Biotinidase Resistant ⁶⁸Gallium-Radioligand Based on Biotin/Avidin Interaction for Pretargeting: Synthesis and Preclinical Evaluation

Abstract

A new macrocyclic system 2,2'-(12-amino-11,13-dioxo-1,4,7,10-tetraazacyclotridecane-4,7-diyl)diacetic acid (ATRIDAT) was designed for coordinating metals in +2 and +3 oxidation states particularly Ga(III), for PET imaging. ATRIDAT was conjugated to d-biotin for pretargeting via biotin-avidin interaction. This model provides high tumor targeting efficiency and stability to biotinidase activity leading to modest signal amplification at the tumor site. Cyclization of triethylenetetramine with protected diethylamino malonate resulted in the formation of 13 membered diamide ring. d-Biotin was then anchored on the pendant amine rendering α-methyne carbon to the biotinamide bond which blocks the biotinidase enzyme activity. Biotinidase stability assay showed remarkable stability toward the action of biotinidase with ∼95% remaining intact after treatment following 4 h. Binding affinity experiments such as HABA assay, competitive displacement studies with d-biotin and CD showed high binding affinity of the molecule with avidin in nanomolar range. Biotin conjugate was successfully radiolabeled withGa(III) with radiolabeling efficiency of ∼70% and then purified to get 99.9% radiochemical yield. IC of the compound was found to be 2.36 mM in HEK cell line and 0.82 mM in A549 as assessed in MTT assay. In biodistribution studies, the major route of excretion was found to be renal. Significant uptake of 4.15 ± 0.35% was observed in tumor in the avidin pretreated mouse at 1 h. μPET images also showed a high tumor to muscle ratio of 26.8 and tumor to kidney ratio of 1.74 at 1 h post-injection after avidin treatment.

Mapping neuroreceptors with metal-labeled radiopharmaceuticals

“…This can be achieved by synthesizing novel chelates or by modifying existing chelates using lipophilic moieties. 66,67 3. Multimodality probes.…”

Radiolabeling and Preclinical Evaluation of a New S-Alkylated Cysteine Derivative Conjugated to C-Substituted Macrocycle for Positron Emission Tomography

“…Synthetic strategy utilizes S-alkylation of N -acetyl cysteine (NAC) with a chloroacetylated derivative of a 13-membered protected macrocycle, di- tert -butyl 2,2′-(12-(( tert -botoxycarbonyl)amino)-11,13-dioxo-1,4,7,10-tetraazatridecane-4,7-diyl)diacetate ( 1 ) 30 ( Scheme 1 ). The Boc group of this macrocycle ( 1 ) was selectively cleaved using HCl gas dissolved in ethyl acetate, exposing the amine group for further reaction to give compound 2 .…”

“…Radiolabeling was performed using the previously published method. 30 68 Ga ( T 1/2 value of 68 min and positron emission of 89%) was fractionated from a 68 Ge to 68 Ga generator. In the generator, 68 Ge (half-life = 270 days) is adsorbed on the titanium dioxide-based inorganic matrix.…”

“…ATRIDAT, already reported by our group to form thermodynamically stable and strong complexes with Ga(III) was used for conjugating the cysteine derivative after slight modification in the pendant arm, keeping the macrocyclic framework intact. 30 PET is a highly sensitive technique which provides a method to visualize and quantify the uptake at the target site. PET aids in early diagnosis, as it detects the changes in the biochemical pathways, allowing detection of disease before the appearance of anatomical changes.…”

Imaging Biotin Trafficking In Vivo with Positron Emission Tomography

“…Biotin has already been radiolabeled using PET and single-photon emission computed tomography (SPECT) radionuclides such as 64 Cu, 18 F, 111 In, 125 I, 131 I, 68 Ga, and 99m Tc 21 − 28 and conjugated with fluorophores for optical imaging. 29 − 31 However, these “biotin-inspired” imaging probes have been developed via the conjugation of the carboxylic group of biotin to chelators or linkers bearing a radionuclide or a fluorophore.…”