Lipidation of KV1.3 Blocker HsTX1[R14A] Extends Plasma Half-Life 2-Fold, Improves Biodistribution

Upregulation of the voltage-gated potassium channel KV1.3 in effector memory T cells is strongly implicated in several autoimmune diseases, including rheumatoid arthritis. Selective blockade of KV1.3 presents an attractive therapeutic strategy. HsTX1[R14A], a 34-residue peptide, demonstrates picomolar potency and high selectivity for KV1.3, proving effective in rodent models of rheumatoid arthritis. However, its in vivo half-life requires improvement to enhance its potential as a viable therapeutic candidate, driving the need for pharmacokinetic optimization strategies.

Researchers investigated the effects of conjugating C14, C16, or C18 acyl chains to HsTX1[R14A] on its binding to serum albumin and lipoproteins, potency at KV1.3, pharmacokinetics (PK), and biodistribution. For biodistribution studies, the fluorophore Cy5 was conjugated to the HsTX1[R14A] analogs. LC-MS/MS assays were developed to quantify the lipidated peptides in mouse plasma. Peptides were administered via subcutaneous (SC) injection to mice, and their plasma exposure and tissue distribution were monitored at various time points, including 4 and 12 hours post-injection.

Conjugation of palmitic (C16) and stearic (C18) acids to HsTX1[R14A] resulted in a significant over 20-fold loss in potency at KV1.3. Despite this, the modification significantly enhanced the peptide's binding to serum albumin, while showing minimal association with plasma lipoproteins. This lipidation strategy profoundly impacted pharmacokinetics: > Lipidation extended the plasma exposure of HsTX1[R14A] by 9-fold and prolonged its elimination half-life by 2-fold after subcutaneous injection to mice. Biodistribution studies using Cy5-labelled lipidated analogs demonstrated enhanced accumulation at the dosing site and draining lymph nodes at 4 and 12 hours after SC injection compared to unlipidated Cy5-HsTX1[R14A]. Furthermore, the lipidated analogs accumulated more in the gastrointestinal tract, while exhibiting significantly less exposure in the kidneys at 4 and 12 hours post-administration, suggesting a favorable shift in organ distribution.