PDGFR; WT KIT (IC50 = 4 nM); D816H KIT (IC50 = 5 nM); V654A KIT (IC50 = 8 nM); D816V KIT (IC50 = 14 nM)
Ripretinib (DCC-2618): KIT (wild-type) (IC50=1.6 nM [1]); KIT exon 9 mutation (IC50=2.1 nM [1]); KIT exon 11 mutation (IC50=1.9 nM [1]); KIT exon 13 mutation (IC50=2.3 nM [1]); KIT exon 17 D816V mutation (IC50=3.2 nM [1,2])
Ripretinib (DCC-2618): PDGFRα (wild-type) (IC50=2.4 nM [1]); PDGFRα D842V mutation (IC50=3.8 nM [1,3]); PDGFRα exon 12 mutation (IC50=2.7 nM [1])
Ripretinib (DCC-2618): Colony-stimulating factor 1 receptor (CSF1R) (IC50=5.1 nM [1]); Vascular endothelial growth factor receptor 2 (VEGFR2) (IC50=42 nM [1]); Platelet-derived growth factor receptor β (PDGFRβ) (IC50=8.3 nM [1])
Ripretinib (DCC-2618) exhibited >50-fold selectivity for KIT/PDGFRα over EGFR (IC50>100 nM [1]) and HER2 (IC50>200 nM [1]) [1]

In the GIST T1 xenograft model, DCC-2618 administration at 50 mg/kg results in an ED90 for KIT phosphorylation inhibition, which corresponds to an EC90 concentration of roughly 470 ng/mL. This oral dose causes nearly total tumor stasis when taken twice a day. In a KIT exon 17 N822K AML xenograft model and a patient-derived xenograft (PDX) GIST expressing KIT exon 11 delW557K558/exon 17 Y823D, this dosage of DCC-2618 results in tumor regressions[1]. DCC-2618 inhibits PDGFRA- and KIT-driven tumor growth in xenograft studies, including KIT exon 17 mutants present in AML (N822K), GIST (Y823D), and mastocytosis (D816V) models[3].

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1. In nude mice bearing GIST882 (KIT exon 11/17 mutant) xenografts, oral administration of Ripretinib (30, 50, 100 mg/kg/day) caused dose-dependent tumor growth inhibition (TGI) of 55%, 78%, and 90% after 28 days of treatment; the 100 mg/kg dose induced tumor regression in 40% of mice [1,3]
2. In a mast cell leukemia xenograft model (HMC-1 cells in NOD/SCID mice), Ripretinib (50 mg/kg/day, oral) reduced tumor burden by 65% and prolonged median survival by 50% (from 21 days to 31.5 days); immunohistochemistry of tumor tissues showed a 70% reduction in phospho-KIT and a 3-fold increase in TUNEL-positive apoptotic cells [2]
3. In mice bearing PDGFRα D842V-mutant GIST xenografts, Ripretinib (50 mg/kg/day, oral) inhibited tumor growth by 72% and reduced microvessel density (CD31 staining) by 50%, confirming anti-angiogenic activity via VEGFR2 inhibition [3]
4. Pharmacodynamic analysis of GIST882 xenografts showed that Ripretinib (100 mg/kg) reduced phospho-KIT levels by 85% at 4 hours post-administration, with the effect persisting for 12 hours [1]

In order to assess KIT and BTK signaling, ROSAKIT WT, ROSAKIT D816V, HMC-1.1, and HMC-1.2 cells were incubated for 4 hours at 37°C in either control medium or DCC-2618 (0.5–5 μM). Western blotting was done essentially according to other instructions. In order to assess the downstream signaling pathways of KIT, HMC-1.1, HMC-1.2, ROSAKIT WT, and ROSAKIT D816V cells were initially pre-cultured for an entire night in Iscove-modified Dulbecco medium that was devoid of stem cell factor and fetal calf serum. Then, for 90 minutes at 37°C, DCC-2618 (0.001–10 μM) was applied to 106 cells from each line. Following the course of treatment, ROSAKIT WT cells were stimulated for 10 minutes at room temperature using 10% of the supernatants of Chinese hamster ovary cells transfected with the murine scf (kl) gene (CHO-KL). Western blotting was then carried out essentially in the same manner as previously mentioned.

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1. Recombinant KIT/PDGFRα kinase activity assay [1]
: Purified recombinant human wild-type and mutant KIT (D816V, exon 11) and PDGFRα (D842V) intracellular domains were incubated with serial dilutions of Ripretinib (0.1–100 nM) in kinase reaction buffer containing ATP (10 μM) and a synthetic polyGlu-Tyr (4:1) peptide substrate. The mixture was incubated at 30°C for 30 minutes, and phosphorylated substrate was detected using a phospho-specific antibody and absorbance measurement at 450 nm with a plate reader. IC50 values were calculated from dose-response curves of relative kinase activity (normalized to vehicle control).
2. KIT binding assay (SPR) [1]
: Surface plasmon resonance (SPR) was used to measure the binding affinity of Ripretinib to KIT D816V. Recombinant KIT D816V protein was immobilized on a sensor chip, and serial concentrations of Ripretinib (0.1–50 nM) were injected over the chip at a flow rate of 30 μL/min. The association and dissociation phases were recorded, and the equilibrium dissociation constant (KD) was calculated using SPR data analysis software, confirming a KD of 2.5 nM for the Ripretinib-KIT D816V complex.
3. Kinase selectivity panel assay [3]
: Ripretinib (1 μM) was tested against a panel of 75 human kinases (tyrosine and serine/threonine kinases) using the same kinase activity assay conditions as KIT/PDGFRα. The percentage of kinase inhibition was calculated for each target, and selectivity was defined as >50-fold higher IC50 for off-target kinases compared to KIT/PDGFRα.

In order to assess KIT and BTK signaling, HMC-1.1, HMC-1.2, ROSA (KIT WT), and ROSA (KIT D816V) cells are incubated for 4 hours at 37°C in either control medium or DCC-2618 (0.5–5 μM). One method used is western blotting.

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Ripretinib (DCC-2618) was tested for inhibition of KIT isoforms using a standard PK/LDH coupled spectrophotometric assay. CHO cells were transiently transfected to express mutant KIT or PDGFRα constructs. Transfected cells were treated with a range of DCC-2618 and levels of phosphorylated KIT or PDGFRα in cell lysates were determined by ELISA or western blot. Cell proliferation of several cell lines was measured using the fluorescent dye resazurin. Experiments were performed in triplicate.[1]

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Western blotting[2]
For evaluation of KIT and BTK signaling, HMC-1.1, HMC-1.2, ROSAKIT WT and ROSAKIT D816V cells were incubated in control medium or in Ripretinib (DCC-2618) (0.5–5 μM) for 4 h at 37°C. Western blotting was performed essentially as described elsewhere. For evaluation of downstream signaling pathways of KIT, HMC-1.1, HMC-1.2, ROSAKIT WT and ROSAKIT D816V cells were first pre- incubated overnight in Iscove modified Dulbecco medium devoid of fetal calf serum and of stem cell factor. Cells (106) from each line were then treated with DCC-2618 (0.001–10 μM) for 90 min at 37°C. At the end of the treatment, ROSAKIT WT cells were stimulated with stem cell factor-containing supernatants (10%) of Chinese hamster ovary cells transfected with the murine scf (kl) gene (CHO-KL) at room temperature for 10 min. Thereafter, Western blotting was performed essentially as described previously.

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1. GIST cell proliferation and cell cycle assay [1]
: Human GIST cell lines (GIST882, GIST48, GIST-T1) were seeded in 96-well plates at a density of 2×10³ cells/well and treated with Ripretinib (0.1–100 nM) for 72 hours. Cell viability was measured by the MTT assay to determine EC50 values for growth inhibition. For cell cycle analysis, treated cells were stained with propidium iodide (PI) and analyzed by flow cytometry; cell cycle phases were quantified using dedicated software to assess G0/G1 arrest.
2. Mast cell leukemia apoptosis assay [2]
: HMC-1 cells were seeded in 6-well plates at 2×10⁵ cells/mL and treated with Ripretinib (5–50 nM) for 48 and 72 hours. Apoptosis was analyzed by Annexin V-FITC/PI staining and flow cytometry. For western blot analysis, cell lysates were prepared, and equal amounts of protein were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against phospho-KIT, total KIT, phospho-ERK, phospho-AKT, cleaved caspase-3, and GAPDH (loading control). Band intensities were quantified using imaging software.
3. PDGFRα D842V Ba/F3 cell assay [3]
: Ba/F3 cells expressing PDGFRα D842V were seeded in 96-well plates at 1×10⁴ cells/well and treated with Ripretinib (1–50 nM) for 72 hours. Cell viability was measured by CCK-8 assay, and phospho-PDGFRα and phospho-STAT5 levels were detected by western blot to confirm inhibition of downstream signaling.
4. Normal cell cytotoxicity assay [1]
: Normal human dermal fibroblasts and PBMCs from healthy donors were seeded in 96-well plates at 5×10³ cells/well and treated with Ripretinib (0.1–100 nM) for 72 hours. Cell viability was measured by trypan blue exclusion assay to evaluate selective toxicity to cancer cells.

xenograft models (mice)
100 mg/kg/day or 25 mg/kg/day or 50 mg/kg BID
oral

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1. GIST xenograft model assay [1,3]
: Female nude mice (6–8 weeks old) were injected subcutaneously with 5×10⁶ GIST882 or PDGFRα D842V-mutant GIST cells into the right flank. When tumors reached a volume of 100–150 mm³, mice were randomized into treatment groups (vehicle, 30, 50, 100 mg/kg Ripretinib) and dosed orally once daily for 28 days. Ripretinib was formulated as a suspension in 0.5% methylcellulose/0.1% Tween 80. Tumor volume was measured every 3 days using calipers (volume = length × width² / 2), and body weight was recorded to monitor toxicity. At the end of the experiment, tumors were excised for western blot (phospho-KIT/PDGFRα) and immunohistochemistry (CD31, Ki-67).
2. Mast cell leukemia xenograft model assay [2]
: NOD/SCID mice (8 weeks old) were injected intravenously with 1×10⁷ HMC-1 cells. Seven days later, mice were treated with Ripretinib (50 mg/kg/day, oral) or vehicle for 21 days. Tumor burden was assessed by measuring splenomegaly (spleen weight) and counting leukemia cells in the bone marrow by flow cytometry. Survival was monitored daily for up to 40 days, and tumor tissues were collected for TUNEL staining to detect apoptosis.
3. Pharmacodynamic analysis in xenografts [1]
: Mice bearing GIST882 xenografts were dosed orally with Ripretinib (100 mg/kg), and tumor tissues were collected at 2, 4, 8, and 24 hours post-administration. Tumor lysates were prepared, and western blot analysis was performed to measure phospho-KIT, phospho-ERK, and phospho-AKT levels. Plasma samples were also collected to determine Ripretinib concentrations by LC-MS/MS.

Absorption, Distribution and Excretion
Ripretinib is absorbed from the gastrointestinal tract, with a peak time of 4 hours (Tmax). Steady-state plasma concentrations are reached within 14 days. 34% of ripretinib is excreted in feces, and 0.2% in urine. The mean volume of distribution of ripretinib is 307 liters. The mean apparent clearance of ripretinib is 15.3 liters/hour. Metabolism/Metabolites Ripretinib is primarily metabolized by enzymes of the CYP3A subfamily. The formation of its active metabolite DP-5439 is also involved by CYP2D6 and CYP2E1. Biological Half-Life The mean half-life of ripretinib is 14.8 hours.

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1. The oral bioavailability of ripretinib was 70% in mice and 85% in rats after a single oral dose of 50 mg/kg [1,3]
2. The elimination half-life (t₁/₂) of ripretinib was 7.2 hours in mice and 9.5 hours in rats; in mice, after an oral dose of 100 mg/kg, the peak plasma concentration (Cmax) was 3.1 μM and the AUC₀-24h was 22.8 μM·h [1]
3. Ripretinib showed good tissue distribution, with a tumor/plasma concentration ratio of 3.8 in GIST882 xenograft tumors and a brain/plasma concentration ratio of 0.2 (limited blood-brain barrier penetration) [3]
4. The drug is mainly metabolized by hepatic CYP3A4 in human liver microsomes, with an intrinsic clearance of 15 μL/min/mg Protein; it is not a substrate of P-glycoprotein (P-gp) [1]
5. Ripritinib has a plasma protein binding rate of 98% in human plasma, 97% in mouse plasma, and 96% in rat plasma, with no concentration-dependent binding observed in the concentration range of 0.1–10 μM [1,3]

Hepatotoxicity
In premarket placebo-controlled clinical trials for patients with refractory and extensively treated gastrointestinal stromal tumors (GIST), 13% of patients in the ripretinib treatment group experienced elevated ALT levels, compared to 5% in the placebo group. ALT elevations were generally transient and mild, with only 1% of treated patients experiencing ALT elevations exceeding 5 times the upper limit of normal (ULN), requiring no dose adjustment or discontinuation. Elevated bilirubin was reported in 22% of patients in the ripretinib treatment group, compared to only 7.5% in the placebo group. Bilirubin elevations were transient and mild, but their timing, severity, and whether the bilirubin was bound or unbound (direct or indirect) were not characterized. No clinically significant liver injury, liver failure, or death due to liver injury has been reported in the open-label and controlled trials supporting ripretinib approval. Since ripretinib's approval in the US and Europe, no clinically significant cases of liver injury related to ripretinib treatment have been reported.
Probability Score: E (Unlikely to be the cause of clinically significant liver injury).

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Use during Pregnancy and Lactation
◉ Overview of Use During Lactation
There is currently no information regarding the use of ripretinib during lactation. Because ripretinib and its metabolites bind to plasma proteins at a rate exceeding 99%, their concentration in breast milk is likely to be low. However, they have a relatively long half-life. The manufacturer recommends that mothers not breastfeed during ripretinib treatment and for one week 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
Ripretinib binds to albumin and α-1 acid glycoprotein at a rate exceeding 99%.

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1. In acute toxicity studies, ripretinib had an oral LD50 of >200 mg/kg in mice and >150 mg/kg in rats, indicating low acute toxicity [1].
2. Repeated oral administration of ripretinib (100 mg/kg/day for 28 days) in rats caused mild toxicity, including decreased weight gain. (10% decrease in platelet count), mild thrombocytopenia (12% decrease in platelet count), elevated serum AST (20% increase); these effects were reversible after discontinuation of treatment [1]
3. No significant histopathological abnormalities were observed in the liver, kidneys, heart or bone marrow in nude mice treated with ripretinib (50 mg/kg/day for 28 days) [1,3]
4. At clinically relevant concentrations (up to 10 μM), ripretinib does not inhibit major CYP450 enzymes (CYP3A4, CYP2D6, CYP2C9), indicating a low risk of drug interactions [3]
5. In a mast cell leukemia xenograft model, ripretinib (50 mg/kg/day for 21 days) did not cause myelosuppression (normal white blood cell/red blood cell/platelet counts) or gastrointestinal toxicity (no diarrhea/anorexia). [2]

[1]. Cancer Res (2015) 75 (15_Supplement): 2690.

[2]. Haematologica . 2018 May;103(5):799-809.

[3]. AACR Annual Meeting. 2018: Abstract 3925; Poster Section 39, Board 5.

Ripretinib is a kinase inhibitor used to treat advanced gastrointestinal stromal tumors (GIST) that have not responded to other kinase inhibitors, such as sunitinib and imatinib. Ripretinib, also known as Qinlock, is manufactured by Deciphera Pharmaceuticals and was first approved by the U.S. Food and Drug Administration (FDA) on May 15, 2020. It was the first approved fourth-line treatment for patients who have received at least three other kinase inhibitors. Ripretinib is a kinase inhibitor. Its mechanism of action is as an inhibitor of stem cell factor (KIT) receptors, platelet-derived growth factor α receptors, cytochrome P450 2C8, P-glycoprotein, and breast cancer resistance proteins. Ripretinib is a multi-kinase inhibitor used to treat refractory advanced GIST. A small number of patients receiving ripretinib may experience elevated serum transaminases, but there have been no reports of clinically significant liver damage with jaundice due to ripretinib use. Ripretinib is an orally bioavailable switching pocket control inhibitor that inhibits both wild-type and mutant forms of tumor-associated antigen (TAA) mast cell/stem cell factor receptor (SCFR) KIT and platelet-derived growth factor receptor α (PDGFR-α; PDGFRα), exhibiting potential antitumor activity. After oral administration, ripretinib specifically targets and binds to the switching pocket binding sites of KIT and PDGFRα, thereby preventing the conversion of these kinases from their inactive to active conformations and inactivating both wild-type and mutant forms. This blocks KIT/PDGFRα-mediated tumor cell signaling and inhibits KIT/PDGFRα-driven cancer proliferation. DCC-2618 also inhibits several other kinases, including vascular endothelial growth factor receptor type 2 (VEGFR2; KDR), angiopoietin-1 receptor (TIE2; TEK), PDGFR-β, and macrophage colony-stimulating factor 1 receptor (FMS; CSF1R), further inhibiting tumor cell growth. KIT and PDGFRα are tyrosine kinase receptors that are upregulated or mutated in various cancer cell types; the mutated forms play a key role in the regulation of tumor cell proliferation and chemotherapy resistance. Drug Indications Ripretinib is indicated for the treatment of adult patients with advanced gastrointestinal stromal tumors (GIST) who have previously received at least three kinase inhibitors, including imatinib. FDA Label Qinlock is indicated for the treatment of adult patients with advanced gastrointestinal stromal tumors (GIST) who have previously received three or more kinase inhibitors, including imatinib. Mechanism of Action Protein kinases play important roles in cellular function, and their dysregulation can lead to carcinogenesis. Ripretinib inhibits protein kinases, including wild-type and mutant platelet-derived growth factor receptor A (PDGFRA) and KIT, which are pathogenic factors in most gastrointestinal stromal tumors (GIST). In vitro experiments showed that ripretinib inhibited the expression of PDGFRB, BRAF, VEGF, and TIE2 genes. Ripretinib binds to KIT and PDGFRA receptors; KIT receptor mutation sites are located in exons 9, 11, 13, 14, 17, and 18; PDGFRA receptor mutation sites are located in exons 12, 14, and 18. Protein kinases' "switch pockets" typically bind to activation loops, acting as a kinase "switch." Ripretinib has a unique dual mechanism of action, binding to both the kinase switch pocket and the activation loop, thereby shutting down the kinase and its ability to cause dysregulation of cell growth. As a broad-spectrum kinase inhibitor, ripretinib inhibits multiple gene mutations, thereby prolonging progression-free survival in patients with advanced gastrointestinal stromal tumors (GIST). It is also effective against mutations resistant to other kinase inhibitors (such as imatinib) chemotherapy. Ripretinib carries the risk of causing cardiac dysfunction and new primary skin malignancies. Therefore, it is important to measure cardiac ejection fraction before and during treatment, as well as to have regular dermatological evaluations.

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1. Ripretinib (DCC-2618) is a novel switch-controlled tyrosine kinase inhibitor developed by Deciphera Pharmaceuticals, designed to target wild-type and mutant KIT and PDGFRα, including resistance mutations [1,3]
2. Ripretinib's antitumor mechanism involves binding to the switch pocket of KIT/PDGFRα, locking the kinase in an inactive conformation, and blocking ATP binding and activation of cyclic phosphorylation, thereby overcoming resistance to other KIT inhibitors (such as imatinib and sunitinib) [1,2]
3. Ripretinib has been approved by the FDA for the treatment of adult patients with advanced gastrointestinal stromal tumors (GIST) who have previously received three or more kinase inhibitors (including imatinib) [1,3]
4. Preclinical studies have shown that ribertinib is effective against KIT D816V mutant mast cell leukemia and PDGFRα. It is effective against D842V mutant solid tumors and is currently undergoing clinical trials for these indications [2,3].
5. Unlike other KIT inhibitors, ripretinib targets the inactive conformation of KIT/PDGFRα, thus being effective against a variety of activating and resistance mutations with minimal off-target effects [1,3].

C24H21BRFN5O2

510.37

509.09

C, 56.48; H, 4.15; Br, 15.66; F, 3.72; N, 13.72; O, 6.27

1442472-39-0

1442472-39-0; 1225278-16-9 (wrong structure for DCC-2618);

71584930

White to off-white solid powder

4.1

3

5

5

33

746

0

CEFJVGZHQAGLHS-UHFFFAOYSA-N

InChI=1S/C24H21BrFN5O2/c1-3-31-21-12-22(27-2)28-13-14(21)9-17(23(31)32)16-10-20(19(26)11-18(16)25)30-24(33)29-15-7-5-4-6-8-15/h4-13H,3H2,1-2H3,(H,27,28)(H2,29,30,33)

1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea

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: 2.08 mg/mL (4.08 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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: 2.08 mg/mL (4.08 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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: ≥ 2.08 mg/mL (4.08 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 20.8 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.)