Glycated ^99mTc‐Tricarbonyl‐Labeled Peptide Conjugates for Tumor Targeting by “Click‐to‐Chelate”

Abstract

Attaching polar pharmacological modifiers to molecular imaging probes is a common strategy to modulate their pharmacokinetic profiles to improve such parameters as the clearance rate of radiotracers and/or metabolites, and to enhance signal-to-background ratios. We combined the tumor-targeting peptide sequence of bombesin (BBN) with glucuronic acid and the single-photon emission computed tomography (SPECT) radionuclide Tc by the "click-to-chelate" methodology. The Tc-tricarbonyl-labeled glucuronated BBN conjugate was compared with a reference compound lacking the carbohydrate. The radiolabeled conjugates displayed similar characteristics in vitro (cell internalization, receptor affinity), but the hydrophilicity of the glycated version was significantly increased. While the tumor uptake of the two radioconjugates in xenografted mice was similar, the glycated peptide exhibited unexpected higher uptake in organs of the hepatobiliary excretion pathway than the more lipophilic reference compound. Control experiments suggest that this may be the result of unspecific accumulation of metabolites in which the glucuronic acid moiety does not act as an innocent pharmacological modifier.

Sorbitol as a Polar Pharmacological Modifier to Enhance the Hydrophilicity of 99mTc-Tricarbonyl-Based Radiopharmaceuticals

“…Therefore, the glycated radiopeptide did not perform significantly better in vivo. Nevertheless, as hypothesized, [ 99m Tc][Tc(CO) 3 (12)] bearing a sorbitol moiety did not show a pronounced accumulation in liver and spleen compared to the previously published glucuronated BBN derivative [18].…”

“…Because this study aims at the investigation of differences in the pharmacokinetics of the radiopeptides (e.g., rate and route of excretion) rather than in tumor uptake, we continued with the evaluation of both compounds in vivo. The receptor affinity (K d ) and maximum receptor occupancy (B max ) were determined by receptor saturation assays using increasing amounts of the radiolabeled peptides ( Figure 3 [18,23,27], yet in this case somewhat surprising as we did not expect the sorbitol moiety to have an influence on these parameters; this in particular because the results of cell binding and internalization experiments were similar for both radiolabeled peptides ( Figure 2). Differences in specific molar activities (MBq/mol) could account for this observation and cannot be entirely excluded even though both 99m Tc-radiopeptides were prepared under identical conditions.…”

“…The receptor affinity (Kd) and maximum receptor occupancy (Bmax) were determined by receptor saturation assays using increasing amounts of the radiolabeled peptides ( Figure 3 (13)] (Kd = 3.7 ± 1.0 nM, Bmax = 0.37 ± 0.02 nM; Table 2) [23]. Some variability in experimentally determined Kd and Bmax of structurally related radiolabeled peptides is not uncommon [18,23,27], yet in this case somewhat surprising as we did not expect the sorbitol moiety to have an influence on these parameters; this in particular because the results of cell binding and internalization experiments were similar for both radiolabeled peptides ( Figure 2). Differences in specific molar activities (MBq/mol) could account for this observation and cannot be entirely excluded even though both 99m Tc-radiopeptides were prepared under identical conditions.…”

“…This strategy includes an application of the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) [15,16] by which an efficient tridentate chelator for the 99m Tc-tricarbonyl core is formed and linked to (bio)molecules of interest while other moieties, e.g., carbohydrates, can be conjugated simultaneously [17]. We have previously applied this methodology to the assembly of a peptide conjugate comprising a modified analog of the binding sequence of the tumor-targeting peptide bombesin (BBN), namely [Nle 14 ]BBN (7)(8)(9)(10)(11)(12)(13)(14), a 99m Tc-tricarbonyl complex and the carbohydrate glucuronic acid [18]. BBN is an attractive peptide for targeting the Gastrin-releasing peptide receptor (GRPR), which is overexpressed by a variety of tumors, including prostate and breast cancer [19].…”

“…While the hydrophilicity of the glycated peptide conjugate was found substantially increased, we were surprised that it accumulated nevertheless to a higher degree in the spleen and liver in comparison to a more lipophilic BBN reference compound. We assumed that glucuronated, negatively charged metabolites of the radiolabeled peptide can be recognized by organic anion transporting polypeptides (OATPs), which are expressed in these organs [18]. We thus resorted to the use of other carbohydrates as pharmacological modifiers.…”