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.

Targeted Delivery of Cyclotides via Conjugation to a Nanobody.

Many naturally occurring peptides have poor proteolytic stability, which limits their therapeutic applications. Cyclotides are plant-derived cyclic peptides that resist proteolysis due to their highly constrained structure, comprising a head-to-tail cyclic backbone and three disulfide bonds that form a cystine-knotted core. This structure makes them useful as scaffolds onto which peptide sequences (epitopes) can be grafted. In this study, VHH7, an alpaca-derived nanobody that targets murine class II MHC molecules, was used for the targeted delivery of cyclotides to antigen-presenting cells (APCs). The cyclotides MCoTI-I, and MCoTI-I with a HA-tag (YPYDVPDYA) grafted into loop 6 (MCoTI-HA), were tested for immunogenic properties. To produce the requisite VHH7-peptide conjugates, a site-specific sortase A-catalyzed reaction in combination with a copper-free strain-promoted cycloaddition reaction was used. MCoTI-I alone did not display any obvious antibody response, thus showing the capacity of cyclotides as immunologically silent scaffolds. By contrast, MCoTI-I conjugated to VHH7 elicited antibodies against cyclic or linear MCoTI-I, thus suggesting a simple and robust approach for targeting cyclotides to APCs, and potentially to other cell types. A similar antibody response was observed when MCoTI-HA was conjugated to VHH7, but there was no reactivity toward a linear HA-tag itself, suggesting differences in conformational constraint between cyclotide-presented and linear epitopes. Studies of commercially available HA antibodies applied to MCoTI-HA confirmed that the conformation of peptide immunogens affects their reactivity. Thus, the production of antibodies that recognize constrained epitopes may benefit from engraftment onto scaffolds such as cyclotides. More broadly, this study validates that a prototypic cyclotide, a member of a peptide family that has proven to be useful as drug design scaffolds in many other studies, can efficiently reach a specific target in vivo.