Design, synthesis and evaluation of ¹¹¹In labeled DOTA-conjugated tetrapeptides having high affinity and selectivity...

Peripheral mu (μ) opioid receptors are implicated in pain, bowel dysfunction and the progression of certain cancers. In an effort to identify radioligands well suited for imaging these peripheral sites, we have prepared and evaluated four hydrophilic ¹¹¹In labeled DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) conjugated μ tetrapeptides.

Peptides were prepared by solid-phase techniques, using orthogonal strategies to achieve branching to DOTA, and then characterized by HPLC, mass spectroscopy and amino acid analysis. Scaffolds included novel peptide H-Dmt-D-Ala-Phe-Orn-NH₂ (DAPO), where Dmt = 2',6'-dimethyltyrosine, and known peptide H-Dmt-D-Arg-Phe-Lys-NH₂ ([Dmt¹]DALDA). Constructs had DOTA conjugation at the Orn⁴ or Lys⁴ side chains, or to the C-terminal through a hexanoic acid-lysine linker. Indium(III) complexation and ¹¹¹In radiolabeling were accomplished by standard methods. Protein binding and Log D_7.4 were determined. Binding and pharmacological profiles were obtained in vitro. Biodistribution and radiometabolite studies were conducted using male CD-1 mice.

All four indium(III)-DOTA conjugates derived from DAPO and [Dmt¹]DALDA showed good selectivity and subnanomolar affinity for μ opioid receptors. One radioligand, H-Dmt-D-Ala-Phe-Orn(δ-[¹¹¹In]In-DOTA)-NH₂, showed 25% specific binding in vivo to μ sites in mouse gut. Notably, this was the least polar of the series, and also showed low sensitivity to modulation of binding by sodium ions. All radioligands showed high kidney uptake of radiometabolites.

Visualizing peripheral μ opioid receptors using ¹¹¹In labeled DOTA-conjugated tetrapeptides appears feasible, but structural modifications to enhance specific binding and metabolic stability, as well as to reduce kidney uptake, will be required.

This study shows in vivo labeling of peripheral μ opioid receptors by a tetrapeptide radioligand, and provides information that should prove useful in the design of peptide radioligands having optimal properties.