CXCR4 and ACKR3 binding azamacrocyclic compounds for targeted PET imaging and therapeutic applications in cancer.

Objectives: The chemokine receptors CXCR4 and ACKR3 (CXCR7) have been shown to be over-expressed in multiple types of cancer [1] and are usually associated with aggressive phenotypes and poor prognosis [2]. Successfully targeting and imaging these receptors could allow earlier diagnosis of some cancers, inform treatment selection and lead to better outcomes for the patients. Our research group has developed a library of high affinity CXCR4 tetraazamacrocyclic antagonists with long residence time on the receptor. We have recently extended the biological validation of these derivatives to ACKR3 and have identified a small sub-set of compounds (with structural features in common) that bind to both of these receptors. These novel CXCR4- and ACKR3-specific agents could offer the opportunity for dual targeting, to better delineate disease and inform treatment.

Methods: Configurationally-restricted multi-macrocyclic derivatives and their Cu(II), Zn(II) and Ni(II) metal complexes were prepared. Their affinity for CXCR4 and ACKR3 was assessed in vitro in U87 glioblastoma cell lines transfected to stably express one receptor type. Affinity for CXCR4 was determined by Ca²⁺ signalling and affinity for ACKR3 was determined by competition binding with fluorescent CXCL12. In vivo affinity for CXCR4 was demonstrated through a series of blocking experiments, using a selected set of compounds to block the CXCR4-specific PET tracer, [⁶⁸Ga]Pentixafor, in mice implanted with CXCR4 over-expressing xenografts.

Results: We have synthesised a library of configurationally-restricted multi-macrocyclic compounds and their corresponding Cu(II), Zn(II) and Ni(II) metal complexes showing high affinity for CXCR4 with IC₅₀ values as low as 4 nM [3]. Among these derivatives, a set of novel compounds was shown to additionally bind to ACKR3. The SAR of these compounds is currently under investigation and preliminary results have already shown affinity for ACKR3 below 100 nM. Successful in vivo block of [⁶⁸Ga]Pentixafor, resulting in an 8-fold reduction in tumour uptake, with doses as low as 4 mg/kg confirmed high specificity of one of these compounds for CXCR4. Two other compounds were assessed in vivo at 1 mg/kg and showed a 2- and 3-fold reduction in tumour uptake, respectively.

Conclusions: We successfully prepared a set of novel multi-macrocyclic metal complexes and assessed their affinity in vitro for both CXCR4 and ACKR3, allowing structure activity relationships to be identified. The affinity of these compounds for CXCR4 was also demonstrated in vivo through blocking studies of a clinically characterized CXCR4 targeting PET tracer. ACKR3 over-expressing tumour models are currently being developed to allow further validation of these derivatives in vivo. Acknowledgements: The authors would like to thank Dr Assem Allam for fellowship funding and his generous contribution to the University of Hull PET Research Centre.

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