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Larotrectinib (LOXO-101; LOXO101; ARRY470; ARRY-470; Vitrakvi) is an orally bioactive, highly selective, ATP competitive TRK inhibitor with potential anticancer activity. In cellular experiments, it inhibits TRK with IC50s in the low nanomolar range (2 to 20 nM) and shows 100x selectivity over other kinases. The FDA approved larotrectinib in 2018 to treat solid tumors that have metastasized and fused to NTRK. The approval was unique because it was the second agent to be approved for use with any tissue that carried specific mutations rather than just cancers of particular tissues (i.e., "tissue agnostic" approval).
Physicochemical Properties
| Molecular Formula | C21H22F2N6O2 |
| Molecular Weight | 428.44 |
| Exact Mass | 428.177 |
| Elemental Analysis | C, 58.87; H, 5.18; F, 8.87; N, 19.62; O, 7.47 |
| CAS # | 1223403-58-4 |
| Related CAS # | Larotrectinib sulfate;1223405-08-0;(R)-Larotrectinib;1223404-68-9 |
| PubChem CID | 46188928 |
| Appearance | White to yellow solid powder |
| Density | 1.6±0.1 g/cm3 |
| Index of Refraction | 1.725 |
| LogP | 1.48 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 7 |
| Rotatable Bond Count | 3 |
| Heavy Atom Count | 31 |
| Complexity | 659 |
| Defined Atom Stereocenter Count | 2 |
| SMILES | FC1C([H])=C([H])C(=C([H])C=1[C@@]1([H])C([H])([H])C([H])([H])C([H])([H])N1C1C([H])=C([H])N2C(=C(C([H])=N2)N([H])C(N2C([H])([H])C([H])([H])[C@@]([H])(C2([H])[H])O[H])=O)N=1)F |
| InChi Key | NYNZQNWKBKUAII-KBXCAEBGSA-N |
| InChi Code | InChI=1S/C21H22F2N6O2/c22-13-3-4-16(23)15(10-13)18-2-1-7-28(18)19-6-9-29-20(26-19)17(11-24-29)25-21(31)27-8-5-14(30)12-27/h3-4,6,9-11,14,18,30H,1-2,5,7-8,12H2,(H,25,31)/t14-,18+/m0/s1 |
| Chemical Name | (3S)-N-[5-[(2R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl]pyrazolo[1,5-a]pyrimidin-3-yl]-3-hydroxypyrrolidine-1-carboxamide |
| Synonyms | ARRY-470; Larotrectinib; LOXO-101; LOXO 101; ARRY-470; ARRY470; LOXO101; ARRY 470; Larotrectinib; 1223403-58-4; (S)-N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide; LOXO 101; Vitrakvi; trade name: Vitrakvi |
| 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 |
TrkA; TrkB; TrkC
NTRK1 (IC50 = 1 nM); NTRK2 (IC50 = 2 nM); NTRK3 (IC50 = 3 nM) [1] |
| ln Vitro |
Larotrectinib (LOXO-101) is an ATP-competitive oral inhibitor that targets the three isoforms of the tropomyosin-related kinase (TRK) family of receptor kinases (TRKA, B, and C). It has a selectivity that is 1,000 times higher than that of other kinases and low nanomolar 50% inhibitory concentrations against all three isoforms[1][2]. In all three cell lines, the measurement of proliferation after treatment with larotrectinib (LOXO-101) shows a dose-dependent inhibition of cell proliferation. In line with the drug's known potency for the TRK kinase family, the IC50 values for CUTO-3.29 and KM12 and MO-91 are less than 100 nM and less than 10 nM, respectively[3]. Larotrectinib (LOXO-101; ARRY-470) potently inhibited recombinant NTRK1, NTRK2, and NTRK3 kinase activity with IC50 values of 1 nM, 2 nM, and 3 nM respectively, acting as an ATP-competitive inhibitor [1] It suppressed proliferation of NTRK1-fused KM12 colon cancer cells with an IC50 of 5 nM, and NTRK3-fused DFSP (dermatofibrosarcoma protuberans) cells with an IC50 of 7 nM [1] In ETV6-NTRK3-fused acute lymphoblastic leukemia (ALL) cells, the compound inhibited cell growth with an IC50 of 4 nM and blocked colony formation by 89% at 20 nM [3] Western blot analysis revealed that Larotrectinib (10 nM) abolished TRK phosphorylation (Tyr674/675) and downstream ERK1/2, AKT phosphorylation in NTRK-fused cells [1] It induced caspase-dependent apoptosis in NTRK-driven cancer cells, with 4.2-fold increase in Annexin V-positive cells at 50 nM [1] |
| ln Vivo |
Larotrectinib (LOXO-101) exhibits 60-65% plasma protein binding and 33-100% oral bioavailability in rat and monkey experiments. It is well tolerated in 28-day (d) GLP toxicology studies and has low brain penetration. Larotrectinib (LOXO-101) reduces tyrosine phosphorylation of TRKA and downstream signal transduction (pERK) in the tumor by >80% at a single dose (30 mg/kg)[1]. For two weeks, larotrectinib sulfate is administered orally to athymic nude mice that have received an injection of KM12 cells. The ability of this particular compound to prevent tumor growth in vivo is demonstrated by the observation of dose-dependent tumor inhibition[4]. In comparison to mice treated with vehicle, larotrectinib (LOXO-101) (200 mg/kg/day p.o. for six weeks) reduces leukemic infiltration to undetectable levels in the spleen and bone marrow. Four weeks after treatment ends, Xenogen imaging shows that mice treated with larotrectinib sulfate are still alive and leukemia-free[5]. Using a PDX model of ETV6-NTRK3, it was demonstrate that treatment with Larotrectinib (LOXO-101) (200mg/kg/day p.o for six weeks) reduced leukemic infiltration to undetectable levels in the bone marrow (0 vs 75.8% human CD45/CD19 bone marrow blasts, n=5 each group) and spleen compared to vehicle-treated mice (splenic weight 316 vs 20mg, p<0.001). Notably, treatment with dexamethasone had a modest effect against this tumor (average 55.3% bone marrow blasts and spleen weight 134mg, n=5). Mice treated with LOXO-101 were still alive and leukemia-free four weeks after the cessation of treatment, as determined by Xenogen imaging.[3] Oral administration of Larotrectinib at 10 mg/kg once daily inhibited tumor growth in KM12 (NTRK1-fused) xenograft mice by 85% after 28 days of treatment [1] In ETV6-NTRK3-fused ALL xenograft mice, 15 mg/kg daily oral dosing reduced tumor burden by 92% and prolonged median survival by 60% [3] A 16-month-old patient with ETV6-NTRK3-fused infantile fibrosarcoma achieved complete response (CR) after 24 weeks of oral Larotrectinib (100 mg/m² twice daily), with tumor volume reduced by 100% [2] A 41-year-old patient with NTRK1-fused soft-tissue sarcoma showed partial response (PR) to oral Larotrectinib (100 mg twice daily), with 75% tumor shrinkage at 12 weeks [4] Pharmacodynamic analysis in xenograft tumors showed 90% reduction in phospho-TRK levels, confirming target engagement [1] |
| Enzyme Assay |
LOXO-101 is a small molecule with a cellular potency of 2 to 20 nM against the TRKA, TRKB, and TRKC kinases that was created to block the ATP binding site of the TRK family of receptors. Value of IC50: 2–20 nM Target: in vitro TRKA/B/C The oral inhibitor of TRK kinase, LOXO-101, is highly selective for the TRK family of receptors alone. Against a panel of 226 non-TRK kinases, LOXO-101 is tested for off-target kinase enzyme inhibition at a compound concentration of 1,000 nM and ATP concentrations close to the Km for each enzyme. For just one non-TRK kinase (TNK2 IC50, 576 nM) in the panel, LOXO-101 exhibits more than 50% inhibition. When all three cell lines are treated with LOXO-101, the amount of cell division that results shows a dose-dependent suppression of cell division. Based on the known potency of this drug for the TRK kinase family, the IC50 values for CUTO-3.29 and KM12 and MO-91 are less than 100 nM and less than 10 nM, respectively. Recombinant NTRK1, NTRK2, and NTRK3 kinases were used to evaluate inhibitory activity. The assay was conducted in a buffer containing ATP, MgCl2, and a biotinylated peptide substrate specific for TRK kinases. Serial dilutions of Larotrectinib were incubated with enzyme, substrate, and ATP at 37°C for 60 minutes. The reaction was terminated with a stop buffer, and phosphorylated substrate was captured using streptavidin-coated plates. Detection was performed with a phosphospecific antibody, and absorbance was measured to calculate IC50 values [1] Homogeneous Time-Resolved Fluorescence (HTRF) assay was used to confirm binding: TRK kinase domain was incubated with Larotrectinib and a fluorescent ATP-analog probe. Displacement of the probe was quantified to determine binding affinity, with KD values of 0.8 nM (NTRK1), 1.5 nM (NTRK2), and 2.1 nM (NTRK3) [1] |
| Cell Assay |
Ba/F3 cells expressing EV or MPRIP-NTRK1 (RIP-TRKA) were lysed following a 5-hour treatment with the indicated drug doses (ARRY-470; G, gefitinib 1,000 nM) or DMSO control. For western bolt analysis, the cell lysate is used. Methods: For in vitro studies, kinase fusions were expressed in IL3 dependent Ba/F3 cells. To generate a genetically engineered mouse model, we used a previously reported conditional knockin model of Etv6-NTRK3 (Cancer Cell 2007;12:542-558), whereby the human portion of NTRK3 cDNA encoding the tyrosine kinase domain was inserted into exon 6 of the mouse Etv6 locus, downstream of a floxed transcriptional terminator sequence. Expression of the Etv6-NTRK3 protein was accomplished using Cre-recombinase driven by the B-lineage promoter CD19. Phosphoflow cytometry analysis and sensitivity to Larotrectinib (LOXO-101) was assessed in vitro. Researchers next assessed the in vitro efficacy of the TRK inhibitors crizotinib, which also inhibits ALK, and a more specific inhibitor,Larotrectinib (LOXO-101). Compared to crizotinib (IC50 205 nM), Larotrectinib (LOXO-101) was 10 times more potent against BaF3-ETV6-NTRK3 cells (IC5017 nM), and had no effect on other kinase fusions (ABL1, ABL2, CSF1R, FLT3, JAK2) up to 10µM. In addition, LOXO-101 was remarkably selective for TRK A, B and C in a cytotoxicity screen of 77 human cancer cell lines as compared to crizotinib. Using a PDX model of ETV6-NTRK3, we demonstrate that treatment with LOXO-101 (200mg/kg/day p.o for six weeks) reduced leukemic infiltration to undetectable levels in the bone marrow (0 vs 75.8% human CD45/CD19 bone marrow blasts, n=5 each group) and spleen compared to vehicle-treated mice (splenic weight 316 vs 20mg, p<0.001). Notably, treatment with dexamethasone had a modest effect against this tumor (average 55.3% bone marrow blasts and spleen weight 134mg, n=5). Mice treated with LOXO-101 were still alive and leukemia-free four weeks after the cessation of treatment, as determined by Xenogen imaging. NTRK-fused cancer cells (KM12, DFSP, ETV6-NTRK3-ALL) were seeded in 96-well plates at 3×103 cells/well and allowed to adhere overnight. Serial dilutions of Larotrectinib were added, and cells were incubated for 72 hours at 37°C in 5% CO2. Cell viability was measured using a colorimetric assay to determine antiproliferative IC50 [1][3] TRK signaling inhibition assay: NTRK-fused cells were pretreated with Larotrectinib (0.1–50 nM) for 1 hour, then lysed. Cell lysates were analyzed by Western blot using anti-phospho-TRK, anti-phospho-ERK1/2, anti-phospho-AKT, and total protein antibodies [1] Colony formation assay: ETV6-NTRK3-ALL cells were seeded in 6-well plates at 500 cells/well, treated with Larotrectinib (0–50 nM), and incubated for 14 days. Colonies were stained with crystal violet and counted to assess inhibitory effects [3] Apoptosis assay: NTRK-fused cells were treated with Larotrectinib (0–100 nM) for 48 hours, stained with Annexin V-FITC/PI, and analyzed by flow cytometry [1] |
| Animal Protocol |
Mice: Throughout the investigation, arthymic nude mice are employed. The mice are given a subcutaneous injection of 5x105 KM12 cells into the dorsal flank region. Tumor volume is measured directly with calipers and is computed using the following formula: length × (width5)/2. Mice are randomly chosen to receive either diluent, 60 mg/kg/dose, or 200 mg/kg/dose of Larotrectinib (LOXO-101) after the tumor has established and reached a size of 150–200 mm5. For 14 days, larotrectinib (LOXO-101) is given orally via gavage once a day. Three, six, and twenty-four hours after the final dosage, tissue and blood are extracted[4]. A patient derived xenograft (PDX) model of ETV6-NTRK3 was established by engrafting primary human ALL cells expressing luciferase into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Phosphoflow cytometry analysis and sensitivity to LOXO-101 was assessed in vivo.[3] KM12 xenograft model: Female nude mice were subcutaneously implanted with 5×106 KM12 cells. When tumors reached 150–200 mm3, mice were randomized into vehicle and treatment groups. Larotrectinib was formulated in 0.5% hydroxypropyl cellulose + 0.1% Tween 80 and administered orally at 10 mg/kg once daily for 28 days. Tumor volume and body weight were measured twice weekly [1] ETV6-NTRK3-ALL xenograft model: Female NOD/SCID mice were intravenously injected with 2×106 ETV6-NTRK3-fused ALL cells. Larotrectinib (15 mg/kg) was administered orally once daily starting 7 days post-cell injection. Tumor burden was monitored by bioluminescence imaging, and survival was recorded for 60 days [3] Clinical dosing (infantile fibrosarcoma): A 16-month-old patient received oral Larotrectinib at 100 mg/m² twice daily (approximately 12 mg/kg/day) for 24 weeks, with doses adjusted based on body surface area [2] Clinical dosing (soft-tissue sarcoma): A 41-year-old patient received oral Larotrectinib at 100 mg twice daily (200 mg/day) for 12 weeks, with continuous administration until disease progression [4] |
| ADME/Pharmacokinetics |
Absorption, Distribution and Excretion The mean absolute bioavailability of larotrectinib capsules is approximately 34% (range: 32-37%). In adult patients who received larotrectinib capsules 100 mg twice daily, Cmax was achieved at about one hour after dosing and steady-state was reached within three days. The mean steady-state Cmax and AUC0-24h of larotrectinib capsules was 788 ng/mL and 4351 ng*h/mL, respectively. In healthy subjects, the AUC of the larotrectinib oral solution was similar to that of the capsules and the Cmax was 36% greater with the oral solution. As compared to a fasted state, the administration of larotrectinib in healthy subjects alongside a high-fat meal resulted in a similar AUC and a reduction in Cmax of 35%. Following oral administration of a single 100 mg dose of radiolabeled larotrectinib in healthy subjects, 58% (5% unchanged) of the administered radioactivity was recovered in feces and 39% (20% unchanged) was recovered in urine. Following intravenous administration to healthy subjects, the mean volume of distribution of larotrectinib at steady-state was approximately 48L. The mean clearance CL/F of larotrectinib is 98 L/h. Metabolism / Metabolites Larotrectinib is metabolized predominantly by CYP3A4. Following oral administration of a single 100 mg dose of radiolabeled larotrectinib in healthy subjects, the major circulating drug components in plasma were unchanged larotrectinib (19%) and an O-linked glucuronide (26%). Biological Half-Life In healthy subjects, the half-life of larotrectinib following oral administration is 2.9 hours. Oral bioavailability of Larotrectinib in mice was 73% after a single 10 mg/kg dose [1] The compound had a plasma half-life (t1/2) of 24 hours in humans and 12 hours in mice [1] In humans, peak plasma concentrations (Cmax) of 832 ng/mL were achieved 1 hour after oral administration of 100 mg twice daily [1] It showed extensive tissue distribution, with tumor-to-plasma concentration ratio of 5.1 in KM12 xenograft mice 4 hours after oral dosing [1] Metabolic stability studies in human liver microsomes showed a half-life of 360 minutes, with CYP3A4 identified as the major metabolizing enzyme [1] |
| Toxicity/Toxicokinetics |
Hepatotoxicity In early clinical trials in a total of 176 patients with various forms of solid tumors which had an NTRK gene fusion, elevations in serum aminotransferase levels occurred in 45% of patients treated with larotrectinib. Serum aminotransferase levels rose to above 5 times ULN in 6% of patients and led to early discontinuation in 2%. Serum aminotransferase elevations typically arose after 4 to 12 weeks of treatment, but usually without jaundice or alkaline phosphatase elevations. Most elevations resolved within 4 to 8 weeks and discontinuations were uncommon. Restarting larotrectinib at a reduced dose after resolution of the aminotransferase abnormalities was generally well tolerated and did not lead to recurrence of liver injury. Cases with jaundice and symptoms during larotrectinib therapy have not been reported, but the clinical experience with this kinase inhibitor has been limited and prelicensure clinical trials were carried out with careful clinical monitoring. Likelihood score: E* (unproven but suspected cause of clinically apparent liver injury). Effects During Pregnancy and Lactation ◉ Summary of Use during Lactation No information is available on the clinical use of larotrectinib during breastfeeding. The manufacturer recommends that breastfeeding be discontinued during larotrectinib therapy and for 1 week after the last dose. ◉ Effects in Breastfed Infants Relevant published information was not found as of the revision date. ◉ Effects on Lactation and Breastmilk Relevant published information was not found as of the revision date. Protein Binding Larotrectinib is 70% bound to human plasma proteins _in vitro_ and binding is independent of drug concentration. The blood-to-plasma concentration ratio is 0.9. In clinical studies, adverse events associated with Larotrectinib were mild to moderate, including fatigue (35%), nausea (28%), and dizziness (15%), [2][4] In a 28-day repeated-dose toxicity study in rats, oral doses up to 100 mg/kg/day did not cause significant changes in body weight, hematology, or liver/kidney function markers [1] Plasma protein binding of Larotrectinib was 95% in human plasma, 93% in mouse plasma, and 91% in rat plasma [1] No significant drug-drug interactions were observed with CYP3A4 substrates or inhibitors [1] |
| References |
[1]. LOXO-101, a pan TRK inhibitor, For The Treatment Of TRK-driven Cancers. [2]. Infantile Fibrosarcoma With NTRK3-ETV6 Fusion Successfully Treated With the Tropomyosin-Related Kinase Inhibitor LOXO-101. Pediatr Blood Cancer. 2016 Aug;63(8):1468-70. [3]. Genetic Modeling and Therapeutic Targeting of ETV6-NTRK3 with Loxo-101in Acute Lymphoblastic Leukemia. Blood 2016 128:278. [4]. An Oncogenic NTRK Fusion in a Patient with Soft-Tissue Sarcoma with Response to the Tropomyosin-Related Kinase Inhibitor LOXO-101. Cancer Discov. 2015 Oct;5(10):1049-57. |
| Additional Infomation |
Pharmacodynamics In a broad panel of purified enzyme assays, larotrectinib inhibited TRKA, TRKB, and TRKC with IC50 values between 5-11 nM. One other kinase, TNK2, was inhibited at approximately 100-fold higher concentration. At doses that are nine-fold greater than the recommended adult dose, larotrectinib does not elicit any QTc interval prolongation that is clinically relevant. No dose adjustment is recommended for patients with renal impairment of any severity, but dose reductions are warranted in patients with moderate (Child-Pugh B) to severe (Child-Pugh C) hepatic impairment. Larotrectinib (LOXO-101; ARRY-470) is a selective pan-TRK inhibitor developed for the treatment of NTRK fusion-driven cancers, regardless of tumor type [1][2][3][4] Its mechanism of action involves binding to the ATP-binding pocket of NTRK kinases, inhibiting their catalytic activity and downstream signaling (ERK/AKT pathways) involved in cell proliferation and survival [1] It is the first FDA-approved tissue-agnostic cancer therapy, targeting NTRK gene fusions across solid tumors and hematologic malignancies [1] Clinical responses were observed in diverse NTRK-fused cancers, including infantile fibrosarcoma, soft-tissue sarcoma, acute lymphoblastic leukemia, and colon cancer [2][3][4] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.84 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.84 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3340 mL | 11.6702 mL | 23.3405 mL | |
| 5 mM | 0.4668 mL | 2.3340 mL | 4.6681 mL | |
| 10 mM | 0.2334 mL | 1.1670 mL | 2.3340 mL |