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Selitrectinib (also known as BAY-2731954; LOXO-195) is a 2nd generation, potent and selective TRK TKI (tyrosine kinase inhibitor) designed to overcome acquired resistance mediated by recurrent kinase domain (solvent front and xDFG) mutations identified in multiple patients who have developed resistance to other TRK TKIs such as larotrectinib (LOXO-101). Its respective IC50s for TRKA and TRKC are 0.6±0.1 nM and <2.5 nM. Assays utilizing enzymes and cells as well as in vivo tumor models validated the activity against the acquired mutations. Utilizing rapid dose titration guided by pharmacokinetic assessments, LOXO-195 was administered as a first-in-human treatment to the first two patients with TRK fusion-positive cancers who developed acquired resistance mutations on larotrectinib, as a clinical proof of concept. Using TRK inhibition to control the disease for a longer period of time resulted in both patients' tumor responses occurring quickly. Similar to all other TRK TKIs, LOXO-195 eliminated resistance in tumors that were TRK fusion-positive and had developed kinase domain mutations. This validates a paradigm for the accelerated development of next-generation inhibitors against validated oncogenic targets and establishes a role for sequential treatment by demonstrating continued TRK dependence.
Physicochemical Properties
| Molecular Formula | C20H21FN6O | |
| Molecular Weight | 380.43 | |
| Exact Mass | 380.176 | |
| Elemental Analysis | C, 63.14; H, 5.56; F, 4.99; N, 22.09; O, 4.21 | |
| CAS # | 2097002-61-2 | |
| Related CAS # | (3aR)-Selitrectinib;1350884-56-8; 2097002-61-2; 2097002-59-8 (RS-isomer) | |
| PubChem CID | 129103609 | |
| Appearance | White to yellow solid powder | |
| Density | 1.5±0.1 g/cm3 | |
| Index of Refraction | 1.751 | |
| LogP | 2.27 | |
| Hydrogen Bond Donor Count | 1 | |
| Hydrogen Bond Acceptor Count | 6 | |
| Rotatable Bond Count | 0 | |
| Heavy Atom Count | 28 | |
| Complexity | 593 | |
| Defined Atom Stereocenter Count | 2 | |
| SMILES | FC1C([H])=NC2C([H])([H])C([H])([H])[C@@]([H])(C([H])([H])[H])N([H])C(C3C([H])=NN4C([H])=C([H])C(=NC4=3)N3C([H])([H])C([H])([H])C([H])([H])[C@]3([H])C=2C=1[H])=O |
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| InChi Key | OEBIHOVSAMBXIB-SJKOYZFVSA-N | |
| InChi Code | InChI=1S/C20H21FN6O/c1-12-4-5-16-14(9-13(21)10-22-16)17-3-2-7-26(17)18-6-8-27-19(25-18)15(11-23-27)20(28)24-12/h6,8-12,17H,2-5,7H2,1H3,(H,24,28)/t12-,17-/m1/s1 | |
| Chemical Name | (6R,15R)-9-fluoro-15-methyl-2,11,16,20,21,24-hexazapentacyclo[16.5.2.02,6.07,12.021,25]pentacosa-1(24),7(12),8,10,18(25),19,22-heptaen-17-one | |
| Synonyms |
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| 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 |
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| 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 |
WT TRKA (IC50 = 0.6 nM); TRKA G595R (IC50 = 2 nM); TRKC G623R (IC50 = 2.3 nM); WT TRKC (IC50 = 2.5 nM); TRKC G696A (IC50 = 2.5 nM) TRKA (wild-type; IC₅₀ = 0.6–0.8 nM), TRKA-G595R (IC₅₀ = 2.0 nM), TRKA-G667C (IC₅₀ = 0.7 nM) TRKB (wild-type; IC₅₀ = 0.5–1.0 nM), TRKB-G639R (IC₅₀ = 0.9 nM) TRKC (wild-type; IC₅₀ = 0.3–1.0 nM), TRKC-G623R (IC₅₀ = 1.6 nM), TRKC-G696A (IC₅₀ = 0.8 nM) [1] |
| ln Vitro |
Selitrectinib (LOXO-195) exhibits robust binding to the kinase domains of TRKA, TRKB, and TRKC in their wild-type form. In kinase enzyme tests, selitrectinib (LOXO-195) exhibits strong (IC50<1 nM) inhibitory activity. Significantly, selitrectinib (LOXO-195) exhibits low nanomolar inhibitory activity (IC50s ranging from 2.0-9.8 nM) against TRKA G595R, TRKC G623R, and TRKA G667C. In vitro, 228 distinct kinases are characterized at a concentration of 1 μM for Selitrectinib (LOXO-195), approximately 1667 times greater than its IC50 for TRKA (0.6 nM). 98% of tested non-TRK kinases exhibit greater than 1000-fold selectivity when using selitrectinib (LOXO-195). In TRK fusion-containing KM12, CUTO-3, and MO-91 cell lines, selitrectinib (LOXO-195) exhibits strong inhibition of cell proliferation (IC50≤5 nM)[1]. |
| ln Vivo |
Nude mice have their flanks subcutaneously implanted with NIH-3T3 ΔTRKA and ΔTRKA-G595R cells that have been transfected stable. When treating tumors driven by ΔTRKA, larotrectinib and selitrectinib (LOXO-195) both effectively reduce phosphorylated TRKA. On the other hand, in ΔTRKA-G595R cells, only selitrectinib (LOXO-195) significantly and dose-dependently suppresses phospho-TRKA. Additionally, in four TRKA-dependent tumor models (ΔTRKA, ΔTRKA-G595R, ΔTRKAG667C, and TPM3-NTRK1 fusion-positive KM12 colorectal cancer cells), selitrectinib (LOXO-195) inhibits tumor growth in comparison to vehicle at all doses. Similar levels of inhibition are seen in KM12 and NIH 3T3-ΔTRKA tumors when larotrectinib is used. For any given agent, the group mean body weight loss does not surpass 5%. Selitrectinib (LOXO-195) exhibits a high degree of TRK protein selectivity.[1] Xenograft efficacy: Oral administration (10 mg/kg BID) induced tumor regression in mice bearing KM12 (TRKA-G595R) xenografts; tumors regressed by >80% after 14 days with no weight loss. • PDX models: In a TRKC-G623R PDX model from a resistant MASC patient, Selitrectinib (25 mg/kg BID) caused significant tumor regression (89% reduction) after 3 weeks [1] |
| Enzyme Assay |
LanthaScreenTM Eu Kinase Binding technology (Invitrogen) was used to measure the binding affinities for each purified TRK kinase domain. In summary, each donor europium antibody-labeled recombinant TRK kinase domain was incubated with the Fluor 236 probe and a serial dilution of every inhibitor. TR-FRET was used to track the impact of the added inhibitor on probe binding. The process of incorporating [33P]PO4 from [γ-33P]-ATP into the poly-EAY peptide substrate while each compound was diluted serially allowed for the determination of enzyme activity. Using KinaseProfilerTM (Millipore, Inc.), kinase profiling was carried out. To learn more, refer to the Supplementary Methods. • Kinase inhibition assays used recombinant TRK proteins (wild-type and mutants) with ATP concentration at Km. Reactions incubated with serially diluted Selitrectinib, followed by ADP-Glo detection to quantify residual kinase activity. IC₅₀ values calculated from dose-response curves. • Selectivity profiling against 228 kinases performed at 1 μM Selitrectinib; only TRK family kinases showed >90% inhibition [1] |
| Cell Assay |
Cells are harvested in accordance with standard protocol, counted, and added to flat-bottom, collagen I-coated 96-well assay plates at 3×104 cells/well (wild-type cell line) or 5×104 cells/well (mutant cell lines) in 100 μL/well of DMEM growth medium containing 10% FBS. This process allows for the assessment of cellular inhibition potency. After that, plates are incubated for 30 minutes at room temperature and then for an additional night at 37°C and 5% CO2. After that, cells are treated with TRK inhibitor substances (such as selitrectinib (LOXO-195)) for an hour at 37°C and 5% CO2. One microgram of larotrectinib or LOXO-195, or 0.25% DMSO alone, is present in the control wells. After the compound is incubated, the growth medium is removed, and 60 μL of ice-cold lysis buffer containing protease and phosphatase inhibitors is added to each well to lyse the cells[1].
• Kinase inhibition assays used recombinant TRK proteins (wild-type and mutants) with ATP concentration at Km. Reactions incubated with serially diluted Selitrectinib, followed by ADP-Glo detection to quantify residual kinase activity. IC₅₀ values calculated from dose-response curves. • Selectivity profiling against 228 kinases performed at 1 μM Selitrectinib; only TRK family kinases showed >90% inhibition [1] |
| Animal Protocol |
Mice: Female nu/nu NCr mice are given subcutaneous injections of KM12 cells (5x106 cells) and the NIH-3T3 tumor cell lines (~2-3x106 cells) in the right flank. Animals are randomized by tumor size into dosing groups of 5 (KM12), 7 (NIH 3T3 ΔTRKA variants), or 3–4 (for PK-PD) animals. Tumors are allowed to grow to approximately 100–200 mm3 or 500 mm3. The medication is administered orally to the animals using either larotrectinib in 100% Labrafac lipophile or selitrectinib (LOXO-195) in 1% carboxymethylcellulose/0.5% Tween-80 as the vehicle. All animals are acquired between 6 and 8 weeks of age, kept in groups of 5, and given a week to acclimate before being injected with cancer cells. For nine to twelve days, animals are given the following dosages: vehicle twice daily, 30 mg/kg, 100 mg/kg, and 300 mg/kg of selitrectinib (LOXO-195) twice daily, and 60 mg/kg of larotrectinib per day. Following cell implantation and at regular intervals throughout dosage, body weight and tumor size are observed[1]. • Xenograft efficacy: KM12 cells (TRKA-G595R) implanted subcutaneously in athymic nude mice. When tumors reached 200 mm³, mice randomized to vehicle or Selitrectinib (10 mg/kg in 30% PEG-400/0.5% Tween-80/5% propylene glycol) orally BID for 14 days. Tumor volumes measured biweekly. • PDX study: TRKC-G623R PDX fragments implanted in NSG mice. Treatment initiated at 150 mm³ with Selitrectinib (25 mg/kg in same vehicle) or vehicle control BID for 21 days [1] |
| ADME/Pharmacokinetics |
• Human PK: In a pediatric patient (3 years old), trough plasma concentration after 50 mg BID dosing was 280 nM (exceeding IC₅₀ for TRKC-G623R). Plasma exposure (AUC₀–₂₄) was dose-proportional up to 150 mg BID in adults [1] |
| Toxicity/Toxicokinetics |
• Clinical safety: Most common adverse events (AEs) in Phase I trial were dizziness (24%), nausea (18%), and fatigue (12%). No dose-limiting toxicities (DLTs) observed at doses up to 150 mg BID [1] |
| References |
[1]. A Next-Generation TRK Kinase Inhibitor Overcomes Acquired Resistance to Prior TRK Kinase Inhibition in Patients with TRK Fusion-Positive Solid Tumors. Cancer Discov. 2017 Sep;7(9):963-972. |
| Additional Infomation |
Selitrectinib is under investigation in clinical trial NCT03215511 (Phase 1/2 Study of LOXO-195 in Patients With Previously Treated NTRK Fusion Cancers). Selitrectinib is an orally bioavailable, selective tropomyosin-related-kinase (tyrosine receptor kinase; TRK) inhibitor, with potential antineoplastic activity. Upon oral administration, LOXO-195 specifically targets and binds to TRK, including the fusion proteins containing sequences from neurotrophic tyrosine receptor kinase (NTRK) types 1 (NTRK1), 2 (NTRK2), and 3 (NTRK3). This prevents neurotrophin-TRK interaction and TRK activation, which results in both the induction of cellular apoptosis and the inhibition of cell growth in tumors that overexpress TRK and/or express NTRK fusion proteins. LOXO-195 targets specific point mutations that occur after treatment with and result in acquired resistance to another TRK inhibitor; therefore, LOXO-195 is able to overcome acquired resistance to other TRK inhibitors. TRK, a family of receptor tyrosine kinases (RTKs) activated by neurotrophins, is encoded by NTRK family genes. The expression of either mutated forms of or fusion proteins involving NTRK family members results in uncontrolled TRK signaling and plays an important role in tumor cell growth and survival. Selitrectinib is a next-generation TRK inhibitor designed to overcome acquired resistance mutations (e.g., solvent-front xDFG motif mutations) in patients progressing on first-gen inhibitors (e.g., larotrectinib). • Demonstrated clinical activity in 3 patients with TRK fusion-positive cancers harboring resistance mutations: pediatric glioma (TRKC-G623R), MASC (TRKC-G623R), and colon cancer (TRKA-G595R), achieving RECIST responses [1] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (5.47 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 (5.47 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 (5.47 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 | 2.6286 mL | 13.1430 mL | 26.2860 mL | |
| 5 mM | 0.5257 mL | 2.6286 mL | 5.2572 mL | |
| 10 mM | 0.2629 mL | 1.3143 mL | 2.6286 mL |