In Vitro Properties and Pharmacokinetics of Temporarily PEGylated Onc72 Prodrugs

Abstract

The favorable properties of antimicrobial peptides (AMPs) to rapidly kill pathogens are often limited by unfavorable pharmacokinetics due to fast degradation and renal clearance rates. Here, a prodrug strategy linking proline‐rich AMP Onc72 to polyethylene glycol (PEGs) with average molecular weights of 5 and 20 kDa via a peptide linker containing a protease cleavage site is tested for the first time in vivo. Onc72 is released from these 5k‐ and 20k‐prodrugs in mouse serum with half‐life times (t1/2) of 8 and 14 h, respectively. Importantly, PEGylation protects Onc72 from proteolytic degradation providing a prolonged release of Onc72, balancing the degradation of free Onc72, and leading to relatively stable Onc72 concentrations and high antibacterial activities. The prodrugs are not hemolytic on human erythrocytes and show only slight cytotoxic effects on human cell lines indicating promising safety margins. When administered subcutaneously to female CD‐1 mice, the prodrugs elimination t1/2 are 66 min and ≈5.5 h, respectively, compared to 43 min of free Onc72. The maximal Onc72 plasma levels are obtained ≈1 and ≈8 h postadministration, respectively. In conclusion, the prodrugs provide extended elimination t1/2 and a constant release of Onc72 in mice, potentially limiting adverse effects and increasing efficacy.

Evaluation of proline-rich antimicrobial peptides as potential lead structures for novel antimycotics against Cryptococcus neoformans

“…All PrAMPs tested, including the reverse and scrambled peptides, showed no significant hemolytic activity compared to the untreated control (addition of PBS) up to concentrations of 600 mg/L (234 to 285 μmol/L), as previously reported for some PrAMPs (Czihal et al, 2012;Krieger et al, 2021;Mohammed et al, 2023), in contrast to a strong hemolytic effect observed for amphotericin B already at the lowest concentration tested (125 mg/L; 135 μmol/L) (Supplementary Figure S5).…”

“…The plates were centrifuged (5 min, 1,000 × g, 4°C), the supernatants (100 μL) were transferred to flat-bottom 96-well plates, and the absorbance was recorded at 405 nm on a microplate reader (PARADIGM™, Molecular Devices). PBS was used as negative control, and a serial dilution of triton X-100 from 0.1 to 0.00078% was used as a positive control ( Czihal et al, 2012 ; Mohammed et al, 2023 ).…”

Harnessing antimicrobial peptides in endodontics

“…Novel design techniques, including peptide sequence modification, development of self‐assembled AMPs, and utilization of biomaterial carriers, hold the potential to address these challenges. For instance, cyclic and PEGylated AMPs demonstrate increased resistance to protease degradation, significantly improving their stability (Malde et al., 2019; Mohammed et al., 2023). Self‐assembled AMPs in the form of nanofibres, nanoparticles and nanoribbons exhibit additional functions whilst enhancing their permeability (Xie et al., 2022).…”