Improved method for quantitative analysis of the cyclotide kalata B1 in plasma and brain homogenate

Abstract

This study provides a new method for quantifying the cyclotide kalata B1 in both plasma and brain homogenate. Cyclotides are ultra‐stable peptides with three disulfide bonds that are interesting from a drug development perspective as they can be used as scaffolds. In this study we describe a new validated LC‐MS/MS method with high sensitivity and specificity for kalata B1. The limit of quantification was 2 ng/mL in plasma and 5 ng/gmL in brain homogenate. The method was linear in the range 2–10,000 ng/mL for plasma and 5–2000 ng/g for brain. Liquid Chromatographic separation was performed on a HyPurity C18 column, 50 × 4.6 mm, 3 µm particle size. The method had inter‐ and intra‐day precision and accuracy levels <15% and 12% respectively. Applying the method to in vivo plasma samples and brain homogenate samples from equilibrium dialysis yielded satisfying results and was able to describe the plasma pharmacokinetics and brain tissue binding of kalata B1. The described method is quick, reproducible and well suited to quantifying kalata B1 in biological matrices.

Pharmacokinetic characterization of kalata B1 and related therapeutics built on the cyclotide scaffold

“…Indeed, the fraction of kB1 unbound in rat plasma determined in previously reported equilibrium dialysis was higher (25%) (Melander et al, 2016), suggesting the plasma protein binding values determined in our experiments might be slightly overestimated. However, whether the equilibrium dialysis method used in the previous study (Melander et al, 2016) was set up to minimize pH change through incubation in a CO2-rich atmosphere was not reported. In addition, we observed extensive binding of the investigated peptides to equilibrium dialysis membranes, which limits assay reliability and prompted our use of the ultracentrifugation method, which is particularly suited to analyses of compounds with high nonspecific binding as it eliminates problems arising from free membrane effects (Damre et al, 2011).…”

“…All the tested cyclotides demonstrated biological (terminal phase) half-lives in the range 1.25−3.2 hours, with kB1 having a biological (terminal phase) half-life of 192 minutes, longer than that recently reported (Melander et al, 2016). The calculated volumes of distribution for the central compartment (Vc) spanned a least an order of magnitude across the native and grafted kB1 cyclotides (kB1, 42.4 mL kg -1 ; ckb-KIN, 114.22 mL kg -1 ; ckb-KAL, 1034.45 mL kg -1 ), and likewise for the volume of distribution at steady state (Vdss) (kB1, 71.14 mL kg -1 ; ckb-KIN, 136.00 mL kg -1 ; ckb-KAL, 1053.60 mL kg -1 ).…”

“…One caveat of the ultracentrifugation method for determining plasma protein binding is that the pH typically increases from initial physiological levels (pH 7.4) in the absence of CO2 buffering, which potentially causes higher binding to plasma proteins (Kochansky et al, 2008). Indeed, the fraction of kB1 unbound in rat plasma determined in previously reported equilibrium dialysis was higher (25%) (Melander et al, 2016), suggesting the plasma protein binding values determined in our experiments might be slightly overestimated. However, whether the equilibrium dialysis method used in the previous study (Melander et al, 2016) was set up to minimize pH change through incubation in a CO2-rich atmosphere was not reported.…”

“…A key metric for assessing the clinical translatability of cyclotides is oral bioavailability (F%) as drugs with a low F% tend to exhibit higher variability in drug exposure among recipients, leading to higher risks of administering sub-therapeutic or toxic doses, particularly when administering drugs with a narrow therapeutic index (Hellriegel et al, 1996). Notwithstanding the variable predictive correlation between human and animal drug bioavailability (Musther et al, 2014), at present there remains a dearth of information regarding the in vivo pharmacokinetics of cyclotides, which have been only minimally explored quantitatively in animal studies (Colgrave et al, 2005;Melander et al, 2016).…”

Cyclic peptide oral bioavailability: Lessons from the past

“…As evident from recent literature on cyclic peptides and the papers published in this issue, there is significant interest in cyclic peptides as therapeutics. The reason for this interest is founded not only on the belief that cyclic peptides have advantages over other drug modalities but also the growing perception that they are a privileged sub‐class among peptides as a whole because of their greater potential to be “drug‐like,” as opposed to linear peptides for example.…”