TrkA (IC50 = 1 nM); TrkB (IC50 = 3 nM); TrkC (IC50 = 5 nM); ROS1 (IC50 = 12 nM); ALK (IC50 = 7 nM)

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]

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 NMS‐E628 for as little as 3 consecutive days induced complete regression of tumor masses observed in the thymus and in lymph nodes.[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]

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.

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).

Absorption, Distribution and Excretion
Entrectinib has a Tmax of 4-5 h after administration of a single 600 mg dose. Food does not produce a significant effect on the extent of absorption.
After a single radio-labeled dose of entrectinib, 83% of radioactivity was present in the feces and 3% in the urine. Of the dose in the feces, 36% was present as entrectinib and 22% as M5.
Entrectinib has an apparent volume of distribution of 551 L. The active metabolite, M5, has an apparent volume of distribution of 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.

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Metabolism / Metabolites
CYP3A4 is responsible for 76% of entrectinib metabolism in humans including metabolism to the active metabolite, M5. M5 has similar pharmacological activity to entrectinib and exists at approximately 40% of the steady state concentration of the parent drug. In rats, six in vivo metabolites have been identified including N-dealkylated, N-oxide, hydroxylated, and glucuronide conjugated metabolites.

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Biological Half-Life
Entrectinib has a half-life of elimination of 20 h. The active metabolite, M5, has a half-life of 40 h.

Hepatotoxicity
In the prelicensure clinical trials of entrectinib in patients with NTRK fusion gene positive solid tumors and ROS1 fusion gene positive non-small cell lung cancer, liver test abnormalities were frequent although usually mild. Some degree of ALT elevation arose in 38% of entrectinib treated patients, but were above 5 times the upper limit of normal (ULN) in only 2% to 3% (although the incidence may have been underestimated as 4.5% of patients had no post-treatment liver function tests). In these trials that enrolled approximately 355 patients, entrectinib was discontinued early due to increased AST or ALT in 0.8% of patients. Thus, in preregistration trials of entrectinib there were no instances of clinically apparent liver injury with jaundice, but therapy was associated with a high rate of serum ALT elevations and the total clinical experience with its use has been limited. The product label for entrectinib recommends monitoring for routine liver tests before, at 2 week intervals during the first month of therapy, and monthly thereafter as clinically indicated.
Likelihood score: E* (unproven but suspect rare cause of clinically apparent liver injury).

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Protein Binding
Entrectinib is over 99% bound to plasma proteins.

[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 metabolite suppress several pathways which contribute to cell survival and proliferation. This suppression shifts the balance in favor of apoptosis thereby preventing cancer cell growth and shrinking tumors.

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.)