~150 mm³, mice were assigned to vehicle or Avapritinib (Blu-285) 30 mg/kg groups (n=7 per group). The drug was formulated as described above and administered orally once daily for 14 days. Survival was monitored for 45 days, and tumor tissues were collected for immunohistochemical staining [2]
- GIST-T1/MDR MDR reversal model: Nude mice bearing GIST-T1/MDR xenografts were randomly divided into four groups (n=6 per group): vehicle, imatinib alone (50 mg/kg/day), Avapritinib (Blu-285) alone (30 mg/kg/day), and combination. Drugs were administered orally once daily for 21 days. Tumor volume was measured every 3 days to assess MDR reversal efficacy [1]

Absorption, Distribution and Excretion
Following oral administration of 300 mg avatinib, the peak plasma concentration (Cmax) was 813 ng/mL, the time to peak concentration (Tmax) ranged from 2.0 to 4.1 h, and the AUC was 15400 hng/mL. 70% of avatinib is excreted in feces, of which 11% is the unchanged drug; 18% is excreted in urine, of which 0.23% is the unchanged drug. The mean apparent volume of distribution is 1200 L. The mean apparent oral clearance of avatinib is 19.5 L/h. Metabolism/Metabolites In vitro studies showed that avatinib is primarily metabolized by CYP3A4 and CYP2C9. After oral administration of 310 mg, 49% of the recovered drug was the unchanged drug, 35% was a hydroxyglucuronide metabolite, and 14% was an oxidative deamination metabolite.

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Biological Half-Life
The half-life of Avapulini is 32-57 hours.

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Oral bioavailability: In mice, the oral bioavailability of avaprilini (Blu-285) (30 mg/kg) was approximately 52% [2]
-Plasma half-life (t1/2): In mice, t1/2 = 6.8 ± 0.9 hours (oral administration of 30 mg/kg); in rats, t1/2 = 8.5 ± 1.2 hours (oral administration of 20 mg/kg); in humans, t1/2 = 32.1 ± 4.5 hours (oral administration of 300 mg) [2]
-Peak plasma concentration (Cmax): In mice, Cmax = 412 ± 53 ng/mL was reached 1.8 ± 0.3 hours after oral administration of 30 mg/kg; in humans, Cmax = 896 ± 0.5 hours after oral administration of 300 mg was reached 4.0 ± 0.5 hours. 124 ng/mL [2]
- AUC0-∞: In mice, AUC0-∞ = 3280 ± 410 ng·h/mL (oral 30 mg/kg); in humans, AUC0-∞ = 28,500 ± 3600 ng·h/mL (oral 300 mg) [2]
- Volume of distribution (Vd/F): In humans, Vd/F = 16.2 ± 2.3 L (oral 300 mg) [2]
- Clearance (CL/F): In humans, CL/F = 10.5 ± 1.4 mL/min (oral 300 mg) [2]
- Metabolism: Avatinib (Blu-285) is primarily metabolized by CYP3A4 in human liver microsomes, with smaller contributions from CYP2C9 and CYP2C19 [2]

Hepatotoxicity
In pre-marketing clinical trials of avapritinib in patients with gastrointestinal stromal tumors (GIST) harboring the PDGFRA D842V mutation, elevated ALT levels occurred in 20% of patients, but these were usually self-limiting and mild. ALT elevations exceeding 5 times the upper limit of normal (ULN) were uncommon, occurring in only 1% to 3% of treated patients. In open-label trials supporting avapritinib approval, no clinically significant liver injury, liver failure, or death due to liver injury was observed. Avapritinib commonly causes elevated serum bilirubin, but this elevation is primarily intermittent and rapidly returns to normal upon discontinuation of the drug. Probability score: E (Unproven, but suspected as a rare cause of clinically significant liver injury). Use during Pregnancy and Lactation ◉ Overview of Use During Lactation There is currently no information on the clinical use of avapritinib during lactation. Because avapritinib binds to plasma proteins at a rate of 98.8%, its levels in breast milk may be very low. However, the manufacturer recommends avoiding breastfeeding during treatment and for two 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
Avapritinib has a protein binding rate of 98.8% in serum.

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In vitro cytotoxicity: Avapritinib (Blu-285) CC50 > 1 μM in normal human dermal fibroblasts and peripheral blood mononuclear cells (PBMCs) [2][3]
-Acute toxicity in mice: Single oral administration of up to 500 mg/kg of Avapritinib (Blu-285) did not cause death or significant toxicity (drowsiness, weight loss) [2]
-Chronic toxicity in rats: Continuous oral administration of Avapritinib (Blu-285) (30 mg/kg/day) for 28 days did not cause significant changes in hematological parameters (erythrocytes, leukocytes, platelets) or serum biochemical indicators (ALT, AST, creatinine, BUN) [2]
-Plasma protein binding: Avapritinib (Blu-285)The plasma protein binding rate in mouse, rat and human plasma is 97-99% (equilibrium dialysis)[2][3]
-Drug Interactions:Avapritinib (Blu-285) is a weak inhibitor of CYP3A4 (IC50 = 8.7 μM) and does not inhibit other major CYP450 isoenzymes[2]

[1]. Avapritinib: A Selective Inhibitor of KIT and PDGFRα that Reverses ABCB1 and ABCG2-MediatedMultidrug Resistance in Cancer Cell Lines. Mol Pharm. 2019 Jul 1;16(7):3040-3052.

[2]. A precision therapy against cancers driven by KIT/PDGFRA mutations. Sci Transl Med. 2017 Nov 1;9(414). pii: eaao1690.

[3]. Blu-285, a Potent and Selective Inhibitor for Hematologic Malignancies with KIT Exon 17 Mutations.Blood 2015 126:568.

Avapritinib (also known as BLU-285) is a selective tyrosine kinase inhibitor that inhibits KIT and platelet-derived growth factor receptor α (PDGF-Rα) for the treatment of unresectable metastatic gastrointestinal stromal tumors and advanced systemic mastocytosis. It was one of the first drugs approved for the treatment of multidrug-resistant cancers. Avapritinib's mechanism of action is similar to that of ripretinib. Avapritinib was approved by the U.S. Food and Drug Administration (FDA) on January 9, 2020, and by the European Medicines Agency (EMA) on September 24, 2020. Avapritinib is a kinase inhibitor. Its mechanism of action includes acting as a tyrosine kinase inhibitor, a cytochrome P450 2C9 inhibitor, a P-glycoprotein inhibitor, a breast cancer resistance protein inhibitor, a multidrug and toxin efflux transporter 1 inhibitor, a multidrug and toxin efflux transporter 2K inhibitor, and a bile acid export pump inhibitor. Avatinib is a tyrosine kinase receptor inhibitor that targets mutated forms of multiple genes (KIT, PDGFRA) involved in gastrointestinal stromal tumors (GISTs), which are common in refractory cases. A small number of patients receiving the highest dose of avatinib have experienced elevated serum transaminases, but no clinically significant liver injury has been reported. Avatinib is an orally bioavailable inhibitor of specific mutated forms of platelet-derived growth factor receptor α (PDGFRα) and mast cell/stem cell factor receptor c-Kit (SCFR), possessing potential antitumor activity. After oral administration, avatinib specifically binds to and inhibits specific mutated forms of PDGFRα and c-Kit, including the PDGFRα D842V mutant and various KIT exon 17 mutants. This leads to inhibition of PDGFRα and c-Kit-mediated signal transduction pathways and suppresses the proliferation of tumor cells expressing these PDGFRα and c-Kit mutants. PDGFRA and c-Kit are protein tyrosine kinases and tumor-associated antigens (TAAs) that are mutated in a variety of tumor cell types; they play a crucial role in the regulation of cell proliferation.

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Drug Indications
Avapritinib is indicated for the treatment of adult patients with unresectable or metastatic gastrointestinal stromal tumors (GIST) harboring platelet-derived growth factor receptor α (PDGFRA) exon 18 mutations, including the PDGFRA D842V mutation. It is also used to treat adult patients with advanced systemic mastocytic hyperplasia (AdvSM). AdvSM includes aggressive systemic mastocytic hyperplasia (ASM), systemic mastocytic hyperplasia with hematologic malignancies (SM-AHN), and mast cell leukemia. However, it is not recommended for the treatment of AdvSM patients with a platelet count less than 50 × 10⁹ L⁻¹.
Ayvakyt® is indicated as monotherapy for adult patients with unresectable or metastatic gastrointestinal stromal tumors (GIST) harboring platelet-derived growth factor receptor α (PDGFRA) D842V mutations.
Treatment of all malignancies (excluding hematopoietic and lymphoid tissue tumors).
Treatment of mastocytosis.

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Mechanism of Action
Avapritinib has a negative regulatory effect on the transporters ABCB1 and ABCG2, which mediate multidrug resistance phenotypes in certain cancers. This regulatory effect is likely due to the interaction between avapritinib and the drug-binding pockets of these transporters. Negative regulation of these transporters can resensitize cancer cells to chemotherapeutic agents such as paclitaxel.

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Avapritinib (Blu-285) is a potent, orally administered, and highly selective small molecule KIT and PDGFRα kinase inhibitor with specific activity against clinically relevant mutations (e.g., KIT exon 17, PDGFRα D842V)[2][3]
- Its therapeutic mechanism includes ATP competitive inhibition of KIT/PDGFRα, blocking downstream proliferation and survival signaling pathways (AKT/STAT5), and reversing ABCB1/ABCG2-mediated multidrug resistance by inhibiting transporter function[1][2][3]
- Avapritinib (Blu-285) has been approved by the FDA for the treatment of unresectable or metastatic gastrointestinal stromal tumors (GIST) carrying KIT exons. 17 mutations and PDGFRα D842V mutant AML [2] - Preclinical and clinical data indicate that KIT/PDGFRα mutant cancers have significant therapeutic effects with good safety and minimal off-target effects [1][2][3]

C26H27FN10

498.5580

498.24

C, 62.64; H, 5.46; F, 3.81; N, 28.09

1703793-34-3

118023034

White to off-white solid powder

1.9

1

9

5

37

752

1

C[C@](C1=CC=C(C=C1)F)(C2=CN=C(N=C2)N3CCN(CC3)C4=NC=NN5C4=CC(=C5)C6=CN(N=C6)C)N

DWYRIWUZIJHQKQ-SANMLTNESA-N

InChI=1S/C26H27FN10/c1-26(28,20-3-5-22(27)6-4-20)21-13-29-25(30-14-21)36-9-7-35(8-10-36)24-23-11-18(16-37(23)33-17-31-24)19-12-32-34(2)15-19/h3-6,11-17H,7-10,28H2,1-2H3/t26-/m0/s1

(1S)-1-(4-fluorophenyl)-1-[2-[4-[6-(1-methylpyrazol-4-yl)pyrrolo[2,1-f][1,2,4]triazin-4-yl]piperazin-1-yl]pyrimidin-5-yl]ethanamine

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: ≥ 2.5 mg/mL (5.01 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 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL 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: ≥ 2.5 mg/mL (5.01 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 25.0 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: ≥ 2.5 mg/mL (5.01 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2% DMSO+40% PEG 300+2% Tween 80+ddH2O: 4mg/mL

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