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Synthesis and Preclinical Evaluation of [Methylpiperazine-11C]brigatinib as a PET Tracer Targeting Both Mutated Epidermal Growth Factor Receptor and Anaplastic Lymphoma Kinase


Antonia A. Högnäsbacka et al, Journal of Medicinal Chemistry, 2023


Lung cancer is one of the most common causes of cancer-related deaths, of which non-small cell lung cancer (NSCLC) accounts for approximately 85%. Part of the standard diagnostic evaluation of NSCLC patients is testing for receptor expression and mutational status in the lung tissue. Commonly found to be mutated in NSCLC adenocarcinomas are the epidermal growth factor receptor (EGFR) and the anaplastic lymphoma kinase (ALK). The most common EGFR mutations found are the tyrosine kinase domain mutations Del19 and L858R resulting in continuous receptor activation independent of ligand interaction. Patients with EGFR mutation-positive NSCLC are usually treated with tyrosine kinase inhibitors (TKIs) like gefitinib, erlotinib, or afatinib. However, treatment resistance in the form of the T790M mutation restores the constitutive activity of the receptor.

NSCLC ALK mutations, on the other hand, are chromosomal rearrangements. The most common ALK mutation in NSCLC is caused by the echinoderm microtubule-associated protein-like 4 (EML4) gene’s 5′ end juxtaposing with the 3′ end of the ALK gene, resulting in the oncogene EML4−ALK. Patients with EML4− ALK mutation-positive NSCLC are treated with TKIs such as crizotinib.

Brigatinib is a tyrosine kinase inhibitor designed to inhibit a broad range of ALK kinase domain mutations, such as the F1174C/V, I1171N, and G120R mutations reported to occur as treatment resistance response to crizotinib or second-generation inhibitors like ceritinib and alectinib.

Brigatinib with its specificity for gene rearranged anaplastic lymphoma kinase (ALK), such as the EML4−ALK mutation-positive NSCLC, has shown a potential to inhibit mutated epidermal growth factor receptor (EGFR). In this study, N-desmethyl brigatinib was successfully synthesized as a precursor in five steps. PET-imaging with 11C-labeled brigatinib may provide a non-invasive method for assessing the mutational status, where the uptake of 11C labeled brigatinib would imply a treatment-sensitive mutation. Radiolabelling with [11C]methyl iodide produced [methylpiperazine-11C]brigatinib, which was then evaluated in non-small cell lung cancer xenografted female nu/nu mice. Significant differences in tumour uptake were observed between the endogenously EML4−ALK mutated H2228 and the control xenograft A549. Tracer uptake in EGFR Del19 mutated HCC827 and EML4−ALK fusion A549 was not significantly different from uptake in A549 xenografts.

Results from nanoScan® PET/CT:

PET/CT imaging was performed on a Mediso nanoScan PET/CT (Mediso Ltd., Hungary). Animals were kept under isoflurane anaesthesia for the whole duration of the scans. Dynamic PET scans were immediately acquired for 60 min after tail vein injection of [methylpiperazine-11C]brigatinib. A computed tomography (CT) was performed just before the PET acquisition. In the blocking study, the PET scan was followed by a contrast enhanced CT scan. The CT scan was acquired while infusing the contrast agent (Iomeron 400, Bracco Diagnostics, UK,) to visualize the cardiovascular structures.

PET scans were acquired in list mode and rebinned dynamically into the following frames: 4 × 5, 4 × 10, 2 × 30, 3 × 60, 2 × 300, 1 × 600, 1 × 900, and 1 × 1200 s, and statically in one frame (0−60 min), reconstruction was performed using a fully 3-dimensional reconstruction algorithm (Tera-Tomo, Mediso Ltd.) with 4 iterations and 6 subsets, and an isotropic 0.4 mm voxel dimension. PET image analysis and quantification were performed using VivoQuant software, and the data were expressed in SUV.

In vivo blocking was performed using the EML4−ALK blocking agent crizotinib and the mutated EGFR blocking agent erlotinib injected 1 h prior to the PET scan. H2228 tumour-bearing nu/nu mice (n = 3 per blocker) underwent two 60 min PET/CT scans, one baseline scan and one PET/CT scan an hour p.i. of the blocker. For each scan, the mice were intravenously injected with 11 ± 2 MBq of [methylpiperazine-11C]brigatinib. The mice were allowed to recuperate for a minimum of one day between the baseline and block scan.

Tumour Uptake Comparison. The tumour-targeting potential of [methylpiperazine-11C]brigatinib was evaluated in four NSCLC cell lines; A549, HCC827, EML4−ALK fusion-A549, and H2228. Time activity curves are depicted in Figure 3A, demonstrating the H2228 xenograft to have the higher uptake.

Figure 3. Tumour uptake of [methylpiperazine-11C]brigatinib in four xenograft models. (A) Time−activity curves assessed by PET.

The T/B and T/M ratios for A549 and A549 EML4−ALK fusion were very similar (A549: T/B 2.60 ± 0.20 and T/M 1.60 ± 0.22 vs EML4−ALK fusion A549 T/B 2.76 ± 0.29 and T/M 1.57 ± 0.18). Only the H2228 xenografts could be visually delineated with PET, and tracer uptake was observed to be heterogeneous within the tumour (Figure 4).

Figure 4. Representative PET images (static 0−60 min time frame) of [methylpiperazine-11C]brigatinib in NSCLC tumours (tumour locations indicated by white arrows).

To determine the specificity of the uptake of [methylpiperazine-11C]brigatinib in the H2228 xenografts, a PET/CT blocking study was performed using the EML4−ALK inhibitor crizotinib and the mutated EGFR inhibitor erlotinib. After scanning, the mice were sacrificed, followed by an ex vivo biodistribution. The ex vivo biodistribution at 60 min p.i. revealed a significant increase in the radioactive concentration in the duodenum, liver, and pancreas following the pretreatment with crizotinib. A significant reduction, however, could be observed in the kidneys (Figure 5A). As the tumour uptake in the ex vivo and in vivo biodistribution was higher after pretreatment with crizotinib (Figure 5B, C), the tumour uptake was corrected to the blood activity concentration derived from the left ventricle of the heart. The tumour-to-blood ratio was significantly lower after pretreatment with crizotinib (Figure 5D).

Figure 5. Biodistribution and tumour uptake of [methylpiperazine-11C]brigatinib in H2228 xenografts at baseline and following pretreatment with 25 mg/kg of crizotinib (N = 3 mice, n = 6 tumours). (A) Ex vivo biodistribution at 60 min p.i., (B) tumour uptake at 60 min p.i., assessed by ex vivo biodistribution, (C) time−activity-curve assessed by PET, and (D) tumour-to-blood ratio calculated from PET results.

Similarly, an ex vivo biodistribution revealed a significant increase in the radioactive concentration in the duodenum and liver when mice were pretreated with erlotinib (Figure 6A). The tumour uptake did not change with erlotinib  locking, indicating the uptake to be less influenced by EGFR binding (Figure 6B,C). Due to experimental difficulties, the left ventricle could only be delineated for  one mouse, which was needed to derive accurate blood activity concentration. Therefore, the effect of erlotinib on the tumour-to-blood ratio could not be determined (Figure 6D).

Figure 6. Biodistribution and tumour uptake of [methylpiperazine-11C]brigatinib in H2228 xenografts at baseline and following pretreatment with 25 mg/kg of erlotinib (N = 3 mice, n = 6 tumours, unless otherwise stated). (A) Ex vivo biodistribution at 60 min p.i., (B) tumour uptake at 60 min p.i., assessed by ex vivo biodistribution, (C) time−activity-curve assessed by PET, and (D) tumour-to-blood ratio calculated from PET results (N= 1, n = 2).

  • [Methylpiperazine-11C]brigatinib was successfully synthetized.
  • There is an ability of [methylpiperazine-11C]brigatinib to differentiate between the EML4−ALK mutation expressing H2228 and the ALK wild-type expressing A549.
  • The tumour-to-blood ratio in H2228 xenografts could be significantly reduced by pretreatment with ALK inhibitor crizotinib, suggesting specific tracer uptake.
  • Crizotinib was used to evaluate the specificity of [methylpiperazine-11C]brigatinib uptake in H2228 xenografts.
  • [Methylpiperazine-11C]brigatinib showed favourable characteristics in clearance from target organs, a low background would favour a high contrast.

Full article on acs.org

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