Distribution and kinetics of the Kv1.3-blocking peptide HsTX1[R14A] in experimental rats

Ralf Bergmann,Raymond S Norton,Rajeev B Tajhya,Holger Stephan,Kristof Zarschler,Michael Bachmann,Sandeep Chhabra,Christine Beeton,Manja Kubeil,Michael W Pennington

doi:10.1038/s41598-017-03998-x

Abstract

The peptide HsTX1[R14A] is a potent and selective blocker of the voltage-gated potassium channel Kv1.3, which is a highly promising target for the treatment of autoimmune diseases and other conditions. In order to assess the biodistribution of this peptide, it was conjugated with NOTA and radiolabelled with copper-64. [64Cu]Cu-NOTA-HsTX1[R14A] was synthesised in high radiochemical purity and yield. The radiotracer was evaluated in vitro and in vivo. The biodistribution and PET studies after intravenous and subcutaneous injections showed similar patterns and kinetics. The hydrophilic peptide was rapidly distributed, showed low accumulation in most of the organs and tissues, and demonstrated high molecular stability in vitro and in vivo. The most prominent accumulation occurred in the epiphyseal plates of trabecular bones. The high stability and bioavailability, low normal-tissue uptake of [64Cu]Cu-NOTA-HsTX1[R14A], and accumulation in regions of up-regulated Kv channels both in vitro and in vivo demonstrate that HsTX1[R14A] represents a valuable lead for conditions treatable by blockade of the voltage-gated potassium channel Kv1.3. The pharmacokinetics shows that both intravenous and subcutaneous applications are viable routes for the delivery of this potent peptide.

Highlights

Voltage-gated potassium (Kv) channels are integral membrane proteins that regulate cell membrane potential and are involved in a variety of cellular functions including apoptosis and cell volume regulation[1]
NOTAHsTX1[R14A] folded rapidly to a single major product, resulting in the typical pattern of a major earlier-eluting peak by RP-HPLC followed by later-eluting misfolded species and side-products (Supplementary Fig. S1)
When tested against the voltage-gated potassium channel Kv1.3 expressed in L929 mouse fibroblast cells, the tagged peptide had an IC50 of 68 ± 12 pM (Supplementary Fig. S2), which was close enough to that of HsTX1[R14A] (IC50 45 ± 3 pM) to confirm that the tagged peptide was an excellent mimic of the parent peptide for the purpose of this study

Summary

Introduction

Voltage-gated potassium (Kv) channels are integral membrane proteins that regulate cell membrane potential and are involved in a variety of cellular functions including apoptosis and cell volume regulation[1]. It was well tolerated and achieved clinical improvement in target lesions in patients with moderate plaque psoriasis[10] These promising preclinical and clinical studies warrant the ongoing development of Kv1.3 channel blockers for the treatment of a variety of immune-related diseases. In delayed-type hypersensitivity, chronic relapsing-remitting experimental autoimmune encephalomyelitis, and pristane-induced arthritis rat models, a single dose of ShK-186 every 2 to 5 days was as effective as daily administration[8]. In view of the high potency and selectivity of HsTX1[R14A] for the target channel Kv1.3, and the importance of this channel as a therapeutic target[6, 21], the persistence of this peptide in vivo strengthens the case for its further development as a therapeutic for the treatment of the above-mentioned immune-related diseases

Methods

Results

Conclusion