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
In cancer patients, after twice-daily administration of repotinib at the approved recommended dose, the geometric mean (CV%) of steady-state peak concentration (Cmax, ss) was 713 (32.5%) ng/mL, and the area under the time-concentration curve (AUC0-24h, ss) was 7210 (40.1%) ng•h/mL. Within a single-dose range of 40 mg to 240 mg (equivalent to 0.25 to 1.5 times the approved recommended dose), the Cmax and AUC0-inf of repotinib increased approximately dose-proportional (but not linearly, with estimated slopes of 0.78 and 0.70, respectively). Steady-state pharmacokinetics are time-dependent and involve CYP3A4 autoinduction. Steady-state concentrations are reached within 14 days after a daily dose of 160 mg. The geometric mean (CV%) absolute bioavailability of repotinib was 45.7% (19.6%). Peak plasma concentrations occurred approximately 2 to 3 hours after a single oral dose of 40 to 240 mg (0.25 to 1.5 times the approved recommended dose) of repotinib in a fasting state. No clinically significant differences in the pharmacokinetics of repotinib were observed after cancer patients consumed a high-fat meal (approximately 800–1000 calories, 50% fat). Following a single oral dose of 160 mg of radiolabeled repotinib, 4.84% was recovered in urine (0.56% unchanged) and 88.8% was recovered in feces (50.6% unchanged). The geometric mean (CV%) of the apparent volume of distribution (Vz/F) after a single oral dose of 160 mg of repotinib in cancer patients was 432 L (55.9%).
Following a single oral dose of 160 mg repotinib in cancer patients, the geometric mean (CV%) of apparent oral clearance (CL/F) was 15.9 L/h (45.5%).
Metabolism/Metabolites

Repotinib is primarily metabolized via CYP3A4, and secondarily via glucuronidation.
Biological Half-Life

The mean terminal half-life of repotinib in cancer patients after a single dose is approximately 50.6 hours. At steady state, the terminal half-life of repotinib in cancer patients is approximately 35.4 hours.

Hepatotoxicity
Liver function abnormalities were common in premarket trials of repotinib for the treatment of ROS1-positive non-small cell lung cancer, but were usually mild to moderate and self-limiting. In the primary registration trial of 264 patients, 34% experienced elevated ALT and 40% experienced elevated AST, with 3.1% and 1.9% of patients having ALT and AST values exceeding 5 times the upper limit of normal, respectively. The median time to onset of transaminase elevations was 15 days (range 1 day to 1 year). 1.1% of patients discontinued treatment due to ALT abnormalities, but permanent discontinuation was rare. No enzyme elevations with jaundice or other symptoms, nor any life-threatening or fatal liver injury, were reported. Since repotinib's approval, no clinically significant cases of liver injury with jaundice have been reported, but clinical experience with its use is limited.
Probability score: E (Unproven but suspected rare cause of clinically significant liver injury).
Effects during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information regarding the use of repotinib during lactation. Because repotinib binds to plasma proteins at a rate of 94.5%, its concentration in breast milk may be low, and its oral bioavailability is less than 50%; however, the half-life of this drug in adults is approximately 50 hours. The manufacturer recommends discontinuing breastfeeding during repotinib treatment and for 10 days after the last dose.
◉ Effects on breastfed infants
As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding
In vitro studies show that repotinib binds to plasma proteins at a rate of 95.4%. In vitro studies show a blood-to-plasma concentration ratio of 0.56.

[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 azahexacyclic macrocyclic compound with the molecular formula C18H18FN5O2. It is a tyrosine kinase inhibitor (highly effective against ROS1, TRKA-C, and ALK) used to treat locally advanced or metastatic ROS1-positive non-small cell lung cancer (NSCLC). It is an EC 2.7.10.1 (receptor protein tyrosine kinase) inhibitor and an antitumor drug. It belongs to the monofluorobenzene, pyrazolopyrimidine, cyclic ether, secondary amide, and azahexacyclic macrocyclic compounds. Repotrectinib is a next-generation tyrosine kinase inhibitor (TKI) specifically designed to treat resistance in NSCLC, particularly resistance caused by ROS1 gene mutations. ROS1 mutations are one of the known oncogenic drivers in NSCLC, with the solvent-front mutation ROS1 G2032R being the cause of 50% to 60% of crizotinib-resistant cases. Riprotinib has a compact macrocyclic structure, which reduces adverse interactions with resistance mutation hotspots and targets mutations in solvent front regions. Although resistance to several tyrosine kinase inhibitors (TKIs) has been reported, including crizotinib, lorlatinib, talectinib, and entrectinib, there have been no reports of resistance to riprotinib. On November 15, 2023, the FDA approved riprotinib (brand name Augtyro) for the treatment of locally advanced or metastatic ROS1-positive NSCLC. This approval was based on favorable results from the TRIDENT-1 study, which showed an objective response rate of 79% in TKI-naïve patients and 38% in TKI-naïve patients. Riprotinib is a kinase inhibitor. The mechanism of action of repotinib is as an inhibitor of proto-oncogene tyrosine protein kinase ROS1, tropomyosin receptor tyrosine kinase A, tropomyosin receptor tyrosine kinase B, tropomyosin receptor tyrosine kinase C, and cytochrome P450 3A4 inducer. Repotinib is an orally administered multi-kinase inhibitor, including receptor tyrosine kinase anaplastic lymphoma kinase (ALK), c-ros oncogene 1 (ROS1), neurotrophic tyrosine receptor kinases (NTRK) types 1, 2, and 3, proto-oncogene SRC, and focal adhesion kinase (FAK), possessing potential antitumor activity. After oral administration, repotinib binds to and inhibits wild-type, point mutant, and fusion proteins of ALK, ROS1, NTRK1-3, SRC, and FAK, with less inhibitory activity against other kinases. Inhibition of these kinases leads to dysregulation of downstream signaling pathways and inhibits the growth of tumor cells with overexpression, rearrangement, or mutation of these kinases. Drug Indications Riprotinib is indicated for the treatment of adult patients with locally advanced or metastatic ROS1-positive non-small cell lung cancer (NSCLC). It is also indicated for the treatment of all malignancies (excluding hematopoietic malignancies). Mechanism of Action Riprotinib is an inhibitor of the proto-oncogene tyrosine protein kinase ROS1 (ROS1) and tropomyosin receptor tyrosine kinases (TRKs) TRKA, TRKB, and TRKC. In summary, we found that riprotinib inhibits ALK activity in some way in in vitro biochemical assays. Compared with crizotinib, riprotinib is superior to crizotinib in inhibiting xenograft tumor growth, which may be related to its pharmacological properties and may also reflect that riprotinib is a potent inhibitor with a broader range of targeted kinases. Immunostaining of tumor tissues showed that both ALK inhibitors significantly increased CD31 expression compared to the control group, indicating increased vascular density. The increase in CD31-positive angiogenesis may be partly due to the overall reduction in tumor volume, leading to a pseudo-increase in CD31-positive angiogenesis expression. However, as shown in Figure 5, we also observed an increase in desmin, a pericyte marker, in tumors treated with repotrectinib, indicating an increase in the number of pericytes. The increase in desmin suggests that the increase in CD31-positive cells is not solely due to tumor shrinkage. Rather, the increase in desmin suggests that tumor stress induced by ALK TKI treatment leads to hypoxia, which in turn triggers angiogenesis and pericyte recruitment, ultimately resulting in an increase in the total number of CD31-positive vessels. Previous studies have reported elevated CD31 levels in treated tumors, which is thought to reflect changes in tumor structure or the response of endothelial cells to the challenge of treatment. In summary, these data suggest that treatment with the recently reported ALK TKI drug repotrectinib inhibits the growth of ALK-driven neuroblastoma cells and xenografts, suggesting that repotrectinib should be further investigated in the treatment of neuroblastoma. [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.)