Abstract View

Background

Development of successful chemotherapies for the aggressive pediatric brain cancer diffuse intrinsic pontine glioma (DIPG) has been hindered by the intact blood-brain barrier.¹ PET-guided drug discovery for DIPG could be beneficial for assessing brain uptake, target engagement, and dose occupancy of small-molecule receptor-targeted compounds.² Activin receptor-like kinase 2 (ALK2) inhibitors have emerged as a chemotherapeutic target for DIPG.^1,3 Building on prior ALK2-targeted radiotracers developed by our lab based on the potent and selective ALK2-inhibitor M4K2009,⁴ here we report next-generation carbon-11 labeled radiotracers for ALK2 which demonstrate markedly improved brain penetration in rodents.

Methods

A series of new ALK2 inhibitors was designed based on physicochemical properties associated with brain permeability and efflux susceptibility, including total polar surface area, hydrogen bond donors, basicity (pKa), and lipophilicity (logP). Radiolabeling of two lead compounds, [¹¹C]M4K2328 and [¹¹C]M4K2329, was accomplished via in-loop [¹¹C]-O-methylation of the corresponding phenol precursors. The resulting radiotracers were purified by semi-preparative HPLC and reformulated into a 10% ethanol/saline solution by C18 solid-phase exchange. Decay-corrected radiochemical yields were calculated from [¹¹C]CO₂, while radiochemical purities and molar activities were determined by radio-HPLC. Preclinical PET imaging was conducted in Sprague-Dawley rats (n = 2, 1 male/1 female) as well as wild-type (n = 4, male) and efflux transporter knock-out mice (n = 4, male) to evaluate brain uptake and efflux liability. Radiometabolite analyses were performed on rat plasma and brain homogenate samples collected 15 minutes post-injection using column-switching HPLC.

Results

Both ¹¹C-labeled tracers were synthesized and isolated in moderate radiochemical yields (6-43%), high radiochemical purity (>99%), and suitable molar activity for PET imaging (15-398 GBq/µmol). Dynamic PET-CT imaging revealed reasonable brain uptake in rodents, with peak standardized uptake values (SUVs) >1.5 for both tracers. Favourable washout kinetics were also observed as reflected by 2:60-minute ratios of 3.14 for [¹¹C]M4K2328 and 1.88 for [¹¹C]M4K2329. Brain uptake increased ~1.5‑fold in efflux transporter knock‑out mice as compared to wild-type mice, indicating limited efflux susceptibility. No brain penetrant radiometabolites were observed 15 minutes post-injection (>98% parent tracer).

Conclusions

[¹¹C]M4K2328 and [¹¹C]M4K2329 demonstrated good brain uptake in rodents, desirable washout kinetics and excellent metabolic stability, and represent the most promising ALK2-targeted PET radiotracers to date. In vitro and in vivo blocking studies to assess binding specificity and selectivity of these tracer candidates are underway, and the lead tracer will be used to measure target engagement of lead drug candidates for ALK2.

Acknowledgements

We thank the Ontario Institute for Cancer Research, Conscience, the Centre for Probe Development and Commercialization, and Mitacs for financial support. MC thanks the Canadian Institute for Health Research for financial support. NV thanks the Azrieli Foundation, Canada Research Chairs Program, Canada Foundation for Innovation, and Ontario Research Fund for support.

References

(1) Smil, D., et al. J. Med. Chem. 2020, 63 (17), 10061–10085. (2) Chassé, M.; Vasdev, N. Neurotherapeutics 2025, 22 (1), e00498. (3) Carvalho, D., et al. Commun. Biol. 2019, 2 (1), 156. (4) Murrell, E., et al. ACS Med. Chem. Lett. 2021, 12 (5), 846–850.