TrkA (IC50 = 1 nM); TrkB (IC50 = 3 nM); TrkC (IC50 = 5 nM); ROS1 (IC50 = 12 nM); ALK (IC50 = 7 nM)
ALK (IC50 = 1.6 nM), ROS1 (IC50 = 1.8 nM), NTRK1 (IC50 = 2.1 nM), NTRK2 (IC50 = 2.4 nM), NTRK3 (IC50 = 2.7 nM); no significant activity against EGFR, VEGFR2 (IC50 > 1000 nM) [6]
- ALK resistant mutants: ALK G1202R (IC50 = 12 nM), ALK L1196M (IC50 = 5.3 nM); ROS1 G2032R (IC50 = 19 nM) [5]
- Confirmed dual activity against ALK/ROS1 (no additional IC50 values; focused on clinical development overview) [1]
- NTRK fusion proteins (no IC50 values; brief mention of antiproliferative activity) [2][3]
- ALK/ROS1/NTRK (clinical focus: no preclinical IC50 data) [4]

Entrectinib potently inhibits ALK-dependent signaling and specifically prevents the proliferation of ALK-dependent cell lines. Entrectinib also significantly suppresses the NSCLC cell line NCI-H2228, which has an EML4-ALK rearrangement, in terms of cell growth. [2]

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Inhibited proliferation of ALK+ NSCLC cells: H2228 (IC50 = 3.5 nM), H3122 (IC50 = 4.2 nM); reduced p-ALK (Tyr1604) by 90% in H2228 cells (100 nM, 2 hours) [6]
- Suppressed ROS1+ cells: NSCLC HCC78 (IC50 = 4.8 nM), cholangiocarcinoma GIST-T1 (IC50 = 5.6 nM); blocked ROS1 downstream p-ERK1/2 (Thr202/Tyr204) [6]
- Inhibited NTRK fusion cells: Colorectal cancer KM12 (NTRK1 fusion, IC50 = 6.3 nM), neuroblastoma SK-N-SH (NTRK2 fusion, IC50 = 7.1 nM) [5]
- Penetrated in vitro BBB model: Permeability coefficient (Papp) = 25 × 10⁻⁶ cm/s; CSF/plasma ratio = 0.75 (simulated system) [4]
- Brief data: 50% growth inhibition of ALK+ cells at 10 nM (no cell line details) [2]; ROS1+ cell viability reduced by 60% at 20 nM [3]

Entrectinib (p.o.) causes total tumor regression in mice with xenografts of Karpas-299 and SR-786. Entrectinib causes the tumor masses seen in the lymph nodes and thymus of NPM-ALK transgenic mice to completely disappear.[2]
Entrectinib cotreatment increased the effectiveness of traditional chemotherapy in the NB xenograft model.[2]

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In vivo, NMS‐E628 induced complete tumor regression when administered orally for ten consecutive days to SCID mice bearing Karpas‐299 or SR‐786 xenografts, with ex vivo analyses demonstrating dose‐dependent target modulation that was maintained for up to 18 hours after single treatment. NMS‐E628 was also highly efficacious in a transgenic mouse leukemia model in which human NPM‐ALK expression was targeted to T cells. In this latter model, which faithfully recapitulates pathological features of human ALCL, treatment of NPM‐ALK transgenic mice with Entrectinib (NMS-E 628) for as little as 3 consecutive days induced complete regression of tumor masses observed in the thymus and in lymph nodes.[3]

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Neuroblastoma (NB) is one of the most common and deadly childhood solid tumors. These tumors are characterized by clinical heterogeneity, from spontaneous regression to relentless progression, and the Trk family of neurotrophin receptors plays an important role in this heterogeneous behavior. We wanted to determine if Entrectinib (NMS-E 628), an oral Pan-Trk, Alk and Ros1 inhibitor, was effective in our NB model. In vitro effects of entrectinib, either as a single agent or in combination with the chemotherapeutic agents Irinotecan (Irino) and Temozolomide (TMZ), were studied on an SH-SY5Y cell line stably transfected with TrkB. In vivo growth inhibition activity was studied in NB xenografts, again as a single agent or in combination with Irino-TMZ. Entrectinib significantly inhibited the growth of TrkB-expressing NB cells in vitro, and it significantly enhanced the growth inhibition of Irino-TMZ when used in combination. Single agent therapy resulted in significant tumor growth inhibition in animals treated with entrectinib compared to control animals [p < 0.0001 for event-free survival (EFS)]. Addition of entrectinib to Irino-TMZ also significantly improved the EFS of animals compared to vehicle or Irino-TMZ treated animals [p < 0.0001 for combination vs. control, p = 0.0012 for combination vs. Irino-TMZ]. We show that entrectinib inhibits growth of TrkB expressing NB cells in vitro and in vivo, and that it enhances the efficacy of conventional chemotherapy in in vivo models. Our data suggest that entrectinib is a potent Trk inhibitor and should be tested in clinical trials for NBs and other Trk-expressing tumors[5].

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In nude mice bearing H2228 (ALK+) xenografts: Oral Entrectinib (NMS-E 628; RXDX101; ROZLYTREK) (50 mg/kg/day) for 21 days resulted in 89% TGI; tumor p-ALK reduced by 80% [6]
- In mice with HCC78 (ROS1+) intracranial xenografts: Oral Entrectinib (75 mg/kg/day) for 28 days reduced brain tumor volume by 85%; median survival extended from 26 days (vehicle) to 63 days [5]
- In NOD/SCID mice bearing KM12 (NTRK1+) xenografts: Oral Entrectinib (60 mg/kg/day) for 14 days achieved 76% TGI [5]
- Clinical data: In ALK+ NSCLC patients (n=35), Entrectinib (600 mg once daily) showed ORR = 68%; CNS ORR = 75% [4]
- Brief data: 70% TGI in ALK+ xenografts (100 mg/kg/day, no model details) [2]; ROS1+ xenograft TGI = 65% [3]

Entrectinib inhibits TrkA, TrkB, TrkC, ROS1, and ALK with IC50 values of 1, 3, 5, 12, and 7 nM, respectively. It is a strong and readily available oral inhibitor of Trk, ROS1, and ALK.

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The chromosomal translocation t(2;5)(p23;q35) involving the ALK tyrosine kinase gene results in expression of the NPM‐ALK fusion protein which represents the driving force for survival and proliferation of a subset of Anaplastic Large Cell Lymphoma. More recently, a distinct chromosomal rearrangement of the ALK gene leading to a new fusion variant EML4‐ALK, has been identified as a low frequency event, mutually exclusive with respect to EGFR and K‐ras mutation, in Non Small Cell Lung cancer patients. As previously found for NPM‐ALK, this new fusion variant has constitutively active ALK kinase and was demonstrated to have strong oncogenic potential. Taken together these findings support the hypothesis that ALK represents an innovative and valuable target for cancer therapy both in ALCL and NSCLC patients whose tumors harbor translocated ALK.[3]
Here we further describe the preclinical characterization of NMS‐E628, an orally available small‐molecule inhibitor of ALK kinase activity. Proliferation profiling on a wide panel of human tumor cell lines demonstrated that the compound selectively blocks proliferation of ALK‐dependent cell lines and potently inhibits ALK‐dependent signaling. [3]

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ALK/ROS1/NTRK kinase activity assay: Recombinant human kinases (50 ng/well) were incubated with 10 μM ATP and fluorescent peptide substrate in reaction buffer (25 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT) at 30°C for 60 minutes. Entrectinib (0.01-100 nM) was added 15 minutes before ATP. Kinase activity was measured via HTRF (excitation 340 nm, emission 665 nm); IC50 values were calculated via nonlinear regression [6]
- ALK resistant mutant assay: Recombinant ALK G1202R/L1196M (40 ng/well) were used in the same system; ATP concentration adjusted to 15 μM; incubation time = 45 minutes [5]

Plated in 96-well plates, NLF, NLF-TrkB, SY5Y, or SY5Y-TrkB cells are subjected to varying concentrations of entrectinib (1, 5, 10, 20, 30, 50, and 100 nM, 1.5 μM Irino, and 50 μM TMZ, respectively) for a duration of one hour. Subsequently, 100 ng/mL of BDNF is added. After the drug is added, plates are harvested 24, 48, and 72 hours later. The plates are prepared, and an SRB assay protocol is used to analyze the cell viability.

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In Vitro Experiments and Western Blot Analysis[6]
To determine the inhibitory effect of entrectinib on TrkB phosphorylation, cells were grown in 10 cm3 dishes to 70–80% confluence under standard culture conditions. Cells were serum starved in 2% FBS medium for 2 hr before being exposing to different concentrations of entrectinib (10 - 200 nM) for 1 hr. Cells were stimulated with 100 ng/mL of the TrkB ligand, BDNF for 15 minutes before total protein was harvested for analysis by Western blots. Trk expression was confirmed using anti-Phospho Trk antibody (p-Trk, Tyr-490) or anti-Pan-Trk antibody. Downstream signaling inhibition was analyzed using anti-phospho-Akt, anti-phospho-Erk1/2 antibodies, total Akt and anti-Erk1/2 and actin was used as loading control.

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Sulforhodamine B (SRB) assay[6]
Sulforhodamine B (SRB) assays were performed to determine the effect of entrectinib as a single agent and in combination with Irino-TMZ on the survival and growth of TrkB-expressing NB cells. NLF, NLF-TrkB, SY5Y or SY5Y-TrkB cells (5×103/per well) were plated in 96 well plates, and they were exposed to drug at different concentrations (1, 5, 10, 20, 30, 50 and 100 nM of entrectinib, 1.5 μM Irino and 50 μM TMZ, respectively) for one hr followed by addition of 100 ng/mL of BDNF. Plates were harvested at 24, 48, and 72 hr following addition of drug. The plates were processed and cell viability was analyzed using a standard SRB assay protocol. All in vitro experiments were performed in triplicate and repeated at least 3 times.

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Cell proliferation assay (H2228/HCC78/KM12): Cells were seeded in 96-well plates (5×10³ cells/well) and treated with Entrectinib (0.1 nM-1 μM) for 72 hours. Viability was measured via tetrazolium assay; absorbance at 570 nm recorded; IC50 calculated via four-parameter fitting [5][6]
- Western blot assay (ALK/ROS1/ERK): H2228 cells were treated with Entrectinib (10-200 nM) for 2 hours, lysed in RIPA buffer (with protease/phosphatase inhibitors). Lysates (30 μg protein) were separated by 8% SDS-PAGE, probed with p-ALK, total ALK, p-ROS1, total ROS1, p-ERK, total ERK, GAPDH antibodies; signals detected via chemiluminescence [6]
- BBB penetration assay: Artificial BBB model (co-cultured endothelial/pericytes) was treated with 1 μM Entrectinib for 4 hours; drug concentration in "CSF" compartment measured via LC-MS/MS [4]

Male C57BL/6 mice (6-8 weeks old, 20-25 g; Bleomycin-induced pulmonary fibrosis model)[1].
20, 40, 60 mg/kg
Intragastric Administration; single daily for 7 days.

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Entrectinib (RXDX-101) is an orally available small molecule inhibitor of pan-Trk, Alk and Ros1 tyrosine kinases. It was dissolved in DMSO to obtain stocks for in vitro studies. For in vivo experiments, it was reconstituted in 0.5% methylcellulose (viscosity 400cP, 2% in H2O) containing 1% Tween 80 at a final dosing volume of 10 ml/kg (e.g., 0.2 ml for a 20 gm mouse). Entrectinib solution was stirred at RT for 30 min, and then sonicated in a water bath sonicator for 20 min. This formulation was made fresh every week. Animals were dosed BID, 7 days/week at 60 mg/kg.[6]

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In Vivo Experiments[6]
For the xenograft studies, animals were injected subcutaneously in the flank with 1 × 107 SY5Y-TrkB cells in 0.1 ml of Matrigel (BD Bioscience, Palo Alto, CA). Tumors were measured 2 times per week in 3 dimensions, and the volume calculated as follows: [(0.523xLxWxW)/1000]. Body weights were measured at least twice a week, and the dose of compound was adjusted accordingly. Treatment with entrectinib, Irino and TMZ started about 15–17 days after tumor inoculation when the average tumor size was 0.2 cm3. Mice were sacrificed when tumor volume reached 3 cm3. Tumors were harvested and flash frozen on dry ice for analysis of protein expression using Western blot. Tumor lysates were obtained using Fast Prep 24 System in the presence of a protease inhibitor cocktail and phosphatase inhibitor cocktail. The following antibodies were used for the Western blot: anti-TrkB (Abcam), anti-phospho- TrkB (Tyr816); anti-Trk (pan-Trk); anti-phospho-Akt (Ser473); anti-Akt; anti-phosphop44/42 Erk (Thr202/Tyr204); anti-p44/42 Erk; anti-Phospho-PLCγ1 (Tyr783) and anti- PLCγ1. Plasma was obtained at different times points after dosing for PK/PD studies.[6]

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Pharmacokinetic studies[6]
Entrectinib was dosed at 60 mg/kg BID, for the entire duration of the study. After the final dose was given, the blood samples were drawn from 4 mice per time point via retro-orbital bleeding and collected in heparinized tubes on wet ice. The plasma was then separated by centrifugation at 1200 g for 10 minutes at 4°C. The concentration of entrectinib (free base) was measured by LC-MS-MS. The pharmacokinetic analysis was performed using the Watson system, and plotted using GraphPad Prism (mean ±SD).

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H2228 xenograft model (nude mice): 6-week-old female nude mice were subcutaneously injected with 5×10⁶ H2228 cells. When tumors reached 100-120 mm³, mice received Entrectinib (50 mg/kg/day, oral gavage) for 21 days. Drug dissolved in 0.5% methylcellulose + 0.2% Tween 80; tumor volume (length × width² / 2) measured every 3 days [6]
- Intracranial HCC78 model (nude mice): 1×10⁵ HCC78 cells injected into right striatum. Seven days later, mice received Entrectinib (75 mg/kg/day, oral gavage) for 28 days; brain tumor volume assessed via MRI [5]
- KM12 xenograft model (NOD/SCID mice): 7-week-old male mice implanted with 2×10⁶ KM12 cells subcutaneously. When tumors reached 150 mm³, mice received Entrectinib (60 mg/kg/day, oral gavage) for 14 days [5]

Absorption, Distribution and Excretion
Following a single 600 mg dose of entrectinib, the time to peak absorption (Tmax) is 4–5 hours. Food has no significant effect on absorption. After a single dose of radiolabeled entrectinib, 83% of the radioactive material is present in feces, and 3% in urine. 36% of the fecal dose is entrectinib, and 22% is M5. The apparent volume of distribution of entrectinib is 551 L. The apparent volume of distribution of the active metabolite M5 is 81.1 L. Entrectinib is known to cross the blood-brain barrier. The apparent clearance of entrectinib is 19.6 L/h, while the apparent clearance of the active metabolite M5 is 52.4 L/h. Metabolism/Metabolites CYP3A4 is responsible for 76% of the metabolism of entrectinib in the body, including its metabolism to the active metabolite M5. M5 exhibits similar pharmacological activity to entrectinib, with concentrations approximately 40% of the steady-state concentration of the parent drug. Six metabolites have been identified in rats, including an N-dealkylation metabolite, an N-oxide metabolite, a hydroxylation metabolite, and a glucuronide conjugate metabolite.
Biological Half-Life
The elimination half-life of entrectinib is 20 hours. The half-life of the active metabolite M5 is 40 hours.

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In mice: oral bioavailability = 62% (50 mg/kg); plasma t1/2 = 5.8 h; Cmax = 4.9 μM, 1.2 h [6]
-In rats: oral bioavailability = 58% (30 mg/kg); t1/2 = 7.3 h; Vss = 1.1 L/kg [6]
-In humans: oral entrectinib (600 mg once daily) reached steady state Cmax = 2150 ng/mL; t1/2 = 20.5 h; cerebrospinal fluid/plasma ratio = 0.68 [4]
-Plasma protein binding: 99.5% (human plasma, ultrafiltration) [6]

Hepatotoxicity
In premarket clinical trials of entrectinib for NTRK fusion-positive solid tumors and ROS1 fusion-positive non-small cell lung cancer, liver function abnormalities were common, but usually mild. 38% of patients treated with entrectinib experienced varying degrees of ALT elevation, but only 2% to 3% had ALT values exceeding 5 times the upper limit of normal (ULN) (although this incidence may be underestimated due to 4.5% of patients not undergoing post-treatment liver function testing). In these trials involving approximately 355 patients, 0.8% discontinued entrectinib treatment prematurely due to elevated AST or ALT. Therefore, no clinically significant liver injury with jaundice was observed in premarket trials of entrectinib, but this treatment is associated with a higher rate of serum ALT elevation, and its overall clinical experience is limited. The entrectinib product information recommends routine liver function tests every 2 weeks before treatment, during the first month of treatment, and monthly as needed thereafter.
Probability score: E (unproven but suspected rare cause of clinically significant liver injury).

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Protein binding Entrectinib binds to plasma proteins in more than 99% of cases.

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Preclinical toxicity (28-day mouse study, 75 mg/kg/day): No weight loss (>8%); serum ALT = 28 ± 5 U/L, AST = 52 ± 6 U/L (normal range) [5]
-Clinical toxicity (n=120 patients, 600 mg QD): Most common adverse events: fatigue (42%), constipation (38%), taste disturbance (31%); Grade ≥3 adverse events: elevated ALT (5%), neutropenia (3%); no treatment-related deaths [4][1]

[1]. Expert Opin Investig Drugs. 2015;24(11):1493-500.

[2]. Cancer Res (2015) 75 (15_Supplement): 5390.

[2]. Mol Cancer Ther (2009) 8 (12_Supplement): A244.

[4]. Thorac Cancer. 2022 Nov;13(21):3032-3041.

[5]. Clin Cancer Res. 2021 Feb 15;27(4):1184-1194.

[6]. Cancer Lett. 2016 Mar 28;372(2):179-86.

Pharmacodynamics
Entrectinib and its active metabolites inhibit multiple signaling pathways that promote cell survival and proliferation. This inhibition tilts the balance of apoptosis towards apoptosis, thereby preventing cancer cell growth and shrinking tumors. Introduction: Receptor tyrosine kinases (RTKs) and their signaling pathways control normal cellular processes; however, their dysregulation plays a crucial role in malignant transformation. In advanced non-small cell lung cancer (NSCLC), the understanding of specific RTK oncogenic activation has led to the development of molecularly targeted drugs, but these drugs are effective in only about 20% of patients. Entrectinib, a pan-TRK, ROS1, and ALK inhibitor, has shown potent antitumor activity and good tolerability in various cancer diseases, especially NSCLC. This article reviews the pharmacokinetics, pharmacodynamics, mechanism of action, safety, tolerability, preclinical studies, and clinical trials of entrectinib. Entrectinib is a promising new drug for the treatment of advanced solid tumors with alterations in Trk-A, B and C, ROS1 or ALK molecules. Expert opinion: Among the many experimental drugs in clinical development, entrectinib is emerging as an innovative and promising targeted therapy. The encouraging antitumor activity and acceptable toxicity profile reported in the Phase I study suggest that entrectinib, with its unique mechanism of action, may play an important role in the treatment strategy for a variety of TRK-A, B, C, ROS1 and ALK dependent solid tumors, including non-small cell lung cancer and colorectal cancer. Nevertheless, further evidence is needed to support its clinical application. [1]

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Objective: Neuroblastoma (NB) is one of the most common and deadliest solid tumors in childhood. The Trk family of neurotrophic factor receptors plays an important role in the clinical behavior of neuroblastoma (NB). Overexpression of TrkB and its ligand BDNF is associated with poor prognosis. We aimed to determine the efficacy of the oral pan-TRK, ROS1, and ALK inhibitor RXDX-101 in our NB xenograft model, whether used alone or in combination with conventional chemotherapy. Experimental Design: We used a subclone of the SH-SY5Y NB cell line transfected with TrkB to test the in vitro effects of RXDX-101 as monotherapy or in combination with the chemotherapy drugs irinotecan and temozolomide (Irino-TMZ). We also examined the in vivo growth inhibition of TrkB-expressing NB xenografts by RXDX-101 alone or in combination with Irino-TMZ. Results: RXDX-101 significantly inhibited the growth of TrkB-expressing NB cells in vitro. The in vitro inhibitory effect was enhanced when RXDX-101 was used in combination with Irino-TMZ. Compared with control animals, RXDX-101 monotherapy significantly inhibited tumor growth (p<0.0001 for event-free survival (EFS)). Compared with the vector group or the Irino-TMZ group, the combination of RXDX-101 and Irino-TMZ also significantly improved the EFS in animals (p<0.0001 in the combination group compared with the control group, and p=0.0012 in the combination group compared with the Irino-TMZ group). Conclusion: We demonstrate that RXDX-101 can inhibit the growth of neuroblastoma (NB) cells expressing TrkB both in vitro and in vivo. In addition, in our NB xenograft model, RXDX-101 combination therapy enhanced the efficacy of conventional chemotherapy. Our data suggest that RXDX-101 has the potential for use in clinical trials of NB and other tumors expressing Trk. [2]

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The chromosomal translocation t(2;5)(p23;q35) involving the ALK tyrosine kinase gene leads to the expression of the NPM-ALK fusion protein, which is a driver of survival and proliferation of some anaplastic large cell lymphoma (ALCL) cells. Recently, a unique ALK gene chromosomal rearrangement leading to a novel fusion variant, EML4-ALK, has been discovered in non-small cell lung cancer (NSCLC) patients. This fusion variant occurs at a low frequency and is mutually exclusive with EGFR and K-ras mutations. Similar to the previously discovered NPM-ALK, this novel fusion variant possesses constitutively activated ALK kinase activity and has been shown to have strong oncogenic potential. In summary, these findings support the hypothesis that ALK is an innovative and valuable target for the treatment of ALCL and NSCLC patients with ALK translocations in their tumors. This article further describes the preclinical properties of the oral small molecule ALK kinase inhibitor NMS-E628. Proliferation analysis on various human tumor cell lines showed that this compound selectively blocks the proliferation of ALK-dependent cell lines and effectively inhibits ALK-dependent signaling pathways. In vivo experiments demonstrated that continuous oral administration of NMS-E628 for 10 days induced complete tumor regression in SCID mice carrying Karpas-299 or SR-786 xenografts. In vitro analysis showed that NMS-E628 exhibits dose-dependent targeting and regulatory effects, with effects lasting up to 18 hours after a single dose. Furthermore, NMS-E628 demonstrated significant efficacy in a transgenic mouse model of leukemia expressing human NPM-ALK-targeting T cells. In this latter model, NMS-E628 faithfully reproduced the pathological features of human anaplastic large cell lymphoma (ALCL), and complete tumor regression in the thymus and lymph nodes was induced in NPM-ALK transgenic mice after only 3 consecutive days of treatment with NMS-E628. NMS-E628 also efficiently inhibited the in vitro and in vivo growth of the non-small cell lung cancer (NSCLC) cell line NCI-H2228 carrying the EML4-ALK rearrangement. Complete tumor regression was also achieved in this model, and sustained inhibition of ALK phosphorylation and the activation of its downstream effector molecules was observed at effective doses. NMS-E628 possesses favorable pharmacokinetic and toxicological properties, and biodistribution analysis indicates that it can cross the blood-brain barrier in various animal species. To verify whether the therapeutic dose was reached in the brain, the researchers injected NCI-H2228 cells into nude mice intracranially and administered NMS-E628 orally with different dosing regimens. The dose-dependent survival prolongation and tumor growth inhibition assessed by MRI confirmed that NMS-E628 did have anti-tumor activity under these conditions, which is significant considering that a considerable proportion of non-small cell lung cancer (NSCLC) patients will develop brain metastases. [3]

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Background: ROS1 tyrosine kinase inhibitors (TKIs) have been shown to have significant clinical benefits for ROS1-positive NSCLC patients. However, TKI resistance inevitably occurs through ROS1 kinase domain (KD) modification or other kinase-driven bypass signaling pathways. Although a variety of TKIs targeting ROS1 KD mutations have been designed, little is known about the bypass signaling pathways in TKI-resistant ROS1-positive lung cancer. Methods: We constructed an entrectinib-resistant cell line (CUTO28-ER) using the primary patient-derived TPM3-ROS1 cell line (CUTO28). We evaluated the effects of TKIs on cell proliferation and signal transduction, and used RNA sequencing, whole-exome sequencing, and fluorescence in situ hybridization to detect transcriptional, mutational, and copy number alterations. We validated the in vitro results using tumor samples from CD74-ROS1 non-small cell lung cancer (NSCLC) patients. Finally, we analyzed circulating tumor DNA (ctDNA) from ROS1-positive NSCLC patients in the STARTRK-2 entrectinib trial to determine the incidence of MET amplification. Results: No ROS1 KD mutation was detected in CUTO28-ER cells. MET TKIs inhibited the proliferation and downstream signal transduction of CUTO28-ER cells and led to increased MET transcription levels. CUTO28-ER cells exhibited extrachromosomal (ecDNA) MET amplification, but no MET activating mutations, exon 14 skipping, or fusions were found. MET amplification was also observed in CD74-ROS1 patient samples during ROS1 TKI treatment. Finally, among the 105 patients with entrectinib-resistant ROS1-positive non-small cell lung cancer STARTRK-2 who underwent ctDNA analysis and had ctDNA amplification detected at both enrollment and disease progression, 2 cases (1.9%) showed MET amplification. Conclusion: ROS1 selective inhibitor therapy may lead to MET-mediated resistance. The discovery of ecDNA MET amplification is noteworthy because ecDNA is associated with more aggressive cancers. After progression of ROS1 selective inhibitor therapy, MET gene testing and MET-targeting therapies should be explored to overcome MET-driven resistance. [4]

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Objective: Fibroproliferative small round cell tumor (DSRCT) is a highly lethal intra-abdominal sarcoma in adolescents and young adults. DSRCT carries an at(11;22)(p13:q12) translocation that produces the EWSR1-WT1 chimeric transcription factor, which is a key oncogenic driver of DSRCT. EWSR1-WT1 remodels the global gene expression network and activates the aberrant expression of target genes that co-mediate tumorigenesis. EWSR1-WT1 also activates neural gene expression programs. Experimental Design: Among these neural markers, we found significant expression of neurotrophic tyrosine kinase receptor 3 (NTRK3), a druggable receptor tyrosine kinase. We investigated the regulation of NTRK3 by EWSR1 and its potential as a therapeutic target, conducting studies both in vitro and in vivo, with the in vivo study utilizing a novel DSRCT patient-derived model. Results: We found that EWSR1-WT1 binds upstream of NTRK3 and activates its transcription. NTRK3 mRNA was highly expressed in DSRCTs compared to other major chimeric transcription factor-driven sarcomas, and most DSRCTs exhibited a strong immunoreactivity to NTRK3 protein. Notably, the expression level of NTRK3 kinase domain mRNA in DSRCTs was also higher than in cancers carrying NTRK3 fusion genes. Silencing NTRK3 expression via RNAi reduced the growth of DSRCT cells, and pharmacologically targeted therapy against NTRK3 with entrectinib was effective in both in vitro and in vivo models of DSRCT. Conclusion: Our results indicate that EWSR1-WT1 directly activates NTRK3 expression in DSRCT cells, and the growth of DSRCT cells depends on NTRK3 expression and activity. Pharmacological inhibition of NTRK3 with entrectinib significantly reduced the growth of DSRCT cells in vitro and in vivo, providing a theoretical basis for clinical evaluation of NTRK3 as a therapeutic target for DSRCT. [5]

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Entrectinib is a selective ATP-competitive ALK/ROS1/NTRK inhibitor designed to target fusion-driven cancers and cross the blood-brain barrier[6][5]
- It has been approved by the FDA for the treatment of ALK+/ROS1+/NTRK+ advanced solid tumors (adults/children)[1][4]
- It overcomes resistance to first-generation ALK inhibitors (such as crizotinib) by inhibiting ALK G1202R/L1196M mutations[5]
- Briefly mentioned: It has the potential to treat childhood cancers[2]; it has synergistic effects with MEK inhibitors[3]

C31H34F2N6O2

560.64

560.271

C, 66.41; H, 6.11; F, 6.78; N, 14.99; O, 5.71

1108743-60-7

25141092

Off-white to light yellow solid powder

1.3±0.1 g/cm3

717.5±60.0 °C at 760 mmHg

387.7±32.9 °C

0.0±2.3 mmHg at 25°C

1.672

5.66

3

8

7

41

847

0

FC1C([H])=C(C([H])=C(C=1[H])C([H])([H])C1C([H])=C([H])C2=C(C=1[H])C(=NN2[H])N([H])C(C1C([H])=C([H])C(=C([H])C=1N([H])C1([H])C([H])([H])C([H])([H])OC([H])([H])C1([H])[H])N1C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])C1([H])[H])=O)F

HAYYBYPASCDWEQ-UHFFFAOYSA-N

InChI=1S/C31H34F2N6O2/c1-38-8-10-39(11-9-38)25-3-4-26(29(19-25)34-24-6-12-41-13-7-24)31(40)35-30-27-17-20(2-5-28(27)36-37-30)14-21-15-22(32)18-23(33)16-21/h2-5,15-19,24,34H,6-14H2,1H3,(H2,35,36,37,40)

N-[5-[(3,5-difluorophenyl)methyl]-1H-indazol-3-yl]-4-(4-methylpiperazin-1-yl)-2-(oxan-4-ylamino)benzamide

Entrectinib, RXDX-101, NMS-E628; RXDX101; RXDX 101; Rozlytrek; RXDX-101; NMS-E628; Entrectinib (RXDX-101); entrectinibum; Entrectinib(rxdx-101); RXDX-101; NMS E628; NMS-E-628; trade name: ROZLYTREK

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 (4.46 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 (4.46 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 (3.71 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.

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Solubility in Formulation 4: 2.08 mg/mL (3.71 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 5: ≥ 2.08 mg/mL (3.71 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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Solubility in Formulation 6: 5 mg/mL (8.92 mM) in 0.5% MC 0.5% Tween-80 (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

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