In vitro and in vivo characterization of an interleukin‐15 antagonist peptide by metabolic stability, ^99mTc‐labeling, and biological activity assays

Abstract

Interleukin (IL)-15 is an inflammatory cytokine that constitutes a validated therapeutic target in some immunopathologies, including rheumatoid arthritis (RA). Previously, we identified an IL-15 antagonist peptide named [K6T]P8, with potential therapeutic application in RA. In the current work, the metabolic stability of this peptide in synovial fluids from RA patients was studied. Moreover, [K6T]P8 peptide was labeled with Tc to investigate its stability in human plasma and its biodistribution pattern in healthy rats. The biological activity of [K6T]P8 peptide and its dimer was evaluated in CTLL-2 cells, using 3 different additives to improve the solubility of these peptides. The half-life of [K6T]P8 in human synovial fluid was 5.88 ± 1.73 minutes, and the major chemical modifications included peptide dimerization, cysteinylation, and methionine oxidation. Radiolabeling of [K6T]P8 with Tc showed a yield of approximately 99.8%. The Tc-labeled peptide was stable in a 30-fold molar excess of cysteine and in human plasma, displaying a low affinity to plasma proteins. Preliminary biodistribution studies in healthy Wistar rats suggested a slow elimination of the peptide through the renal and hepatic pathways. Although citric acid, sucrose, and Tween 80 enhanced the solubility of [K6T]P8 peptide and its dimer, only the sucrose did not interfere with the in vitro proliferation assay used to assess their biological activity. The results here presented, reinforce nonclinical characterization of the [K6T]P8 peptide, a potential agent for the treatment of RA and other diseases associated with IL-15 overexpression.

Peptides as Therapeutic Agents for Inflammatory-Related Diseases

“…Thus, Lys 41 was substituted with two polar amino acids, glutamic acid and threonine, containing a charged and an uncharged side chain, in peptides named [K6E]P8 and [K6T]P8, respectively. [K6T]P8 peptide showed, in CTLL-2 cells proliferation assays, higher antagonist activity with respect to P8, while [K6E]P8 was inactive and the dimer of [K6T]P8 showed the highest inhibitory activity, in CTLL-2 proliferation assays [ 81 ].…”

d‐Amino acid substitutions and dimerization increase the biological activity and stability of an IL‐15 antagonist peptide

“…In this experiment, the P8 peptide could not be included as a control because one of the main modifications of this peptide is the spontaneous dimerization through free cysteine residues by disulfide bond formation (unpublished results). This transformation was detected for the [K6T]P8 peptide in synovial fluid of patients with RA 38 …”

“…In general, the dimerization by formation of a disulfide bond introduces conformational constraints that can improve the biological activity, selectivity, and stability of those peptides that are directly involved in receptor binding. 37 In fact, we demonstrated that the P8 peptide The [A4a]P8 peptide has a lysine residue at the N-terminal, which is associated with a lower half-life in plasma. 39 Moreover, the presence of basic residues (lysine or arginine) in the peptide sequence constitutes cleavage sites of trypsin, plasmin, and kallikrein.…”