www.nature.com/scientificreports OPEN Distribution and kinetics of the Kv1.3-blocking peptide HsTX1[R14A] in experimental rats Received: 19 January 2017 Ralf Bergmann1, Manja Kubeil1,2, Kristof Zarschler1, Sandeep Chhabra3, Rajeev B. Tajhya4, Accepted: 9 May 2017 Christine Beeton 4, Michael W. Pennington5, Michael Bachmann1, Raymond S. Norton 3 & Published: xx xx xxxx Holger Stephan 1 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. 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 regulation1. Elevated expression of the voltage-gated channel Kv1.3 in effector memory T (TEM) lymphocytes is implicated in the pathology of a range of autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus and type 1 diabetes2, as well as non-autoimmune conditions such as asthma, chronic obstructive pulmonary disease and graft-versus-host disease, and therefore Kv1.3 channels are a highly promising therapeutic target for the treatment of such diseases3, 4. The sea anemone-derived peptide ShK, from Stichodactyla helianthus, exhibits 5 high affinity for these channels, with an IC50 value of 11 pM . ShK and its analogues significantly reduce disease severity in several animal models of TEM lymphocyte-related diseases including delayed-type hypersensitivity, chronic relapsing-remitting experimental autoimmune encephalomyelitis, pristane-induced arthritis and asthma 6–9 via blockade of Kv1.3 channels in TEM cells . One ShK analogue, ShK-186 (dalazatide), has completed phase I human clinical trials following subcutaneous administration. It was well tolerated and achieved clinical improve- ment in target lesions in patients with moderate plaque psoriasis10. These promising preclinical and clinical stud- ies warrant the ongoing development of Kv1.3 channel blockers for the treatment of a variety of immune-related diseases. HsTX1 toxin, from the scorpion Heterometrus spinnifer, is a 34-residue, C-terminally amidated peptide cross-linked by four disulfide bridges. The native peptide blocks Kv1.3 channels with similar affinity to ShK pep- tide analogues11, 12, and an analogue, HsTX1[R14A], has been developed recently that not only retains the high affinity of the naturally-occurring peptide, but also exhibits an approximate 2000-fold greater selectivity for Kv1.3 channels over other potassium channels13, 14. HsTX1[R14A] therefore represents a very promising therapeutic candidate for the treatment of the above-mentioned diseases. 1Helmholtz-Zentrum Dresden - Rossendorf, Institute of Radiopharmaceutical Cancer Research, Dresden, D-01328, Germany. 2School of Chemistry, Monash University, Melbourne, Victoria, 3800, Australia. 3Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, 3052, Australia. 4Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX, 77030, USA. 5Peptides International, Louisville, KY, 40299, USA. Correspondence and requests for materials should be addressed to R.S.N. (email: ray. [email protected]) or H.S. (email: [email protected]) SCIENTIFIC REPORTS | 7: 3756 | DOI:10.1038/s41598-017-03998-x 1 www.nature.com/scientificreports/ Figure 1. Structure of HsTX1[R14A] conjugate. (a) Simulated structure of HsTX1[R14A]14 with the NOTA tag at the N-terminus shown schematically. (b) Amino acid sequence of HsTX1[R14A] showing the positions of the four disulfide bridges11. Although HsTX1[R14A] adopts a very stable structure that is resistant to proteolysis14, an oral route of admin- istration appears unlikely. We have therefore explored buccal15–17 and pulmonary18 delivery of this peptide, both of which proved to be effective. We are also exploring slow-release formulations (unpublished). An important question that arises in considering the optimal route of administration and frequency of dosing, however, is the lifetime of the peptide in vivo and its tissue distribution. In the case of ShK-186, for example, a 111In-labelled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid conjugate was used to assess whole-blood pharmacoki- netic parameters as well as peptide absorption, distribution, and excretion. ShK-186 was absorbed slowly from the injection site, resulting in blood concentrations above the Kv1.3 channel-blocking IC50 value for up to 7 days in monkeys8. In delayed-type hypersensitivity, chronic relapsing-remitting experimental autoimmune encepha- lomyelitis, and pristane-induced arthritis rat models, a single dose of ShK-186 every 2 to 5 days was as effective as daily administration8. The slow dissemination of ShK-186 from the injection site and its long residence time on the Kv1.3 channel contribute to its prolonged therapeutic effect in animal models of autoimmune disease. In this study we have used HsTX1[R14A] modified at its N-terminus with a 1,4,7-triazacyclononane-triacetic acid (NOTA) tag for labelling with 64Cu as an ideal positron emitter19, 20, enabling positron emission tomography (PET) studies of peptide distribution in rats over a period of days. The results show a long in vivo half-life as a result of slow renal clearance of the peptide. 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 target6, 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. Results Peptide characterisation. HsTX1[R14A] was synthesised as described previously14 but with a NOTA tag coupled to the N-terminus via an aminoethyloxyethyloxyacetyl (AeeA) linker, as shown in Fig. 1. NOTA- HsTX1[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. Radiolabelling. The peptide HsTX1[R14A] with a NOTA tag was efficiently labelled with64 CuII (half-life 12.7 h). The 64CuII complex was formed in a concentration range of 5 to 100 µg/100 µL NOTA-peptide conju- 64 gate within 30 min at 37 °C, employing 35–55 MBq (0.95–1.5 mCi) of [ Cu]CuCl2. Radio-TLC and radio-HPLC exhibit a single peak of [64Cu]Cu-NOTA-HsTX1[R14A] and no trace of free 64CuII (Supplementary Figs S3 and S4). The64 CuII complex formed was stable in the presence of 0.1 M aqueous EDTA solution for at least 24 h. These results verify that NOTA is an appropriate bifunctional chelating agent for 64Cu-labelling of peptides22, 23, and the corresponding 64Cu-labelled peptide HsTX1[R14A] can be utilised to obtain reliable information about the biodistribution. Biodistribution. [64Cu]Cu-NOTA-HsTX1[R14A] peptide biodistribution was studied by organ and tissue extraction (Fig. 2; Supplementary Tables S1 and S2) and by small animal PET. [64Cu]Cu-NOTA-HsTX1[R14A] was injected intravenously (i.v.) to study the biodistribution and kinetics after direct injection into the circulation. A subcutaneous (s.c.) injection was carried out to evaluate the distribution and kinetics of the radiotracer with first contact to the lymphatic system and in direct comparison to i.v. administration. As a relatively low molecular mass peptide (4164 Da before complexation with Cu), the [64Cu] Cu-NOTA-HsTX1[R14A] was rapidly distributed in the circulation after i.v. injection. The s.c. injection caused SCIENTIFIC REPORTS | 7: 3756 | DOI:10.1038/s41598-017-03998-x 2 www.nature.com/scientificreports/ Figure 2. Biodistribution of the [64Cu]Cu-NOTA-HsTX1[R14A] in rats after single injection (i.v., black bars, n = 12; s.c., gray bars, n = 4). The data are presented as %ID (a–d) or SUV (e–h) in means ± SEM; BAT – brown adipose tissue, WAT – white adipose tissue, Hard. Gl. – Harderian glands, Sub. gl. – submandibular glands, lymph nodes – submandibular lymph nodes, urine calc. – urine calculated as difference between injected dose and recovery. a delay in the 64Cu activity accumulation in several tissues. After 60 min this effect was significant in the spleen, pancreas, kidneys, heart and the remaining body (Supplementary Table S1). However, due to the fast diffusion from the injection site into the circulation, no significant difference in the whole blood concentration between the two injection types could be detected after 1 h. After 4 h, the spleen and femur represent the only organs where the SCIENTIFIC REPORTS | 7: 3756 | DOI:10.1038/s41598-017-03998-x 3 www.nature.com/scientificreports/ Figure 3.