Bozitinib (Vebreltinib; formerly APL-101/PLB-1001, CBT-101) is a novel, an ATP-competitive, and highly selective c-MET kinase inhibitor with blood-brain barrier permeability. Bozitinib (PLB-1001) attaches itself to the tyrosine kinase superfamily's typical ATP-binding pocket. Phase I/II trials are presently investigating it as a single agent in patients with solid tumors, non-small cell lung cancer (NSCLC), and glioblastomas; additionally, it is being investigated in patients with hepatocellular carcinoma and renal cell carcinoma in combination with PD-L1 inhibitors. Due to its ability to cross the blood–brain barrier, the medication has shown promise in treating glioblastoma and has shown anticancer effects in a number of human xenograft tumor models with MET dysregulation. A small number of patients with chemotherapy-resistant, MET-altered gliomas have shown the drug's safety and preliminary efficacy. In China, a phase II trial (NCT04258033) is assessing bozitinib in patients with non-small cell lung cancer (NSCLC) who have skipped MET exon 14.

Physicochemical Properties

Molecular Formula	C20H15F3N8
Molecular Weight	424.381912469864
Exact Mass	424.14
Elemental Analysis	C, 56.60; H, 3.56; F, 13.43; N, 26.40
CAS #	1440964-89-5
Related CAS #	1440964-89-5
PubChem CID	72202701
Appearance	Light yellow to yellow solid powder
LogP	2.1
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	8
Rotatable Bond Count	4
Heavy Atom Count	31
Complexity	681
Defined Atom Stereocenter Count	0
InChi Key	QHXLXUIZUCJRKV-UHFFFAOYSA-N
InChi Code	InChI=1S/C20H15F3N8/c1-29-9-11-6-14(15(21)7-17(11)27-29)20(22,23)19-26-25-18-5-4-16(28-31(18)19)12-8-24-30(10-12)13-2-3-13/h4-10,13H,2-3H2,1H3
Chemical Name	6-(1-cyclopropylpyrazol-4-yl)-3-[difluoro-(6-fluoro-2-methylindazol-5-yl)methyl]-[1,2,4]triazolo[4,3-b]pyridazine
Synonyms	PLB 1001; Bozitinib; PLB-1001; PLB1001; CBT101; CBT 101; CBT-101; Vebreltinib
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	c-Met The phosphorylation of MET and STAT3 is inhibited by bozitinib (PLB-1001) (30 μM; 6 hours), and PLB-1001 has more inhibitory effects on MET and its downstream signaling buffers.
ln Vitro	The phosphorylation of MET and STAT3 is inhibited by bozitinib (PLB-1001) (30 μM; 6 hours), and PLB-1001 has more inhibitory effects on MET and its downstream signaling buffers. PLB-1001 treatment (30 nM, 6 hr) inhibited phosphorylation of MET (Y1234/Y1235) and its downstream effector STAT3 (Y705) in human astrocytes transiently overexpressing ZM fusion variants (E1-E2 or E2-E2), METex14, or wild-type MET, as demonstrated by Western blotting. This inhibitory effect was comparable to that of crizotinib (3 µM, 6 hr). [1] In a drug sensitivity assay, four MET inhibitors (including PLB-1001) demonstrated significantly better growth inhibition effects (lower normalized Area Under the Curve, AUC) on a ZM fusion-positive patient-derived glioma cell line and a METex14-positive patient-derived cell line compared to 56 non-MET inhibitors. [1]
ln Vivo	In subcutaneous xenograft models using U87 MG cells stably expressing the ZM E2-E2 fusion, daily oral administration of PLB-1001 (100 mg/kg/day) significantly reduced tumor growth and final tumor weight compared to vehicle or crizotinib (50 mg/kg/day) treatment. [1] In an intracranial orthotopic xenograft model with ZM E2-E2-expressing U87 MG cells, PLB-1001 treatment (50 mg/kg/day, oral gavage) initiated 14 days post-implantation suppressed tumor growth as assessed by MRI at 30 days. Withdrawal of the drug led to resumed tumor growth. PLB-1001 treatment significantly prolonged overall survival of mice compared to the vehicle-treated group. Immunohistochemical analysis of tumors from treated mice showed diminished staining intensities of p-MET, p-STAT3, Ki-67, CD31, and pHH3. [1]
Enzyme Assay	The selectivity of PLB-1001 for MET kinase was assessed using a LANCE enzyme assay. The inhibition rate of 100 different kinases was tested by detecting the level of ULight/CREBtide-substrate phosphorylation after incubation with 2 µM of PLB-1001. MET kinase activity was inhibited by 95.1%, while other kinases were barely inhibited. The list of assayed kinases is provided in the supplementary table of the literature. [1]
Cell Assay	Western Blot analysis [1] Cell Types: human astrocytes (HA) Cell Tested Concentrations: 30 μM Incubation Duration: 6 hrs (hours) Experimental Results: p-STAT3, p-MET expression diminished. For cell viability assays, cells were seeded in 96-well plates. Cell viability was assessed daily over 6 days using an MTS assay according to the standard protocol. [1] For drug sensitivity analysis in patient-derived cells (PDCs), cells were seeded in 384-well plates and treated with a seven-point serial dilution series of drugs. After 6 days of treatment, cell viability was analyzed using an ATP-monitoring system. Dose-response curves were fitted, and the Area Under the Curve (AUC) was calculated for evaluation. [1] Western blotting was performed to analyze protein expression and phosphorylation. Tissue or cell lysates were prepared, and equal amounts of total protein were separated by SDS/PAGE, transferred to PVDF membranes, and detected using an ECL system. Beta-Tubulin or GAPDH was used as a loading control. [1] Immunohistochemical staining was performed on paraffin-embedded tissue sections. After deparaffinization, rehydration, and antigen retrieval, sections were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies and 3,3'-diaminobenzidine staining. [1]
Animal Protocol	For subcutaneous xenograft studies, U87 MG cells transduced with ZM E2-E2 fusion or control vector were injected subcutaneously into the flank of female BALB/c nude mice. When tumors reached approximately 100 mm³, mice were randomized to receive daily oral gavage of PLB-1001 (100 mg/kg), crizotinib (50 mg/kg), or vehicle (0.9% normal saline). Tumor dimensions were measured periodically, and volumes were calculated. [1] For intracranial orthotopic models, U87 MG cells expressing ZM E2-E2 fusion were implanted into the brains of female BALB/c nude mice using a stereotactic apparatus. Tumor growth was monitored by MRI. Mice with established tumors (∼80 mm³) were treated daily with PLB-1001 (50 mg/kg, oral gavage) or vehicle. [1] For pharmacokinetic studies in rats, Sprague-Dawley rats were fasted for 16 hours before intragastric administration of PLB-1001 at 4.5 mg/kg body weight. Blood and brain tissue were collected at specified time points post-administration for drug concentration analysis. [1]
ADME/Pharmacokinetics	In an MDCK-MDR1 cell permeability assay, PLB-1001 demonstrated higher apparent permeability and a lower efflux rate compared to other MET inhibitors (crizotinib, cabozantinib, foretinib) and temozolomide. The efflux rate of PLB-1001 was similar to non-substrates of the P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), indicating it is not a substrate for these major blood-brain barrier efflux transporters. [1] Following intragastric administration of 4.5 mg/kg PLB-1001 to Sprague-Dawley rats, the drug concentration in brain tissue peaked at 0.207 µg/g tissue weight (brain/plasma ratio ~1.75%) at 3 hours and then gradually declined. [1] In a Phase I clinical trial, after a single oral dose in glioma patients, PLB-1001 reached stable plasma concentrations between 1,000 to 6,000 ng/mL over 24 hours. The concentration in cerebrospinal fluid (CSF) collected on day 15 was approximately 3–8% of the concurrent plasma concentration. [1]
Toxicity/Toxicokinetics	In the Phase I clinical trial involving 18 recurrent high-grade glioma patients, no dose-limiting toxicities (DLT) or deaths related to PLB-1001 were observed within the 4-week DLT observation period. The maximum tolerated dose (MTD) was not reached. [1] Grade 3 adverse events (AEs) of hepatotoxicity were observed in two patients with grade III glioma: one patient (200 mg bid dosage) had increased alanine aminotransferase (ALT) at week 12, and another (300 mg bid dosage) had increased total bilirubin at week 7. These did not occur within the DLT window. Common grade 1–2 AEs included increased serum lipase and glutamic oxaloacetic transaminase, and decreased albumin. [1]
References	[1]. Mutational Landscape of Secondary Glioblastoma Guides MET-Targeted Trial in Brain Tumor. Cell. 2018 Nov 29;175(6):1665-1678.e18.
Additional Infomation	Vebreltinib is an orally bioavailable inhibitor of the proto-oncogene c-Met (hepatocyte growth factor receptor; HGFR) with potential antineoplastic activity. Upon administration, vebreltinib selectively binds to c-Met, thereby inhibiting c-Met phosphorylation and disrupting c-Met signal transduction pathways. This may induce cell death in tumor cells overexpressing or expressing constitutively activated c-Met protein. c-Met, a receptor tyrosine kinase overexpressed or mutated in many tumor cell types, plays key roles in tumor cell proliferation, survival, invasion, metastasis, and tumor angiogenesis. Bozitinib (PLB-1001) is also referred to as CBI-3103 or IDD-100. Its chemical name is (6-(1-cyclopropyl-1H-pyrazol-4-yl)-3-[difluoro(6-fluoro-2-methyl-2H-indazol-5-yl)methyl]-[1,2,4]triazolo[4,3-b]pyridazine). [1] Molecular dynamics simulation suggests that compared to crizotinib, PLB-1001 has additional interaction sites within the MET kinase ATP-binding pocket, potentially contributing to higher binding affinity and specificity. [1] The drug was evaluated in a Phase I, open-label, 3+3 dose-escalation study (NCT02978261) in recurrent high-grade glioma patients harboring ZM fusion and/or METex14. The recommended Phase II dose (RP2D) for monotherapy was determined to be 300 mg twice daily. [1] Among evaluable patients, 2 out of 6 sGBM patients achieved a partial response (PR), and 2 out of 6 sGBM and 5 out of 9 grade III glioma patients achieved stable disease (SD) according to RANO criteria. [1] Analysis of a recurrent tumor from a patient who initially responded to PLB-1001 but later progressed revealed acquired mutations in the PI3K-Akt-mTOR pathway genes (PIK3CA, PIK3CG, PTEN), suggesting a potential resistance mechanism to MET inhibitor monotherapy. [1]

Solubility Data

Solubility (In Vitro)

DMSO: 50~85 mg/mL (200.3~117.8 mM) Ethanol: ~2.5 mg/mL (~5.9 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.90 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 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (4.90 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 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. Solubility in Formulation 3: ≥ 2.08 mg/mL (4.90 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.3564 mL	11.7819 mL	23.5638 mL
	5 mM	0.4713 mL	2.3564 mL	4.7128 mL
	10 mM	0.2356 mL	1.1782 mL	2.3564 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.