Synthesis, radiolabeling, and evaluation of a 68Ga-labeled tyrosine kinase inhibitor for detecting EGFRT790M mutations in vivo

Radiolabeled tyrosine kinase inhibitors (TKIs) have been used in clinics for the detection of epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer (NSCLC) patients by positron emission tomography (PET), and the potential responder for TKI targeted therapy can be selected and benefited in subsequent TKI-based anti-tumor treatment. However, as activated T790M resistance mutation is often observed in NSCLC patients underwent EGFR TKI based therapies, the detection of EGFR mutation status is of great importance to evaluate therapeutic efficacy and prolongs overall survival with these patients. In addition, no radiotracer has been reported with the ability to detect EGFR^T790M mutation in vivo at present. Based on the chemical structure of Rociletinib, a novel radiolabeled PET tracer (⁶⁸Ga-1) was developed with potent binding affinity in vitro (with IC₅₀ value of 9.21 nM). In addition, ⁶⁸Ga-1 also showed promising properties in detection EGFR^T790M mutation in the subsequent in vitro and in vivo evaluations. Herein we report the synthesis, radiolabeling, and preclinical evaluation of ⁶⁸Ga-1 for detecting EGFR^T790M mutation, and our result indicated that Rociletinib may be regarded as a lead compound to develop a selective EGFR^T790M PET tracer with further modification and optimizations.

Introduction

It is considered that the EGFR mutation is the driving force in NSCLC patients since a great percentage of patients can be detected with EGFR gene alteration and overexpression (Sharma et al., 2007; Sequist et al., 2015; Zheng et al., 2016). As a subfamily member of receptor tyrosine kinase, EGFR plays crucial roles in signaling pathways related to cellular survival, multiplication, differentiation and metastasis (Zheng et al., 2016; Bahce et al., 2017). Activating EGFR was thus regarded as an attractive biomarker for tumor imaging and drug development in recent years (Bernard-Gauthier et al., 2015; Bahce et al., 2017). During the last decades, EGFR TKIs have demonstrated their potential to improve clinical outcomes in targeted therapies, with an overall response rate of about 80% in NSCLC patients harboring activating EGFR alterations (Yeh et al., 2011; Sun et al., 2018). The EGFR mutation status is of great importance to predict the clinical outcome of TKI based therapy. Therefore, multiple technical platforms have been developed for genotyping EGFR, including non-invasive PET imaging (Yeh et al., 2011; Bernard-Gauthier et al., 2015; Bahce et al., 2017; Sun et al., 2018). Based on highly specific radiotracers, non-invasive PET/CT images have been used to detect the sensitive EGFR mutation status before and/or after TKI treatment, which showed superior properties compared with other techniques. Over 30 TKI based PET tracers have been reported with the ability to detect EGFR sensitive mutations at present, and several tracers have been evaluated in clinical (see Figure 1) (Bernard-Gauthier et al., 2015; Bahce et al., 2017; Zhu et al., 2022).

Figure 1

Figure 1. Structures of representative reported radiotracers based on first-generation EGFR TKIs and Rociletinib with its analogues.

Although favorable clinical outcome can be achieved with first-and second-generation EGFR TKI based therapies, a majority of patients (up to 70%) experience disease progression due to acquired resistance following a period of 9–15 months of EGFR TKI treatment (Wu et al., 2023). The predominant mechanism behind TKI resistance is a secondary EGFR alteration known as T790M, located at the gatekeeper site (position 790) within exon 20. This specific mutation accounts for nearly 60% of acquired resistance instances in NSCLC cases (Yun et al., 2008; Husain et al., 2017; Leonetti et al., 2019). Therefore, the detection and monitoring of EGFR^T790M mutation status during EGFR-TKI treatment is essential to maintain and predict therapeutic efficacy (Ward et al., 2013; Yu et al., 2013; Finlay et al., 2014). Nevertheless, only countable radiotracers that targeted EGFR^T790M have been developed and reported to date (Goggi et al., 2019; Fawwaz et al., 2021) (see Figure 1).

Derived from highly potent inhibitor (WZ002) of EGFR^T790M, ¹⁸F-FEWZ displayed limited specificity and low binding to the target due to the rapid blood clearance. Although radiobrominated Rociletinib displayed specificity in cell uptake studies, the low in vivo tumor accumulation in biodistribution studies indicated the tracer was insufficient for tumor visualization with PET. In addition, no in vivo PET studies were performed with EGFR^T790M targeted PET tracers at present.

Held same “Aniline-Pyrimidine-Aniline” scaffold, current EGFR^T790M targeted PET tracers with higher lipophilicity displayed limited tumor accumulation, we believe the introduction of more hydrophilicity moiety to the molecule would enhance the ability to cross/pass through multiple membranes for tumor engagement and retention.

To develop potent PET tracers that selectively target EGFR^T790M for invasive detection and visualization, Rociletinib attracted our attention. Rociletinib showed potent and selective activity against EGFR^T790M in both pre-clinical and clinical studies (Walter et al., 2013; Sequist et al., 2015). Although Rociletinib has not been approved by FDA for multiple reasons, the pyrimidine-based scaffold can be regarded as an ideal “warhead” with high affinity for developing EGFR^T790M targeted PET tracers. Bearing several hydrophilicity groups such as carboxylic acid and amine, current chelators such as DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), and NOTA(2,2′,2''- (1,4,7-triazonane-1,4,7-triyl)triacetic Acid) display higher hydrophilicity, and the introduction of these chelators would improve the pharmacokinetics properties of desired molecules. In addition, the introduction of chelators moiety enables the therapeutic radionuclide labeling (such as ¹⁷⁷Lu and ¹⁶¹Tb) to the potent “warhead” for the development of Targeted-Radionuclide-Therapy pharmaceuticals. In this investigation, a potent ⁶⁸Ga radiolabeled Rociletinib derivative (⁶⁸Ga-1) was successfully designed and prepared with the introduction of DOTA moiety (for radiometal labeling) and evaluated in vitro, and further in vivo evaluations for detecting EFGR^T790M were also performed and discussed.

Materials and methods

Molecular docking studies

To evaluate the binding model of the designed compound with EGFR^T790M, molecular docking was performed. All calculations were performed on Discovery Studio (Accelrys Inc., Version 3.1, United States). The protein structure of EGFR^T790M was prepared with a previously published crystal structure (PDB ID: 2JIT) (Yan et al., 2017). The binding of the compound with the target protein was allocated as a sphere from its original reported ligand, and the space size is large enough to incorporate the binding site of EGFR^T790M with the ligand. All docking calculations were performed on GOLD program.

General information for chemical synthesis

All regents, solvents and consumables used in this study were obtained from commercial sources and used without further processing unless indicated. Thin-layer chromatography (TLC) with silica gel plates for monitoring the organic reactions, and flash column chromatography was used for the separation and purification of crude products. ¹H and ¹³C NMR tests were performed on an AV-400 Bruker spectrometer in the Analysis and testing center of Sichuan university. Mass spectra were obtained and recorded on an Agilent 6125 LC/MSD system. Compound 1 was synthesized following an identical procedure for Rociletinib, with minor modifications (Walter et al., 2013; Ward et al., 2013; Fawwaz et al., 2021).

The synthesis route is outlined in Scheme 1 and all detailed information about the synthesis procedures was provided in the supporting materials, as well as the characterization data.

Scheme 1

Scheme 1. Synthesis of DOTA-Rociletinib (compound 1). Conditions: (a) Trifluoroacetic Acid, 2-Butanol, overnight; (b) Trifluoroacetic Acid, dichloromethane, r. t 4h; (c) Triethylamine, DOTAGA-anhydride, dimethyl sulfoxide, overnight.

Biochemical activity test

EGFR Kinase inhibition tests (Lantha screen Assay) for compound 1 were performed by ChemPartner Co., Ltd. (Shanghai, China) with Staurosproine and Rociletinib as positive control.

⁶⁸Ga radiolabeling

⁶⁸GaCl₃ was eluted from a⁶⁸Ge/⁶⁸Ga generator (Eckert & Ziegler, Germany) by 5 mL of 0.1 N HCl solution and used for radiolabeling according to a classic DOTA based radiolabeling procedure published previously (Lindner et al., 2018; Giesel et al., 2019). Briefly, the generator solution containing ⁶⁸GaCl₃ (400 μL) was added into the reaction mixture of 40 μL of compound 1 and 40 μL of sodium acetate (1 N) in a reaction vial (pH 4–4.5). The radiolabeling reaction was carried out under 90 °C for 15 min. The reaction mixture was then diluted with 5 mL of distilled water and passed through a C-18 cartridge (Waters, United States). Trapped on the cartridge, the radioactivity was first washed by 20 mL of distilled water and 20 mL of air. The radioactivity was then eluted into a product vial by 500 μL of ethanol, and 4.5 mL of water was added into the product vial for final formulation. The radiochemical purity of ⁶⁸Ga-1 was determined on an Agilent 1100 system (Agilent, United States) equipped with a FC3200 gamma detector (Bioscan, United States). A Phenomenex C-18 Luna column was used for HPLC elution with MeCN (Phase A) and 0.1% TFA in water (Phase B) based on a gradient method set as follows (flowrate: 1 mL/min): 0–9 min, 30% A to 70% A; 9–10 min, 70% A to 30% A.

In vitro physicochemical property tests

The stability profile of ⁶⁸Ga-1 was evaluated in 80% EtOH solution, PBS saline and rat serum as described in previously published literature (Chen et al., 2023). Briefly, about 600 μL of freshly prepared product solutions were added into 2 mL of these test solutions with each 100 μL. These test solutions were kept in a water bath (37 °C) for 4 h, and the samples for stability test were obtained from these solutions at 1, 2, 3 and 4-h after water bath incubation. Samples were loaded into the HPLC system and eluted with the mobile phase as described in the radiolabeling section previously. The protein was denatured by acetonitrile and removed by centrifugation before loading to HPLC for samples from rat serum.

Lipophilicity of a radiotracer is crucial for the biodistribution and Pharmacokinetics properties. The lipophilicity of the radiolabeled compound 1 (⁶⁸Ga-1) was determined according to previously published literature and presented as the negative logarithms of the partition coefficient using PBS saline with pH 7.4 (Log D_7.4) (Li et al., 2019). Briefly, radioactivity concentrations in PBS saline layer and 1-octanol layer were obtained from a mixture of radiotracer mixture of PBS saline and 1-octanol and were used to calculate the partition coefficient by the ratios of the two concentrations. A series of radiotracer solutions were produced, and the corresponding partition coefficient was tested until a constant value of log D_7.4 was reached.

Cell uptake experiments

H1975^L858R/T790M, H3255^L858R and A549^WT cells were purchased from American Type Culture Collection and cultured in the correspond medium recommended by American Type Culture Collection (ATCC). Plasmid-transfected A549 cells that express EGFR^T790M mutation were obtained from Haixing Biosciences (Suzhou, China). In cellular uptake studies, cells were cultured and seeded into 60-mm culture dishes with a density of 5 * 10⁵ cells per well overnight for attachment before use. Freshly prepared ⁶⁸Ga-1 (∼370 KBq, ∼50–100 uL) was added into the medium of cells and incubated at 37 °C for testing. At designated timepoints, i.e., 15-, 30-, 45-, 60-min, the medium was collected, and the well was washed with cold PBS (2 mL). The PBS and medium were combined and tested in the wizard (2470, Perkin Elmer, United States) for radioactivity counts. Cells in the well were treated with trypsin and then collected, counted in the wizard. In cell blocking studies, Rociletinib was added into the well (final concentration of 1 μM was reached in the well) 1 h before the addition of radiolabeled ⁶⁸Ga-1, and the subsequent operation procedure was kept identical as described above. Tracer uptake ratios in the cell can be calculated by the following formula: Uptake radios = (radio-counts for the cells)/(radio-counts for the cells + radio-counts for the medium and PBS) * 100%.

Biodistribution studies

To further evaluate the in vivo pharmacokinetics profile of ⁶⁸Ga-1, a tissue biodistribution assessment was conducted in normal Kunming mice. All protocols and procedures in the biodistribution study were reviewed and approved by the animal care and use committee of Sichuan University. Briefly, all animals were administered with 30–40 μCi of ⁶⁸Ga-1 by tail vein injection under isoflurane-induced anesthesia and grouped by timepoints, i.e., 5, 15, 30, 60, 90 and 120 min (n = 5). A gentle stream of air (0.8 L/min) containing 2% of isoflurane gas were generated by a compact anesthesia module (MINERVE, France) and fed into the anesthesia chamber. At designated timepoints, animals were sacrificed by carbon dioxide inhalation, and the organs and tissues of interest were harvested, weighted, and tested in the gamma-counter (2470, Perkin Elmer, United States) for radio-counts. The tissue biodistribution profile of ⁶⁸Ga-1 can be calculated with the decay-corrected injected dose and presented as the percentage of the injected dose per gram of tissue (%ID/g).

Micro-PET/CT imaging studies

Preparation of animal models: tumor cells were cultured, harvested and suspended in 100 μL of PBS (approximately 5 * 10⁶ cells). The cells (100 μL suspension) were then injected subcutaneously into the left or right axilla of the BALB/c nude mice (16–20 g, 4–6 weeks). The length of the long axis (a) and short axis of (b) the tumor was measured every 2 days, and the volume of the tumor was estimated by the following formula: Tumor volume = a*b*b/2. Once the volume of the tumor reached 500 mm³ (approximately 10–15 days after injection of tumor cells), the PET imaging study was carried out immediately. Briefly, 3.74 MBq of freshly prepared ⁶⁸Ga-1 was administered into the tumor-bearing mice via tail vein injection with isoflurane-induced anesthesia (details were provided in biodistribution studies section). Static PET imaging was performed at 15-, 30-, 60-, and 120-min post-injection on a micro-PET/CT scanner (IRIS, Inviscan, France), and the data were reconstructed by 3D-OSEM algorithm with a Monte Carlo-base accurate model for attenuation correction. The anesthesia of animals during the scan was maintained by the inhalation of air containing 2% of isoflurane gas (also generated by the compact anesthesia module) by a mice ventilation mask. The regions of interest (ROIs) for organs of interest were manually drawn on the static PET images, and the radio-concentration (KBq/cc) for all ROIs were obtained from software. For blocking studies, subjects were pre-injected with Rociletinib (1 mg/kg) 30 min before the tracer administration.

Statistical analysis

All data in this study are presented as mean ± standard deviation (Mean ± SD). Statistical analysis was performed with GraphPad Prism 10.1.2 using unpaired independent sample t-tests, and a P value <0.05 was considered statistically significant.

Results

Molecular docking studies

Based on the cocrystal structure of EGFR^T790M with Rociletinib (PDB: 2JIT), a number of important ligand-protein interactions can be noticed, such as two hydrogen bonds with Met793 carbonyl and amide of the EGFR^T790M kinase with the anilinopyrimidine core of Rociletinib, and a hydrophobic interaction of the mutant gatekeeper residue Met790 and the trifluoromethyl substituent on the pyrimidine ring of Rociletinib. In addition, we also noticed the amide that attached to the piperazine ring, which oriented towards the external space of the kinase binding pocket. Therefore, the introduction of a bulky group with significant steric hindrance may not affect the binding potency of compounds with target kinase. In docking studies, compound 1 displayed similar binding mode with Rociletinib with the introduction of DOTA, and overlaid well with Rociletinib, suggesting a comparable high affinity of compound 1 with EGFR^T790M. In addition, the introduction of DOTA would improve the pharmacokinetics properties of compound 1 compared with Rociletinib as a radiotracer, since DOTA moiety contains multiple hydrophilic centers such as carboxyl groups and nitrogen atoms (Figure 2).

Figure 2

Figure 2. Crystal complex of rociletinib with (PDB: 2JIT) (A) and predicated binding of compound 1 with EGFR^T790M (B).

Chemical synthesis

Compound 1 was successfully prepared following the previously published literature with minor modifications, as described above in Scheme 1 (Walter et al., 2013; Ward et al., 2013; Fawwaz et al., 2021). Briefly, tert-butyl 4-(4-amino-3-methoxyphenyl)piperazine-1-carboxylate, as a commercially available starting material, reacts with intermediate N-(3-((2-chloro-5-(trifluoromethyl)pyrimidin-4-yl)amino)phenyl)acrylamide for Rociletinib to produce compound 2. After the deprotection reaction under acidic condition, the unprotected amino group at compound 3 was used for the amide formation reaction with DOTA-GA anhydride to generate the final target molecule (compound 1, DOTA- Rociletinib). The details for the chemical synthesis procedure and the characterization profile were provided in supporting materials.

Results for biochemical activity test

Based on the Lantha screen Assay, compound 1 displayed similar enzymatic inhibition activity with Rociletinib. As shown in Table 1, the IC₅₀ values for Rociletinib and compound 1 were 8.24 nM and 9.21 nM, respectively. The results indicate that the incorporation of the DOTA as chelating group with larger steric hindrance on the N atom of the piperazinyl group in compound 1 showed nearly no impact on the enzymatic inhibitory activity against EGFR^T790M. Based on molecular docking studies and biochemical testing, compound 1 was identified as a promising radiolabeled PET tracer targeting EGFR^T790M and the subsequent radiolabeling and evaluations were carried out thereafter.

Table 1

Table 1. The IC₅₀ values for rociletinib and compound 1 against EGFR^T790M in lantha screen assay.

⁶⁸Ga radiolabeling

Radiolabeling of Compound 1 was successfully achieved with high radiochemical yield (92.93% ± 2.57%, n = 5) under 90 °C for 10 min. The radiochemical purity of ⁶⁸Ga-1 was greater than 95% after the C18 cartridge based SPE purification procedure as displayed in Figure 3. The retention time of ⁶⁸Ga-1 was 7.8 min and the unlabeled precursor of compound 1 is 7.6 min, respectively.

Figure 3

Figure 3. ⁶⁸Ga Radiolabeling reaction for compound 1 (A) and the co-injection of final product with the precursor (B).

In vitro physicochemical property tests

Based on the analytical HPLC analysis of samples from ⁶⁸Ga-1 solutions of 80% EtOH, PBS saline and rat serum, ⁶⁸Ga-1 displayed high in vitro stability. Over 90% intact ⁶⁸Ga-1 was identified from the HPLC diagram with these test solutions incubated at 37 °C. According to the radio-concentrations for the PBS and 1-octanol, the log D_7.4 value for ⁶⁸Ga-1 was −1.68 ± 0.24, indicating a more hydrophilic property compared to Rociletinib, which displayed a log P value of 3.95 from the ChemBioDraw 20.0 predictions.

Cell uptake experiments

The tracer uptake values for all cells are expressed as the ratios of radio-counts in cells to the sum of radio-counts added into the well, as presented in Figure 4. ⁶⁸Ga-1 displayed higher accumulation in H1975 cells (bearing EGFR^T790M/L858R) and A549-EGFR^790M (with EGFR^T790M) compared with H3255 cells (EGFR^L858R) and A549 (EGFR^WT). After 2-h incubation, tracer uptake ratio for A549^WT, H3255^L858R, H1975^T790M/L858R and A549^T790M cells were 0.74% ± 0.10%, 1.29% ± 0.21%, 3.96% ± 0.59% and 3.65% ± 0.42% (n = 3), respectively. In addition, with the addition of Rociletinib as blocking agent, a remarkable decrease in tracer uptake ratios was observed in these cells, as also displayed Figure 4. In H1975^T790M/L858R and A549^T790M cells, tracer uptake ratios decreased to 2.57% ± 0.53% and 1.86% ± 0.59% (n = 3), with blocking efficiencies of 35% and 49% for two cells, respectively. However, only 3% and 14% blocking efficiencies were observed in A549^WT and H3255 cells. These results indicated a strong correlation between the EGFR^T790M expression and tracer uptake in cell experiments.

Figure 4

Figure 4. Results for cell uptake experiments of ⁶⁸Ga-1 (n = 3).

Biodistribution studies

The tissue biodistribution properties of ⁶⁸Ga-1 in Kunming mice are expressed as %/ID/g in each time point, as displayed in Figure 5 (n = 5, and the detailed quantitative data were provided in SI). ⁶⁸Ga-1 showed a fast pharmacokinetics profile, and the radioactivity in blood, liver, kidney and other major organs showed a fast clearance during the 120-min experiment. The liver displayed highest radioactivity accumulation at 15 min post injection (p.i.) with 8.77% ± 2.78% ID/g, and the radioactivity accumulation decreased to 5.33% ± 2.08% ID/g at 120 min p.i. In addition, small intestine and spleen displayed a significant radioactivity uptake during the experiment, with peak uptake value of 4.11% ± 0.79% ID/g and 3.14% ± 1.21% ID/g at 60 min p.i., suggesting a major hepatobiliary excretion of this compound in mice. A significant accumulation of radioactivity in kidney was also noticed, with peak uptake value of 4.98% ± 1.94% ID/g at 60 min p.i. and 3.81% ± 1.31% ID/g at 120 min p.i., respectively. Thus, a minor urinary excretion of ⁶⁸Ga-1 can be inferred according to the accumulation of radioactivity in the kidney. Other major organs, such as the lung, heart, muscle and brain, showed no abnormal radioactivity accumulation, with peak uptake values of 1.74% ± 0.75%, 2.51% ± 0.82%, 0.62% ± 0.21% and 0.18% ± 0.09% ID/g at 60 min p.i., respectively. A small amount accumulation of radioactivity in the bone was observed, indicating a minor decomposition of ⁶⁸Ga-1 in vivo. With the biodistribution profile of ⁶⁸Ga-1 in hand, the subsequent PET imaging study was performed thereafter.

Figure 5

Figure 5. Biodistribution profile of ⁶⁸Ga-1 in normal Kunming mice (n = 5).

Micro-PET/CT imaging studies

The representative PET images for ⁶⁸Ga-1 in different tumor models and the corresponding Time-activity curves are presented in Figure 6 (n = 3). Radioactivity accumulation in tumor mice well agreed with the biodistribution profile, with significant initial accumulation of radio-signal in the liver, kidney, lower digestive tract, spleen and bladder observed in all kinds of tumor mouse models investigated in this study. The bladder, gallbladder, kidney and liver displayed the highest tracer accumulation, indicating a major urinary excretion and minor hepatobiliary metabolism of ⁶⁸Ga-1. For A549-EGFR^T790M and H1975^L858R/T790M mice models, tumor regions were clearly visualized. However, in H3255^L858R and A549^WT mice models, lower radioactivity accumulation in tumors were observed as well as mice from blocking studies (A549-EGFR^T790M mice models blocked with Rociletinib).

Figure 6

Figure 6. Micro-PET studies for ⁶⁸Ga-1 in mice models (n = 3). (A) Static PET images of A549-EGFR^T790M mice models at different time points; (B) Time-activity curves generated from A549-EGFR^T790M mice PET images; (C) Representative static PET images for H1975, H3255, A549^WT and A549-EGFR^T790M (blocked with Rociletinib) mice models at 60 min post-injection; (D) Selected organ uptake value for different tumor models at 60 min post-injection. Red arrows denote the tumor.

As displayed in Figures 6A,B, an initial high uptake in gallbladder, kidney and liver were observed in A549-EGFRT^790M mice models with %ID/g value of 5.11 ± 0.73, 3.10 ± 0.59 and 4.60 ± 0.58, respectively. A fast uptake of radiotracer in tumor can be noticed and maintained afterwards, with %ID/g values of 1.62 ± 0.38 at 15 min p.i. and 1.56 ± 0.16 at 60 min p.i., respectively. With the gradually clearance of radio-signal from major organs and background such as lung and muscle, the “Tumor-to-muscle (T/M)” ratio peaked at 60 min p.i. with highest value of 3.82 during the entire scan. The tumor uptake displayed a decrease after 60 min p.i., with %ID/g value of 1.06 ± 0.10 and T/M ratio of 2.46 obtained at 120 min p.i., respectively. However, the gallbladder exhibited highest uptake (peaked at 60 min p.i. with 6.67% ± 0.78% ID/g) which may due to the lipophilicity of the pyrimidine scaffold at all time points. Based on the PET images and in vivo distribution profile of ⁶⁸Ga-1 obtained in A549-EGFR^T790M mice models, the subsequent PET image studies were performed at 60 min p.i. for H1975, H3255 and A549^WT mice models and in blocking studies.

In EGFR^T790M positive H1975 mice models, a similar tumor uptake was observed with %ID/g value of 1.65 ± 0.13 at 60 min p.i., as well as a T/M ratio of 3.96. The other major organs also displayed similar tracer uptake profiles, as displayed in Figures 6C,D. In blocking studies with A549-EGFR^T790M mice models, the pre-injection of Rociletinib significantly inhibited the tumor uptake of ⁶⁸Ga-1 (P < 0.05), with the uptake value decreasing to 0.83 ± 0.12 at 60 min p.i., while other major organs displayed comparable uptake values compared with non-blocking subjects. In addition, ⁶⁸Ga-1 also exhibited a lower tumor uptake in H3255 mice models with EGFR^L858R mutations and A549 mice models with EGFR^WT. As also displayed in Figures 6C,D, an average uptake value of 1.10% ± 0.04% ID/g (H3255 mice models) and 1.12% ± 0.12% ID/g (A549 mice models) was respectively observed at 60 min p.i., compared with that of A549-EGFR^T790M mice models (1.62% ± 0.38% ID/g) and H1975 mice models (1.65% ± 0.13% ID/g). Together with the uptake profile obtained in blocking studies, the results in A549-EGFR^T790M mice models indicated the tumor uptake of ⁶⁸Ga-1 is selective. In all subjects investigated in these studies, no abnormal uptake of radio-signal in other major organs was observed, indicating the in vivo stability of ⁶⁸Ga-1.

Discussion

As a subfamily member of receptor tyrosine kinase, EGFR was regarded as the most important target for anti-tumor therapies for NSCLC patients in last decades. The genotyping of EGFR is also of great importance for personalizing NSCLC therapy and predicting therapy efficacy. Although over 30 radiotracers have been reported to possess clinical potential to visualize EGFR mutations (such as ex20ins, 19-Del and 21-L858R) in vivo, few PET tracers have been reported with the ability to detect EGFR^T790M mutation. Developing highly sensitive and selective EGFR^T790M PET tracers for the complement of current EGFR targeted PET tracers is of great importance for scientific research and clinical applications. Bearing the pyrimidine scaffold, Rociletinib held potent affinity against EGFR^T790M, and our interest in this compound lies in the convenient modification and conversion of Rociletinib, to obtain the target molecule as a⁶⁸Ga-labeled PET tracer using DOTA moiety as a chelator group. ⁶⁸Ga-1 was produced with high radiochemical yield, and the in vitro evaluations indicated it possessed strong stability and suitable lipophilicity, which is important for further in vivo evaluations. The selective binding of ⁶⁸Ga-1 with EGFR^790M was confirmed by cell uptake experiments with EGFR^T790M positive and wild type cells, as well as blocking studies. In H1975 (EGFRT^790M/L858R) and A549-EGFR^T790M cells, tracer uptake is twofold higher than that of EGFR^WT cells, and the uptake can be effectively blocked by Rociletinib. Compared with maximum cell uptake of 6% with ¹⁸F-FEA-Erlotinib (Huang et al., 2018) and 5% of ¹⁸F-MPG (Sun et al., 2018), 4% of ⁶⁸Ga-1 seems reasonable for this type of TKI tracer. The relatively low absolute percentage of cell uptake may be associated with cell membrane permeability and membrane protein expression (Kell, 2021).

Biodistribution studies revealed the hepatobiliary/urinary excretion of ⁶⁸Ga-1 in vivo, as well as a moderate pharmacokinetics property. In micro-PET/CT studies, ⁶⁸Ga-1 also displayed selective accumulation in EGFRT790M positive tumors with highest uptake observed (1.62% ± 0.38% ID/g in A549-EGFR^T790M tumors and 1.65% ± 0.13% ID/g in H1975 tumors) at 60 min p.i., and an effective blocking efficacy (49.7%) was observed in blocking studies. However, tracer accumulation was also observed in A549 EGFR^WT tumor models (1.12% ± 0.12% ID/g) and H3255 EGFR^L858R tumor models (1.10% ± 0.04% ID/g), compared with 1.62%–1.63% ID/g in EGFR^T790M positive tumors, making it challenging to identify EGFR^T790M from other EGFR mutations or wild type EGFR. In addition, a relatively higher background uptake in bladder, gallbladder, liver, kidney and lung was noticed in both biodistribution and micro-PET/CT imaging studies, suggesting a higher plasma protein binding, nonspecific binding and lack of target engagement of ⁶⁸Ga-1.

Suitable hydrophilicity of a PET tracer is essential for the in vivo pharmacokinetics properties, and most current successful 68Ga labeled PET tracer held strong hydrophilicity, such as ⁶⁸Ga-PSMA-11 (log P: −2.91 ± 0.0631) and ⁶⁸Ga-FAPI-46 (log D_7.4: −3.38 ± 0.01) (Baranski et al., 2018; Kogler et al., 2025). Adequate hydrophilicity of a compound would lead to fast background clearance via kidney and potential membrane penetration, which is important for target engagement. Insufficient hydrophilicity of a compound usually related to high plasma protein binding and nonspecific binding, which would lead to higher and longer retention of background uptake in liver, kidney, gallbladder and lower gastrointestinal tract. Therefore, according to the log P value of ⁶⁸Ga-1 and the PET images obtained in this study, we conclude that the insufficient hydrophilicity of compound 1 impairs both background clearance and the membrane penetration necessary for target engagement. In addition, although compound 1 and Rociletinib displayed potent in vitro enzymatic activity against EGFR^T790M, Rociletinib exhibited limited specificity against EGFR^WT with a Ki value of 303.3 nM in a previously reported literature (compared with that of 21.5 nM against EGFR^T790M/L858R in same study) (Walter et al., 2013). Thus, as an analogue of Rociletinib, the in vivo specificity of ⁶⁸Ga-1 would be comprised by the insufficient selectivity against EGFR^WT and other alterations of EGFR.

Therefore, further structural modification and optimization of this compound should be focused on the improvement of binding potency on EGFR^T790M and selectivity against EGFR^WT and other EGFR alterations, and the following options will be beneficial: 1. extensive structural-activity-relationship studies for screening of ideal compounds; 2. chelator/linker optimization to improve affinity; 3. other strategies to enhance the tumor retention and “Tumor to background” ratios such as covalent targeted radioligand technology (Cui et al., 2024). In addition, the optimization of physicochemical properties by enhancement of hydrophilicity would also be of great importance for this tracer to improve the target accumulation and background clearance. Moreover, with DOTA moiety as a chelator to enable radiometal labeling in compound 1, further therapeutic radionuclides may also be introduced to the “Aniline-Pyrimidine-Aniline” scaffold to form theragnostic radiopharmaceuticals.

Conclusion

In conclusion, ⁶⁸Ga-1 was successfully synthesized and demonstrated promising preliminary characteristics; however, its specificity toward EGFR^T790M mutations requires further investigation and validation. ⁶⁸Ga-1 displayed in vitro and in vivo specificity toward EGFR^T790M compared with EGFR^WT and EGFR^L858R. However, the insufficient selectivity against EGFR^WT and other EGFR alterations of ⁶⁸Ga-1 making it challenging to identify EGFR^T790M in vivo. Therefore, further structural modification and optimization, focused on the selectivity and physicochemical parameters of this compound, would be needed to develop more successful EGFR^T790M selective targeted PET tracer for NSCLC diagnosis and treatment.

Data availability statement

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

Ethics statement

The animal study was approved by West China Hospital of Sichuan University Ethics Committee. The study was conducted in accordance with the local legislation and institutional requirements.

Author contributions

JX: Investigation, Methodology, Writing – review and editing. WX: Writing – review and editing, Investigation, Supervision. HD: Methodology, Writing – review and editing. XW: Funding acquisition, Investigation, Writing – original draft. JZ: Conceptualization, Funding acquisition, Project administration, Writing – review and editing.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This work was supported by the NHC Key Laboratory of Nuclear Technology Medical Transformation (MIANYANG CENTRAL HOSPITAL, Grant No. 2021HYX002) and the Precision Medicine Key Laboratory of Sichuan Province with Grant No. 2023KF-01.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fbioe.2026.1741512/full#supplementary-material

References

Bahce, I., Yaqub, M., Smit, E. F., Lammertsma, A. A., van Dongen, G. A., and Hendrikse, N. H. (2017). Personalizing NSCLC therapy by characterizing tumors using TKI-PET and immuno-PET. Lung Cancer 107, 1–13. doi:10.1016/j.lungcan.2016.05.025

PubMed Abstract | CrossRef Full Text | Google Scholar

Baranski, A. C., Schäfer, M., Bauder-Wüst, U., Roscher, M., Schmidt, J., Stenau, E., et al. (2018). PSMA-11-Derived dual-labeled PSMA inhibitors for preoperative PET imaging and precise fluorescence-guided surgery of prostate cancer. J. Nucl. Med. 59 (4), 639–645. doi:10.2967/jnumed.117.201293

PubMed Abstract | CrossRef Full Text | Google Scholar

Bernard-Gauthier, V., Bailey, J. J., Berke, S., and Schirrmacher, R. (2015). Recent advances in the development and application of radiolabeled kinase inhibitors for PET imaging. Molecules 20 (12), 22000–22027. doi:10.3390/molecules201219816

PubMed Abstract | CrossRef Full Text | Google Scholar

Chen, F., Wu, Y., Ma, Y., Yin, H., Su, F., Huang, R., et al. (2023). Synthesis, radiolabeling, and evaluation of 68Ga-labeled aminoquinoxaline derivative as a potent PFKFB3-targeted PET tracer. Front. Chem. 11, 1158503. doi:10.3389/fchem.2023.1158503

PubMed Abstract | CrossRef Full Text | Google Scholar

Cui, X.-Y., Li, Z., Kong, Z., Liu, Y., Meng, H., Wen, Z., et al. (2024). Covalent targeted radioligands potentiate radionuclide therapy. Nature 630 (8015), 206–213. doi:10.1038/s41586-024-07461-6

PubMed Abstract | CrossRef Full Text | Google Scholar

Fawwaz, M., Mishiro, K., Nishii, R., Makino, A., Kiyono, Y., Shiba, K., et al. (2021). A radiobrominated tyrosine kinase inhibitor for EGFR with L858R/T790M mutations in lung carcinoma. Pharmaceuticals (Basel) 14 (3), 256. doi:10.3390/ph14030256

PubMed Abstract | CrossRef Full Text | Google Scholar

Finlay, M. R., Anderton, M., Ashton, S., Ballard, P., Bethel, P. A., Box, M. R., et al. (2014). Discovery of a potent and selective EGFR inhibitor (AZD9291) of both sensitizing and T790M resistance mutations that spares the wild type form of the receptor. J. Med. Chem. 57 (20), 8249–8267. doi:10.1021/jm500973a

PubMed Abstract | CrossRef Full Text | Google Scholar

Giesel, F. L., Kratochwil, C., Lindner, T., Marschalek, M. M., Loktev, A., Lehnert, W., et al. (2019). (68)Ga-FAPI PET/CT: biodistribution and preliminary dosimetry estimate of 2 DOTA-containing FAP-targeting agents in patients with various cancers. J. Nucl. Med. 60 (3), 386–392. doi:10.2967/jnumed.118.215913

PubMed Abstract | CrossRef Full Text | Google Scholar

Goggi, J. L., Haslop, A., Ramasamy, B., Cheng, P., Jiang, L., Soh, V., et al. (2019). Identifying nonsmall-cell lung tumours bearing the T790M EGFR TKI resistance mutation using PET imaging. J. Label. Comp. Radiopharm. 62 (9), 596–603. doi:10.1002/jlcr.3771

PubMed Abstract | CrossRef Full Text | Google Scholar

Huang, S., Han, Y., Chen, M., Hu, K., Qi, Y., Sun, P., et al. (2018). Radiosynthesis and biological evaluation of (18)F-labeled 4-anilinoquinazoline derivative ((18)F-FEA-Erlotinib) as a potential EGFR PET agent. Bioorg Med. Chem. Lett. 28 (6), 1143–1148. doi:10.1016/j.bmcl.2017.08.066

PubMed Abstract | CrossRef Full Text | Google Scholar

Husain, H., Scur, M., Murtuza, A., Bui, N., Woodward, B., and Kurzrock, R. (2017). Strategies to overcome bypass mechanisms mediating clinical resistance to EGFR tyrosine kinase inhibition in lung cancer. Mol. Cancer Ther. 16 (2), 265–272. doi:10.1158/1535-7163.Mct-16-0105

PubMed Abstract | CrossRef Full Text | Google Scholar

Kell, D. B. (2021). The transporter-mediated cellular uptake and efflux of pharmaceutical drugs and biotechnology products: how and why phospholipid bilayer transport is negligible in real biomembranes. Molecules 26 (18), 5629. doi:10.3390/molecules26185629

PubMed Abstract | CrossRef Full Text | Google Scholar

Kogler, J., Donat, C. K., Trommer, J., Kopka, K., and Stadlbauer, S. (2025). Synthesis and preclinical evaluation of FAP-targeting radiotracers for PET and optical imaging. EJNMMI Radiopharm. Chem. 10 (1), 77. doi:10.1186/s41181-025-00398-9

PubMed Abstract | CrossRef Full Text | Google Scholar

Leonetti, A., Sharma, S., Minari, R., Perego, P., Giovannetti, E., and Tiseo, M. (2019). Resistance mechanisms to osimertinib in EGFR-mutated non-small cell lung cancer. Br. J. Cancer 121 (9), 725–737. doi:10.1038/s41416-019-0573-8

PubMed Abstract | CrossRef Full Text | Google Scholar

Li, S., Cai, Z., Wu, X., Holden, D., Pracitto, R., Kapinos, M., et al. (2019). Synthesis and in vivo evaluation of a novel PET radiotracer for imaging of synaptic vesicle glycoprotein 2A (SV2A) in nonhuman Primates. ACS Chem. Neurosci. 10 (3), 1544–1554. doi:10.1021/acschemneuro.8b00526

PubMed Abstract | CrossRef Full Text | Google Scholar

Lindner, T., Loktev, A., Altmann, A., Giesel, F., Kratochwil, C., Debus, J., et al. (2018). Development of quinoline-based Theranostic ligands for the targeting of fibroblast activation protein. J. Nucl. Med. 59 (9), 1415–1422. doi:10.2967/jnumed.118.210443

PubMed Abstract | CrossRef Full Text | Google Scholar

Sequist, L. V., Soria, J. C., Goldman, J. W., Wakelee, H. A., Gadgeel, S. M., Varga, A., et al. (2015). Rociletinib in EGFR-mutated non-small-cell lung cancer. N. Engl. J. Med. 372 (18), 1700–1709. doi:10.1056/NEJMoa1413654

PubMed Abstract | CrossRef Full Text | Google Scholar

Sharma, S. V., Bell, D. W., Settleman, J., and Haber, D. A. (2007). Epidermal growth factor receptor mutations in lung cancer. Nat. Rev. Cancer 7 (3), 169–181. doi:10.1038/nrc2088

PubMed Abstract | CrossRef Full Text | Google Scholar

Sun, X., Xiao, Z., Chen, G., Han, Z., Liu, Y., Zhang, C., et al. (2018). A PET imaging approach for determining EGFR mutation status for improved lung cancer patient management. Sci. Transl. Med. 10 (431), eaan8840. doi:10.1126/scitranslmed.aan8840

PubMed Abstract | CrossRef Full Text | Google Scholar

Walter, A. O., Sjin, R. T., Haringsma, H. J., Ohashi, K., Sun, J., Lee, K., et al. (2013). Discovery of a mutant-selective covalent inhibitor of EGFR that overcomes T790M-mediated resistance in NSCLC. Cancer Discov. 3 (12), 1404–1415. doi:10.1158/2159-8290.Cd-13-0314

PubMed Abstract | CrossRef Full Text | Google Scholar

Ward, R. A., Anderton, M. J., Ashton, S., Bethel, P. A., Box, M., Butterworth, S., et al. (2013). Structure- and reactivity-based development of covalent inhibitors of the activating and gatekeeper mutant forms of the epidermal growth factor receptor (EGFR). J. Med. Chem. 56 (17), 7025–7048. doi:10.1021/jm400822z

PubMed Abstract | CrossRef Full Text | Google Scholar

Wu, P. S., Lin, M. H., Hsiao, J. C., Lin, P. Y., Pan, S. H., and Chen, Y. J. (2023). EGFR-T790M mutation-derived interactome rerouted EGFR translocation contributing to gefitinib resistance in non-small cell lung cancer. Mol. Cell Proteomics 22 (9), 100624. doi:10.1016/j.mcpro.2023.100624

PubMed Abstract | CrossRef Full Text | Google Scholar

Yan, X. E., Zhu, S. J., Liang, L., Zhao, P., Choi, H. G., and Yun, C. H. (2017). Structural basis of mutant-selectivity and drug-resistance related to CO-1686. Oncotarget 8 (32), 53508–53517. doi:10.18632/oncotarget.18588

PubMed Abstract | CrossRef Full Text | Google Scholar

Yeh, H. H., Ogawa, K., Balatoni, J., Mukhapadhyay, U., Pal, A., Gonzalez-Lepera, C., et al. (2011). Molecular imaging of active mutant L858R EGF receptor (EGFR) kinase-expressing nonsmall cell lung carcinomas using PET/CT. Proc. Natl. Acad. Sci. U. S. A. 108 (4), 1603–1608. doi:10.1073/pnas.1010744108

PubMed Abstract | CrossRef Full Text | Google Scholar

Yu, H. A., Arcila, M. E., Rekhtman, N., Sima, C. S., Zakowski, M. F., Pao, W., et al. (2013). Analysis of tumor specimens at the time of acquired resistance to EGFR-TKI therapy in 155 patients with EGFR-mutant lung cancers. Clin. Cancer Res. 19 (8), 2240–2247. doi:10.1158/1078-0432.Ccr-12-2246

PubMed Abstract | CrossRef Full Text | Google Scholar

Yun, C. H., Mengwasser, K. E., Toms, A. V., Woo, M. S., Greulich, H., Wong, K. K., et al. (2008). The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc. Natl. Acad. Sci. U. S. A. 105 (6), 2070–2075. doi:10.1073/pnas.0709662105

PubMed Abstract | CrossRef Full Text | Google Scholar

Zheng, D., Wang, R., Ye, T., Yu, S., Hu, H., Shen, X., et al. (2016). MET exon 14 skipping defines a unique molecular class of non-small cell lung cancer. Oncotarget 7 (27), 41691–41702. doi:10.18632/oncotarget.9541

PubMed Abstract | CrossRef Full Text | Google Scholar

Zhu, J., Li, Y., Wu, X., Li, Y., Wang, L., and Fan, H. (2022). In vivo PET imaging of EGFR expression: an overview of radiolabeled EGFR TKIs. Curr. Top. Med. Chem. 22 (28), 2329–2342. doi:10.2174/1568026622666220903142416

PubMed Abstract | CrossRef Full Text | Google Scholar