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. Elevated expression of the voltage-gated channel Kv1.3 in effector memory T (T EM) lymphocytes is implicated in the pathology of a range of autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus and type 1 diabetes, 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 diseases. The sea anemone-derived peptide ShK, from Stichodactyla helianthus, exhibits high affinity for these channels, with an IC 50 value of 11 pM. ShK and its analogues significantly reduce disease severity in several animal models of T EM lymphocyte-related diseases including delayed-type hypersensitivity, chronic relapsing-remitting experimental autoimmune encephalomyelitis, pristane-induced arthritis and asthma via blockade of Kv1.3 channels in T EM cells. One ShK analogue, ShK-186 (dalazatide), has completed phase I human clinical trials following subcutaneous administration. It was well tolerated and achieved clinical improvement in target lesions in patients with moderate plaque psoriasis. 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.
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 peptide analogues, 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 channels. HsTX1[R14A] therefore represents a very promising therapeutic candidate for the treatment of the above-mentioned diseases.
Although HsTX1[R14A] adopts a very stable structure that is resistant to proteolysis, an oral route of administration appears unlikely. We have therefore explored buccal and pulmonary 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 We are also exploring slow-release formulations (unpublished).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 111 In-labelled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid conjugate was used to assess whole-blood pharmacokinetic 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 IC 50 value for up to 7 days in monkeys. 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. 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 64 Cu as an ideal positron emitter, 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 target, 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.
HsTX1[R14A] was synthesised as described previously 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 IC 50 of 68 ± 12 pM (Supplementary Fig.S2), which was close enough to that of HsTX1[R14A] (IC 50 45 ± 3 pM) to confirm that the tagged peptide was an excellent mimic of the parent peptide for the purpose of this study.
The peptide HsTX1[R14A] with a NOTA tag was efficiently labelled with 64 Cu II (half-life 12.7 h). The 64 Cu II complex was formed in a concentration range of 5 to 100 µg/100 µL NOTA-peptide conjugate within 30 min at 37 °C, employing 35–55 MBq (0.95–1.5 mCi) of [64 Cu]CuCl 2. Radio-TLC and radio-HPLC exhibit a single peak of [64 Cu]Cu-NOTA-HsTX1[R14A] and no trace of free 64 Cu II (Supplementary FigsS3 and S4). The 64 Cu II 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 64 Cu-labelling of peptides, and the corresponding 64 Cu-labelled peptide HsTX1[R14A] can be utilised to obtain reliable information about the b
