Development and Modification of a Novel Pharmacophore for a CXCR4-Targeting Radiopharmaceutical

Introduction: Previously, we reported a series of radiotracers that target the C-X-C chemokine receptor 4 (CXCR4) for PET imaging ([⁶⁸Ga]Ga-BL02, [¹⁸F]BL08 and [⁶⁸Ga]Ga-BL31) and radipharmaceutical therapy ([¹⁷⁷Lu]Lu-BL02) based on LY2510924, a cyclic peptide antagonist. Given their capability for high contrast imaging and high therapeutic index, we optimized the cyclic peptide pharmacophore via rational modification of its sequence, for clinical translation efforts. We identified several candidates that show excellent binding affinities to CXCR4 and structurally modified them with a linker-chelator conjugate. These novel radioligands were then labeled and evaluated in preclinical mantle cell lymphoma xenograft models.

Methods: Each peptide was synthesized via solid-phase peptide synthesis and the inhibition constant was measured by a cell-based competitive binding assays using [¹²⁵I]SDF-1, with LY2510924, a potent CXCR4-targeting antagonist, as a standard. An anionic (Lys(Cysteic Acid)) or cationic (Lys(D-Arg)) linker and the DOTA chelator were then conjugated to peptide candidates. These peptides were labeled with [⁶⁸Ga]GaCl₃ and evaluated in Z138 xenograft-bearing NRG male mice via PET imaging and/or biodistribution studies.

Results: All radiotracers were labeled with ⁶⁸Gaat >50% decay-corrected radiochemical yields and >99% radiochemical purity. From a small targeted screen of cyclic peptides, we identified 4 cyclic peptides with enhanced affinity to CXCR4 as compared to LY2510924 (IC₅₀=20 nM): DKW01 (IC₅₀=6.7 nM), DKW02 (IC₅₀=14.0 nM), DKW03 (IC₅₀=4.9 nM) and DKW04 (IC₅₀=9.2 nM). These were modified to carry the Lys(Cysteic acid)-based linker and the DOTA chelator (BL34, BL36, BL37 and BL43, respectively). Evaluation in Z138 xenograft mice showed tumor uptakes of 15.1±3.1, 12.2±0.8, 6.2±0.6 and 11.8±2.4 %ID/g at 1 h post-injection (p.i). All radiotracers showed relatively low non-target organ uptake values. Given its higher tumor uptake, BL34 was selected for further modification. The Lys(D-Arg) linker (BL42) was conjugated and compared to the anionic linker. Comparison between [⁶⁸Ga]Ga-BL34 and [⁶⁸Ga]Ga-BL42 showed comparable tumor uptake values (15.1±3.1 vs 16.3±1.3 %ID/g). However, [⁶⁸Ga]Ga-BL42 had high liver (15.5±2.3 %ID/g) and kidney (6.1±1.4 %ID/g) uptake values, precluding further assessment of cationic-based linkers. Finally, comparison with Orn- (BL44) and Dap- (BL45) based linkers over the Lys showed similar results, with high tumor uptake values (18.4±1.8 and 14.4±5.0 %ID/g), and low non-target organ values.

Conclusions: Derived from a new and potent CXCR4-targeting pharmacophore, DKW01, a series of radiotracers were synthesized, labeled and assessed. BL34 had high tumor uptake and low non-target organ uptake and, therefore, is a promising candidate for further evaluation as a CXCR4-targeting radiotheranostic.

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