Oxidant and Antioxidant Effects of Gentisic Acid in a ¹⁷⁷Lu-Labelled Methionine-Containing Minigastrin Analogue

Abstract

Background: The radiolabelling of receptor-binding peptides for therapy is a challenge since the peptide itself is exposed (during labelling, storage and transport) to radiation-induced damage, directly or indirectly, in aqueous solution. Hence, the use of radiostabilizers seems to be mandatory, especially in peptide molecules that contain radiation-sensitive amino acids Objective: The aim of this study was to investigate the effect of two stabilizers, gentisic acid and methionine, to delve into how each of them affects the radiolabelling and stability of the minigastrin analogue [177Lu]Lu-DOTA-His-His-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 through the analysis of the 22 species distinguished over time by an optimized HPLC system. Method: The stabilizers, in different combinations, were present from the beginning of the labelling process that was carried out at 96 °C for 15 min. The stability was studied up to 7 days without any dilution, to better detect the radiation effects. Results: The unexpected selective oxidation of the methionine residue of the radiolabelled peptide, promoted by gentisic acid, led to studying the effect of pH, from 3.5 to 6.0, in the presence of only this stabilizer. A pH-dependent antioxidant behavior of gentisic acid was revealed, reaching optimum results at pH 5.0-5.5. Conclusion: The selective oxidation of the methionine residue could be induced by the H2O2 produced in the reaction between gentisic acid and free radicals of water, during the protection of the radiolabelled peptide from the attack of these harmful species. Therefore, the addition of methionine becomes necessary to effectively decrease this selective oxidation in the methionine-containing peptide.

Radiolysis-Associated Decrease in Radiochemical Purity of 177Lu-Radiopharmaceuticals and Comparison of the Effectiveness of Selected Quenchers against This Process

“…This difference cannot be substantiated by the non-specific sorption of unbound [ 177 Lu]Lu 3+ on the HPLC column [ 43 , 57 , 58 , 59 ] because, in this particular case, the values determined by the HPLC methods significantly exceed the values obtained by the TLC methods. This has also been observed in several other studies [ 45 , 46 ]. To confirm that this impurity is not a cationic form of 177 Lu or its weak associate with other possible ligands in the sample (such as acetate-anion), we conducted a comparative experiment using a CM cation-exchange cartridge (Sep-Pak Accell Plus CM Plus Light Cartridge (Milford, MA, USA)), which is often used for the final purification of radiopharmaceuticals with metal radionuclides in manual and automated synthesis.…”

“…Notably, the [ 177 Lu]Lu-PSMA-617 samples already obtained with gentisic acid during the first day after synthesis began to acquire a brown color (which became more saturated during further storage— Figure 15 C). This color change is most likely due to the formation of gentisic acid oxidation products (such as 1,4-benzoquinone-2-carboxylic acid and others [ 46 , 48 , 79 ]) upon interaction with radicals during radiolysis. The samples prepared with the mixture of cysteine and DMSA remained clear and colorless throughout the monitoring period (up to 144 h), and no eye-visible precipitate formation was observed.…”

“…At the same time, the presence of 0.1 M ascorbic acid did not stabilize the 225 Ac-labeled conjugates, leading to lower complex stability and transchelation. Ghosh et al [ 45 ], using the compound [ 177 Lu]Lu-RM1 (antagonist to gastrin-releasing peptide receptor) as an example, showed that samples (0.1 M acetate buffer, 0.74 GBq/mL activity concentration, 10.175–12.765 GBq/mmol molar activity) with ascorbic acid (500 μg) have a half-life of degradation around 39.2 h. Under the same conditions, the average half-life of the degradation of [ 177 Lu]Lu-RM1 in the presence of 500 μg gentisic acid was 142.5 h. However, Trindade et al used a DOTA-conjugated minigastrin analog—[ 177 Lu]Lu-DOTA-H2MG11—and reported an undesired oxidative effect of gentisic acid [ 46 ]. The reduction of this effect, together with the suppression of radiolysis, was achieved using a mixture of gentisic acid (110 mM) and methionine (78 mM) as stabilizers.…”

Automated Radiosynthesis, Preliminary In Vitro/In Vivo Characterization of OncoFAP-Based Radiopharmaceuticals for Cancer Imaging and Therapy

“…All [ 68 Ga]Ga-OncoFAP and [ 18 F]AlF-OncoFAP radio-conjugates were highly stable (>99% of intact compound) in 0.9% saline solution and in human plasma at 37 • C for 2 h. On the other hand, a stability test of [ 177 Lu]Lu-DOTAGA-OncoFAP radiolabeled batch showed the presence of radiolysis of about 10% every 24 h, which was significantly reduced to about 2% within 8 days by the addition of 20 mg of gentisic acid as a radical scavenger (Figure 3b) [15,16]. All the radiolabeled preparations of OncoFAP derivatives exhibited high hydrophilicity.…”

The Puzzle of Aspirin and Iron Deficiency: The Vital Missing Link of the Iron-Chelating Metabolites

“…Several studies have been reported related to the interactions of 2,5-dihydroxybenoic acid with other metal ions in addition to iron [146,147]. However, there are not as many reports on the iron-binding and metabolic effects of 2,5-dihydroxybenzoic acid in comparison to those of 2,3-DHB and salicylic acid.…”