WT ALK (IC50 = 1.01 nM); ALK(L1196M) (IC50 = 1.08 nM); ALK(G1202R) (IC50 = 1.26 nM); Trk receptor; ROS1

TPX-0005 is an orally available and potent ATP-competitive inhibitor that can be taken orally and is effective against clinically resistant mutants of ALK, ROS1, TRKA, TRKB, and TRKC recombinant kinases. Through the inhibition of target phosphorylation and concurrent inactivation of downstream effectors like ERK, AKT, and STAT3, TPX-0005 exhibits strong anti-proliferative activity in the range of sub-nanomolar to low nanomolar in a number of human cancer cell lines and engineered stable cell lines expressing the targeted oncogenes or their solvent front mutants[2]. Similar to saracatinib, TPX-0005 also inhibits H2228 cell migration in a wound healing assay. In addition to inhibiting a wide range of mutant ALKs and the wild-type ALK, TPX-0005 can suppress metastatic features by inhibiting SRC, which also helps it overcome primary resistance[1].

TPX-0005 treatment results in significant regression of tumors harboring the oncogenic ALK, ROS1 and TRKC fusions in patient derived xenograft tumor models. Additionally, via inhibition of target phosphorylation, TPX-0005 demonstrates strong anti-tumor activity in a number of mouse xenograft tumor models in tumors containing solvent front mutation-carrying oncogenes as well as tumors containing wildtype oncogenic targets[2].

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Repotrectinib inhibits tumor growth in a xenograft model of neuroblastoma [3]
To further investigate repotrectinib in neuroblastoma we employed a mouse xenograft model. CLB-BAR neuroblastoma cells were injected subcutaneously and the resulting tumors treated with either repotrectinib (20 mg/kg, twice daily), crizotinib (80 mg/kg, once daily) or vehicle control. Animals treated with repotrectinib displayed minor increases in tumor volume during the 14 day treatment (Fig. 4a). Tumor growth inhibition (TGI) values of 87.07% and 66.4% were observed with repotrectinib and crizotinib, respectively (Fig. 4a). Upon repotrectinib drug release after 14 days tumor growth resumed (Fig. 4a). Tumors in the vehicle control group continued to grow reaching a significant increase compared to repotrectinib treatment after day 6 (p = 0.008) (Fig. 4a). As expected, crizotinib displayed antitumor activity in agreement with previous reports. Tumor volume and weight were significantly decreased at day 14 in both repotrectinib and crizotinib groups, however, crizotinib was less effective in inhibition of tumor growth than repotrectinib (Fig. 4a). In addition to effective inhibition of tumor growth, animals treated with repotrectinib exhibited an increase in weight, showing a significant weight gain over the 14 day experiment (p < 0.0001 at day 14) (Fig. 4b).

TPX-0005/repotrectinib is a novel, rationally-designed, highly potent ALK/ROS1/TRK inhibitor, has IC50 values of 5.3 nM, 1.01 nM, 1.26 nM, and 1.08 nM for SRC, WT ALK, ALK G1202R, and ALK L1196M, in that order. It might possess anticancer properties. By significantly inhibiting the phosphorylation of EML4-ALK (IC50 13 nM) and the SRC substrate paxillin (IC50 107 nM), it successfully overcomes this primary resistance (IC50 100 nM in the cell proliferation assay). In a wound healing assay, PX-0005 inhibits H2228 cell migration with activity comparable to that of saracatinib. All things considered, TPX-0005 has a very promising profile and is capable of defeating several ALK resistance mechanisms, such as secondary mutations, bypass signaling activation, and EMT. As such, it deserves further clinical research.

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Inhibition of ALK activity in neuroblastoma cell lines [3]
CLB-BAR and CLB-GE cells were plated in 10 cm dishes and treated with either 200 or 300 nM of repotrectinib or crizotinib as described previously. Cell lysates were collected after 1 h treatment and protein concentration was determined by BCA assay. Protein lysates were analyzed by western blotting and visualized using ECLTM Prime Western Blotting Derection Reagent. Each membrane of primary phospho-antibodies was stripped using 0.5 M NaOH for 30 min and re-blotted for total protein. β-actin was used to verify equality of sample loading. Experiments were performed in triplicates. Images were cropped using Adobe Photoshop CS6 and the final version was done using Illustrator CS6.

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ALK phosphorylation IC50 in PC-12 cells [3]
Cells were transiently transfected, as described previously with ALK mutant constructs or the wild type ALK construct as specified. Constructs were confirmed by sequencing. Briefly, 3 × 106 cells were electroporated using 100 µL of Ingenio electroporation solution and 0.75 μg of mutant ALK constructs or 1.5 μg of the wild type variant in an Amaxa electroporator. Transfections (four) were pooled in a final volume of 10.5 mL, and 500 µL were plated per well into 24-well plates. After 48 h, cells were treated with serial dilutions of either repotrectinib or crizotinib for four hours. Cell lysates were collected and analyzed by immunoblotting. Actin, phospho-ALK-Y1604 and pan-ALK band intensity were determined using Image Studio Lite, actin was used for normalization of phospho-ALK-Y1604. pan-ALK was performed to corroborate equal loading. Images were cropped and contrast adjusted using Adobe Photoshop CS6. The IC50 of the ALK phosphorylation was defined as the concentration of drug that resulted in 50% levels of ALK-Y1604 phosphorylation with respect to non-treated cells.

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Neurite outgrowth assay [3]
ALK constructs, either mutant (0.75 μg) or wild type (1.5 μg), and pEGFPN1 (0.5 μg) were co-transfected into 2 × 106 PC-12 cells. After transfection, cells were diluted in 7.5 mL of medium, mixed and 300 µL were seeded into 24-well plates. The next day cells were treated with either 200 nM repotrectinib or 250 nM crizotinib, wild type ALK was stimulated with 1 µg/mL of ALKAL111,50. Neurite outgrowth was analyzed 48 h post transfection. Neurite formation was determined with a Zeiss Axiovert 40 CFL microscope, GFP-positive cells carrying neurites double the size of the cell body were considered positive. Experiments were performed in triplicate.

TPX-0005 is also a potent SRC inhibitor (IC50 5.3 nM). In tests of cell proliferation, the increased SRC kinase activity in the H2228 lung cancer cell line confers resistance to crizotinib (IC50 1200 nM) and ceritinib (IC50 1000 nM). With strong inhibition of the phosphorylation of EML4-ALK (IC50 13 nM) and the SRC substrate paxillin (IC50 107 nM), as well as other downstream signaling targets, TPX-0005 effectively overcame this primary resistance (IC50 100 nM in cell proliferation assay). Similar to saracatinib, TPX-0005 inhibited H2228 cell migration in a wound healing assay.

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Proliferation assay [3]
Neuroblastoma cell lines were seeded into 48-well plates to achieve 30–40% confluency at the time of treatment. Repotrectinib and crizotinib were dissolved in DMSO and prepared freshly prior to addition. The concentrations of repotrectinib and crizotinib used for proliferation assays were 50, 100, 200, 300, 400 and 500 nM. The amount of DMSO did not exceed 0.1% of total medium volume. Plates were placed in an Incucyte and 16 images/well were taken every 24 h for 5 days. Each experiment was repeated three independent times and performed in triplicate. Images were taken using the 10x magnification objective for the phase contrast channel and were processed and analyzed using the Incucyte live-cell imaging system. Analysis definition was created by selecting basic analyzer, phase contrast channel and selecting 6–8 representative images. The segmentation and the minimum area (µm2) filters were adjusted to achieve a maximum detection of cells excluding debris. The analysis definition was done for each cell line separately and those specific parameters were used for all the images in each cell line group.

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Apoptosis determination [3]
Cells were seeded in 6-well plates and treated with repotrectinib or crizotinib with the indicated concentrations for 24 h. For western blotting, cell lysates were collected using RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP40, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1x phosphoSTOP, 1x cOmplete EDTA-free) and protein concentration was determined with the Pierce®BCA protein assay kit. Samples were immunoblotted with PARP antibody, which recognizes both full length and cleaved PARP1. Actin was used to normalize cleaved PARP1 in three independent experiments. Signal for PARP1 and actin was visualized simultaneously with immobilon Forte Western HRP substrate in an Odyssey Fc system, band intensity was determined using Image Studio Lite software. Flow cytometry was employed to analyze cells stained with Annexin V and propidium iodide as a complementary assay to PARP cleavage. Cells were collected and stained after treatment according to the manufacture’s protocol (Dead cell apoptosis kit) and deposited in a 5 mL tube through cell strainer cap before analysis using an LSRII flow cytometer. Data analysis was performed using FlowJo v9.6 software. Image processing was done using Adobe Photoshop CS6 and Illustrator C6S.

Xenograft neuroblastoma model [3]
In order to study the efficacy of repotrectinib we used a xenograft model of neuroblastoma. Female BALB/cAnNRj-Foxn1nu mice (Janvier Laboratory) 4–6 weeks old were housed with access to food and water ad libitum in a 12:12 light-dark cycle. The animals were allowed to acclimatize for 1 week prior to being subcutaneously injected into the left flank with 1 × 106 CLB-BAR cells in serum-free medium mixed with Matrigel Matrix at a ratio of 1:1. The total injection volume was 100 µL. Once the tumor reached a volume of 150 mm3, mice were randomized to treatment groups using 10 animals per group. Tumor tissues treated with crizotinib or the vehicle presented in this paper have previous been used as controls in experiments by Alam et al. Compounds were administered orally at 80 mg/kg bodyweight daily for crizotinib and 20 mg/kg bodyweight twice daily (40 mg/kg per day) for repotrectinib for 14 days, crizotinib treatment was 4 fold higher than repotrectinib. The control group was treated with the vehicle solution; a mix of 1% carboxymethylcellulose sodium salt and 0.5% Tween-80. Tumor volume was measured by caliper every two days and calculated by the following equation: V = (p/6) × L × W2 (V, volume; p, pi; L, length; W, width). Tumor Growth Inhibition (TGI) was calculated according to: TGI = 100% x (1-((TVt-TV0)/(CVt-CV0))) where TVt was the tumor volume in the treated group at the end of the experiment, TV0 was the tumor volume in the treated group at the beginning of the study, CVt was the tumor volume in the control group at the end of the study, and CV0 was the tumor volume in the control group at the beginning of the treatment. Animal weight was recorded every two days.

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repotrectinib/TPX-0005(30 mg/kg); oral

Mice with established PC9 tumors

Absorption, Distribution and Excretion
The geometric mean (CV%) of repotrectinib steady state peak concentration (Cmax,ss) is 713 (32.5%) ng/mL and the area under the time concentration curve (AUC0-24h,ss) is 7210 (40.1%) ng•h/mL following the approved recommended twice daily dosage in patients with cancer. Repotrectinib Cmax and AUC0-inf increases approximately dose-proportional (but less than linear with estimated slopes of 0.78 and 0.70, respectively) over the single dose range of 40 mg to 240 mg (0.25 to 1.5 times the approved recommended dosage). Steady-state PK was time-dependent with an autoinduction of CYP3A4. Steady-state is achieved within 14 days of daily administration of 160 mg. The geometric mean (CV%) absolute bioavailability of repotrectinib is 45.7% (19.6%). Peak repotrectinib concentration occurred at approximately 2 to 3 hours post a single oral dose of 40 mg to 240 mg (0.25 to 1.5 times the approved recommended dosage) under fasted conditions. No clinically significant differences in repotrectinib pharmacokinetics were observed in patients with cancer following administration of a high-fat meal (approximately 800-1000 calories, 50% fat).
Following a single oral 160 mg dose of radiolabeled repotrectinib, 4.84% (0.56% as unchanged) was recovered in urine and 88.8% (50.6% unchanged) in feces.
The geometric mean (CV%) apparent volume of distribution (Vz/F) was 432 L (55.9%) in patients with cancer following a single 160 mg oral dose of repotrectinib.
The geometric mean (CV%) apparent oral clearance (CL/F) was 15.9 L/h (45.5%) in patients with cancer following a single 160 mg oral dose of repotrectinib.

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Metabolism / Metabolites
Repotrectinib is primarily metabolized by CYP3A4 followed by secondary glucuronidation.

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Biological Half-Life
The repotrectinib mean terminal half-life is approximately 50.6 h for patients with cancer following a single dose. The steady-state repotrectinib terminal half-life is approximately 35.4 h for patients with cancer.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the use of repotrectinib during breastfeeding. Because repotrectinib is 94.5% bound to plasma proteins, the amount in milk is likely to be low and oral bioavailability is less than 50%; however, the drug’s half-life is about 50 hours in adults. The manufacturer recommends that breastfeeding be discontinued during repotrectinib therapy and for 10 days after the final 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.

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Protein Binding
Repotrectinib binding to plasma protein was 95.4% in vitro. The blood-to-plasma ratio was 0.56 in vitro.

[1]. Cancer Res (2016) 76 (14_Supplement): 2132.

[2]. Cancer Res (2017) 77 (13_Supplement): 3168.

[3]. Sci Rep. 2019 Dec 18;9:19353.

Repotrectinib is an azamacrocycle with formula C18H18FN5O2. It is a tyrosine kinase inhibitor (highly potent against ROS1, TRKA-C, and ALK) used for the treatment of locally advanced or metastatic ROS1-positive non-small cell lung cancer. It has a role as an EC 2.7.10.1 (receptor protein-tyrosine kinase) inhibitor and an antineoplastic agent. It is a member of monofluorobenzenes, a pyrazolopyrimidine, a cyclic ether, a secondary carboxamide and an azamacrocycle.
Repotrectinib is a next-generation tyrosine kinase inhibitor (TKI) specifically designed to address resistance in the treatment of non-small cell lung cancer (NSCLC), specifically due to mutations in the ROS1 gene. ROS1 mutations are one of the defined oncogenic drives of NSCLC, and the solvent-front mutation ROS1 G2032R is responsible for 50 to 60% of [crizotinib]-resistant cases. Repotrectinib possesses a compact macrocyclic structure that both limits adverse interactions with resistance mutation hotspots and targets mutations in the solvent-front region. Although resistance to multiple TKI has been reported, including [crizotinib], [lorlatinib], [taletrectinib], and [entrectinib], there has been no reported case of repotrectinib resistance. On November 15th, 2023, the FDA approved repotrectinib under the brand name Augtyro for the treatment of locally advanced or metastatic ROS1-Positive NSCLC. This approval is based on favorable results from the TRIDENT-1 study, where the objective response rate was 79% in TKI-naive patients and 38% in TKI-pretreated patients respectively.
Repotrectinib is a Kinase Inhibitor. The mechanism of action of repotrectinib is as a Proto-Oncogene Tyrosine-Protein Kinase ROS1 Inhibitor, and Tropomyosin Receptor Tyrosine Kinase A Inhibitor, and Tropomyosin Receptor Tyrosine Kinase B Inhibitor, and Tropomyosin Receptor Tyrosine Kinase C Inhibitor, and Cytochrome P450 3A4 Inducer.
Repotrectinib is an orally available inhibitor of multiple kinases, including the receptor tyrosine kinase anaplastic lymphoma kinase (ALK), c-ros oncogene 1 (ROS1), the neurotrophic tyrosine receptor kinase (NTRK) types 1, 2 and 3, the proto-oncogene SRC, and focal adhesion kinase (FAK), with potential antineoplastic activity. Upon oral administration, repotrectinib binds to and inhibits wild-type, point mutants and fusion proteins of ALK, ROS1, NTRK1-3, SRC, FAK and, to a lesser extent, other kinases. Inhibition of these kinases leads to the disruption of downstream signaling pathways and the inhibition of cell growth of tumors in which these kinases are overexpressed, rearranged or mutated.

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Drug Indication
Repotrectinib is indicated for the treatment of adult patients with locally advanced or metastatic ROS1-positive non-small cell lung cancer (NSCLC).
Treatment of all conditions included in the category of malignant neoplasms (except haematopoietic neoplasms)

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Mechanism of Action
Repotrectinib is an inhibitor of proto-oncogene tyrosine-protein kinase ROS1 (ROS1) and of the tropomyosin receptor tyrosine kinases (TRKs) TRKA, TRKB, and TRKC.

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In summary, we show that repotrectinib abrogates ALK activity in in vitro biochemical assays, in a manner comparable to crizotinib. However, repotrectinib is superior to crizotinib in abrogating xenograft tumor growth, likely due to its pharmacology properties, and also perhaps reflecting that repotrectinib is a potent inhibitor with a broader target kinase range. Immunostaining of the tumor material showed a significant increase in CD31 for both ALK inhibitors compared to the control group, indicating increased density of blood vessels. The increase of CD31-positive vessels could be in part due to the overall decrease in tumor volume and lead to a perceived increase of expression of CD31-positive vessels. However, we also observe an increase of the pericyte marker desmin in repotrectinib treated tumors, as shown in Fig. 5, indicating an increase in pericyte number. The increase in desmin would suggest that it is not simply tumor shrinkage that leads to an increase of CD31-positive cells. Instead, the increased desmin suggests that tumor stress by ALK TKI treatment leads to hypoxia and subsequent angiogenesis and the recruitment of pericytes that bring about the overall increase of CD31-positive vessels. Increased CD31 levels have been noted before in treated tumors and have been considered to reflect changing architecture in the tumor or an endothelial cell response to the therapeutic challenge. Altogether, these data suggest that upon treatment with the recently described ALK TKI repotrectinib, growth of ALK-driven neuroblastoma cells and xenografts are inhibited, suggesting that repotrectinib should be further explored in a neuroblastoma setting.[3]

C18H18FN5O2

355.37

355.144

C, 60.84; H, 5.11; F, 5.35; N, 19.71; O, 9.00

1802220-02-5

1802220-02-5; 2058227-19-1

135565923

White to off-white solid powder

1.5±0.1 g/cm3

1.694

1.71

2

6

0

26

524

2

FC1C=CC2=C(C=1)[C@@H](C)NC1C=CN3C(=C(C=N3)C(NC[C@H](C)O2)=O)N=1

FIKPXCOQUIZNHB-WDEREUQCSA-N

InChI=1S/C18H18FN5O2/c1-10-8-20-18(25)14-9-21-24-6-5-16(23-17(14)24)22-11(2)13-7-12(19)3-4-15(13)26-10/h3-7,9-11H,8H2,1-2H3,(H,20,25)(H,22,23)/t10-,11+/m0/s1

(3R,11S)-6-fluoro-3,11-dimethyl-10-oxa-2,13,17,18,21-pentazatetracyclo[13.5.2.04,9.018,22]docosa-1(21),4(9),5,7,15(22),16,19-heptaen-14-one

Ropotrectinib; TPX0005; 1802220-02-5; Ropotrectinib; Augtyro; 08O3FQ4UNP; Repotrectinib [USAN]; repotrectinibum; TPX-0005; TPX 0005; Augtyro

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 (7.03 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.03 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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