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

Oral activity has been described for cyclotide-containing traditional medicines, and demonstrated for reengineered cyclotides bearing grafted therapeutic epitopes, highlighting their potential for translation to the clinic. Here we report preclinical pharmacokinetic parameters for the prototypic cyclotide kalata B1 (kB1) and two orally active grafted analogues, ckb-KAL and ckb-KIN, to provide the first in vivo dose-exposure metrics for cyclotides. Native and grafted kB1 molecules exhibited multiple compartment kinetics and measurable but limited oral bioavailability of similar magnitude to several orally administered peptide drugs in the clinic. Cyclotides are mostly associated with the central compartment, and display small (0.07-0.13 L kg-1 for kB1 and ckb-KIN) to moderate (1 L kg-1 for ckb-KAL) steady state volumes of distribution. This study provides new data essential to the evaluation of cyclotides as therapeutics, validating them as a viable drug design scaffold with tunable pharmacokinetic properties.

Novel approaches to the design of bioavailable melanotropins.

INTRODUCTION: The melanocortin system is a primordial and critical system for survival, involved in a wide variety of physiological functions. It includes melanocortin receptors (MCRs) and melanotropin ligands (MCLs). MCRs are important drug targets that can regulate several key physiological processes. Extensive efforts have been made to develop peptide and peptidomimetics targeting melanocortin receptors including MC1R, MC3R, MC4R and MC5R. Most research is focused on developing potent and selective melanotropins. However, developing bioavailable melanotropins remains challenging. Areas covered: Herein, the authors summarize promising strategies for developing bioavailable MCLs by using cyclized N-methylated melanotropins, and using cyclotide and tetrapeptide as templates. They discuss their unique advantages in oral availability and targeting MCRs in the central nervous system or in peripheral tissues. Finally, they discuss the observed differences in thepharmacology of MCRs between in vitro and in vivo tests. Expert opinion: N-methylated cyclized melanotropins have great potential to become bio- available drugs targeting MCRs in the brain, while MCR-grafted cyclotides tend to target MCRs in peripheral tissue. A better understanding of the biased signaling process is a new challenge and opportunity for the future discovery of bioavailable MCLs.

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

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.

Biodistribution of the cyclotide MCoTI-II, a cyclic disulfide-rich peptide drug scaffold.

Disulfide-rich macrocyclic peptides are promising templates for drug design because of their unique topology and remarkable stability. However, little is known about their pharmacokinetics. In this study, we characterize the biodistribution in mice of Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II), a cyclic three-disulfide-containing peptide that has been used in a number of studies as a drug scaffold. The distribution of MCoTI-II was compared with that of chlorotoxin, which is a four-disulfide-containing peptide that has been used to develop brain tumor imaging agents; dermorphin, which is a disulfide-less peptide; and bovine serum albumin, a large protein. Both MCoTI-II and chlorotoxin distributed predominantly to the serum and kidneys, confirming that they are stable in serum and suggesting that they are eliminated from the blood through renal clearance. Although cell-penetrating peptides have been reported to be able to transport across the blood-brain barrier, MCoTI-II, which is a cell-penetrating peptide, showed no uptake into the brain. The uptake of chlorotoxin was higher than that of MCoTI-II but lower than that of dermorphin, which is considered to have low uptake into the brain. This study provides insight into the behavior of disulfide-rich peptides in vivo. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

The Prototypic Cyclotide Kalata B1 Has a Unique Mechanism of Entering Cells.

Cyclotides combine the stability of disulfide-rich peptides with the intracellular accessibility of cell-penetrating peptides, giving them outstanding potential as drug scaffolds with an ability to inhibit intracellular protein-protein interactions. To realize and optimize the application of cyclotides as a drug framework and delivery system, we studied the ability of the prototypic cyclotide, kalata B1, to enter mammalian cells. We show that kalata B1 can enter cells via both endocytosis and direct membrane translocation. Both pathways are initiated by targeting phosphatidylethanolamine phospholipids at the cell surface and inducing membrane curvature. This unusual approach to initiate internalization might be harnessed to deliver drugs into cells and, in particular, cancer cells, which present a higher proportion of surface-exposed phosphatidylethanolamine phospholipids. Our findings highlight the potential of these peptides as drug leads for the modulation of traditionally "undruggable" targets, such as intracellular protein-protein interactions.

Structural parameters modulating the cellular uptake of disulfide-rich cyclic cell-penetrating peptides: MCoTI-II and SFTI-1.

Peptides are emerging as a new class of therapeutics due to their high potency and specificity for a range of targets, including the inhibition of protein-protein interactions. Disulfide-rich cyclic peptides, in particular, have attracted much attention in drug design due to their ultra-stable structure. Moreover, some of them have been shown to internalize into cells, which makes them potential scaffolds to deliver pharmaceutically bioactive sequences to intracellular targets. Here we examined the effects of structural modifications on the cell-penetrating properties of two disulfide-rich cyclic cell-penetrating peptides, Momordica cochinchinensis trypsin inhibitor II (MCoTI-II) and sunflower trypsin inhibitor-1 (SFTI-1). We found that the cellular uptake of MCoTI-II can be improved by increasing the overall positive charge of the native sequence. On the other hand, mutations to SFTI-1 did not significantly influence its cellular uptake, suggesting a non-specific endocytosis-dependent mechanism of cellular uptake. This study provides an understanding of the structural features affecting the internalization of MCoTI-II and SFTI-1, and hence provides a guide for the development of these disulfide-rich cyclic scaffolds into potential drug leads.