A Biodistribution Study of the Radiolabeled Kv1.3-Blocking Peptide DOTA-HsTX1[R14A] Demonstrates Brain Uptake in a Mouse Model of Neuroinflammation

Abstract

The voltage-gated potassium channel Kv1.3 regulates the pro-inflammatory function of microglia and is highly expressed in the post-mortem brains of individuals with Alzheimer’s and Parkinson’s diseases. HsTX1[R14A] is a selective and potent peptide inhibitor of the Kv1.3 channel (IC50 ∼ 45 pM) that has been shown to decrease cytokine levels in a lipopolysaccharide (LPS)-induced mouse model of inflammation. Central nervous system exposure to HsTX1[R14A] was previously detected in this mouse model using liquid chromatography with tandem mass spectrometry, but this technique does not report on the spatial distribution of the peptide in the different brain regions or peripheral organs. Herein, the in vivo distribution of a [64Cu]Cu-labeled DOTA conjugate of HsTX1[R14A] was observed for up to 48 h by positron emission tomography (PET) in mice. After subcutaneous administration to untreated C57BL/6J mice, considerable uptake of the radiolabeled peptide was observed in the kidney, but it was undetectable in the brain. Biodistribution of a [68Ga]Ga-DOTA conjugate of HsTX1[R14A] was then investigated in the LPS-induced mouse model of neuroinflammation to assess the effects of inflammation on uptake of the peptide in the brain. A control peptide with very weak Kv1.3 binding, [68Ga]Ga-DOTA-HsTX1[R14A,Y21A,K23A] (IC50 ∼ 6 μM), was also tested. Significantly increased uptake of [68Ga]Ga-DOTA-HsTX1[R14A] was observed in the brains of LPS-treated mice compared to mice treated with control peptide, implying that the enhanced uptake was due to increased Kv1.3 expression rather than simply increased blood–brain barrier disruption. PET imaging also showed accumulation of [68Ga]Ga-DOTA-HsTX1[R14A] in inflamed joints and decreased clearance from the kidneys in LPS-treated mice. These biodistribution data highlight the potential of HsTX1[R14A] as a therapeutic for the treatment of neuroinflammatory diseases mediated by overexpression of Kv1.3.

Immunocytoprotection after reperfusion with Kv1.3 inhibitors has an extended treatment window for ischemic stroke

“…Opening of the blood brain barrier (BBB) in tMCAO has been reported to be biphasic, once at 6 h and once at 72 h, with no Evans blue or fluorescein extravasation occurring at 24 or 48 h ( Hone et al, 2018 ). However, even under conditions where the brain is inflamed, and the BBB confirmed to be compromised, only very small amounts of ShK-sized peptides (4 kDa) penetrate into the brain as a recently published PET imaging study using HsTX1 labeled with a long-lived 64 Cu-isotope demonstrated ( Reddiar et al, 2022 ). Similar to ShK-223, HsTX1 [R14A] is a 34-residue, C-terminally amidated peptide cross-linked by four disulfide bridges, that inhibits K V 1.3 with picomolar affinity.…”

“…Similar to ShK-223, HsTX1 [R14A] is a 34-residue, C-terminally amidated peptide cross-linked by four disulfide bridges, that inhibits K V 1.3 with picomolar affinity. When LPS treatment was used to open-up the blood brain barrier, brain uptake of the 64 Cu-labeled HsTX1 [R14A] only increased from less than 0.1 SUV (standard uptake value) to 0.25 SUV compared to SUVs of 40-95 in the kidney, demonstrating very limited bioavailability in the brain even with a compromised BBB ( Reddiar et al, 2022 ). Similar results had been obtained with an LS-MS/MS assay ( Reddiar et al, 2021 ).…”

Peripheral Administration of the Kv1.3-Blocking Peptide HsTX1[R14A] Improves Cognitive Performance in Senescence Accelerated SAMP8 Mice

“…Our work has demonstrated that HsTX1[R14A] effectively inhibits microglia-mediated neurotoxicity by attenuating the release of pro-inflammatory mediators such as tumor necrosis factor-alpha (TNF-α) and nitric oxide from lipopolysaccharide (LPS)-activated microglia in vitro [ 23 ]. Following peripheral administration in a mouse model of neuroinflammation induced by LPS, HsTX1[R14A] readily crosses the blood–brain barrier (BBB) [ 24 ] to achieve microglial Kv1.3 blockade, with a substantial reduction in plasma and brain levels of cytokines, including TNF-α, interleukin 1β (IL-1β) and interleukin-6 (IL-6) [ 23 , 25 ]. It should be noted that the reduction of cytokines observed in the brain could be a combined effect of HsTX1[R14A] acting on both microglia in the brain and peripheral immune cells.…”