Synthesis and antimicrobial evaluation of two peptide LyeTx I derivatives modified with the chelating agent HYNIC for radiolabeling with technetium-99m

Abstract

BackgroundCurrent diagnostic methods and imaging techniques are not able to differentiate septic and aseptic inflammation. Thus, reliable methods are sought to provide this distinction and scintigraphic imaging is an interesting option, since it is based on physiological changes. In this context, radiolabeled antimicrobial peptides have been investigated as they accumulate in infectious sites instead of aseptic inflammation. The peptide LyeTx I, from the venom of Lycosa erythrognatha, has potent antimicrobial activity. Therefore, this study aimed to synthesize LyeTx I derivatives with the chelating compound HYNIC, to evaluate their antimicrobial activity and to radiolabel them with 99mTc.MethodsTwo LyeTx I derivatives, HYNIC-LyeTx I (N-terminal modification) and LyeTx I-K-HYNIC (C-terminal modification), were synthesized by Fmoc strategy and purified by RP-HPLC. The purified products were assessed by RP-HPLC and MALDI-ToF-MS analysis. Microbiological assays were performed against S. aureus (ATCC® 6538) and E. coli (ATCC® 10536) in liquid medium to calculate the MIC. The radiolabeling procedure of LyeTx I-K-HYNIC with 99mTc was performed in the presence of co-ligands (tricine and EDDA) and reducing agent (SnCl2.2H2O), and standardized taking into account the amount of peptide, reducing agent, pH and heating. Radiochemical purity analysis was performed by thin-layer chromatography on silica gel strips and the radiolabeled compound was assessed by RP-HPLC and radioactivity measurement of the collected fractions. Data were analyzed by ANOVA, followed by Tukey test (p-values < 0.05).ResultsBoth LyeTx I derivatives were suitably synthesized and purified, as shown by RP-HPLC and MALDI-ToF-MS analysis. The microbiological test showed that HYNIC-LyeTx I (N-terminal modification) did not inhibit bacterial growth, whereas LyeTx I-K-HYNIC (C-terminal modification) showed a MIC of 5.05 μmol.L−1 (S. aureus) and 10.10 μmol.L−1 (E. coli). Thus, only the latter was radiolabeled with 99mTc. The radiochemical purity analysis of LyeTx I-K-HYNIC-99mTc showed that the optimal radiolabeling conditions (10 μg of LyeTx I-K-HYNIC; 250 μg of SnCl2.2H2O; pH = 7; heating for 15 min) yielded a radiochemical purity of 87 ± 1 % (n = 3). However, RP-HPLC data suggested 99mTc transchelation from LyeTx I-K-HYNIC to the co-ligands (tricine and EDDA).ConclusionsThe binding of HYNIC to the N-terminal portion of LyeTx I seems to affect its activity against bacteria. Nevertheless, the radiolabeling of the C-terminal derivative, LyeTx I-K-HYNIC, must be better investigated to optimize the radiolabeled compound, in order to use it as a specific imaging agent to distinguish septic and aseptic inflammation.

Paclitaxel-loaded folate-coated long circulating and pH-sensitive liposomes as a potential drug delivery system: A biodistribution study

“…The results showed a high radiochemical purity and stability (over to 90%), up to 24 h after radiolabeling. Radiochemical purity above 90% is required since the presence of high amount of impurities leads to poor image quality, which might result in a biodistribution profile that no longer reflects the nanoparticle behavior [26,45]. …”

Technetium-99 m radiolabeled paclitaxel as an imaging probe for breast cancer in vivo

“…It is well described that 99m Tc-labeling reactions generate two main impurities, 99m TcO 2 and 99m TcO 4 − [24,25]. The presence, in high concentration, of these entities leads to images in poor quality that might impair the correct data interpretation.…”

“…Therefore, by using the optimized protocol described in this study less than 5% of impurities were detected indicating that 99m Tc-PTX can be used in further in vivo assays. Furthermore, the physiological pH obtained after labeling (pH = 7.4) is optimal for intravenous administration [24]. The presence of —OH groups in the polyethoxylated castor oil, a constituent of the Taxol formulation might explain the high labeling efficiency.…”

“…Additionally, a significant uptake in the intestines was observed suggesting that fecal route also contribute to the excretion of 99m Tc-PTX [44,45]. Noteworthy is the low uptake in thyroid and stomach, which confirms the high labeling stability showed by in vitro assays [24,25]. …”

Shortened derivatives from native antimicrobial peptide LyeTx I: In vitro and in vivo biological activity assessment

“…9,10 Further studies suggested that LyeTx I amino-terminal side is indispensable to the secondary structure conformation of the peptide, influencing its interaction with bacteria, once either the modification of LyeTx I carboxyl-terminal side (LyeTx I-K-HYNIC) or the suppression of a histidine residue in the same portion (LyeTx I-b) does not compromise its antimicrobial activity. 11,12 Recently, LyeTx I-b was formulated with carboxymethylcellulose and showed activity against a strain of Staphylococcus aureus resistant to penicillin, erythromycin, and ampicillin, acting in planktonic condition and reducing biofilm viability in 90%. Furthermore, it is very effective in the treatment of keratitis, with no signs of ocular toxicity in an in vivo eye infection model induced by the same bacterium.…”