(T1230-08-52) Lipidation of Kv1.3 Blocking Peptide HsTX1[R14A] Modulates Its Pharmacokinetics and Biodistribution to Target Tissues

Purpose: Autoimmune diseases currently impact over 250 million people globally. Current therapeutics for autoimmune diseases typically provide symptomatic relief or halt progression temporarily, but side effects and relapses are still significant challenges for autoimmune patients. Previous studies have shown that the pathogenesis of several autoimmune diseases is associated with upregulation of the voltage-gated potassium channel Kv1.3 in effector memory T cells (1-3). Inhibition of Kv1.3 with HsTX1[R14A], a 34-residue disulfide-stabilised peptide, is a promising treatment approach for autoimmune disease due to its high potency (IC50 = 45 pM) and target selectivity (4). HsTx1[R14A] is highly stable and resistant to a range of proteases in in vitro studies. More importantly, it was found to be effective when dosed weekly in rodent models of rheumatoid arthritis and delayed-type hypersensitivity (5). However, for patients with autoimmune diseases requiring long-term medication, the dosing interval of HsTX1[R14A] could be further optimised to reduce dosing frequency and enhance its suitability as a drug candidate. Lipidation of therapeutics is one of the approaches to prolong their systemic exposure and alter their biodistribution. Lipid moieties may enhance the ability of the modified drugs to associate with endogenous macromolecules such as albumin and lipoproteins, in the interstitial spaces following subcutaneous administration (6). This could allow the drug to hitchhike on albumin or lipoprotein tracking pathways, leading to protection against rapid renal clearance. Furthermore, lipidation of drugs might improve their circulation time in vivo by forming a depot at the subcutaneous injection site to allow slow release to the systemic circulation (7). Here we explored how lipid conjugation of HsTX1[R14A] affected its circulating half-life and its distribution in organs and draining lymph nodes.

Methods: HsTX1[R14A] was conjugated to two different lipid moieties (Lip1 and Lip2) on the N terminus. Subsequently, the impact of lipid conjugation on pharmacokinetics was evaluated in C57B/L6J mice after subcutaneous administration (2 mg/kg). LC-MS/MS assays were developed and validated to quantify HsTX1[R14A] and lipidated analogues in mouse plasma. Cy5-abelled HsTX1[R14A] analogues were used for biodistribution and plasma protein binding studies. Cy5 was conjugated to a lysine residue in native and lipidated HsTX1[R14A] using click chemistry (8). Ultracentrifugation was used to investigate the association between peptide analogues and plasma proteins and lipoproteins. For biodistribution studies, C57B/L6J mice were administered 2 mg/kg Cy5-labelled HsTX1[R14A] analogues subcutaneously. Vital organs, tissues at the injection site and primary draining lymph nodes were collected 4 and 12 h post-dosing, then imaged using an AMI HT. Data were analysed using ImageJ, Microsoft Excel and GraphPad Prism . Statistical analysis of pharmacokinetic parameters was done using one-way ANOVA with post hoc Tukey’s test. Comparison of biodistribution data was performed using a Student’s t-test.

Results: The LCMS/MS assay for quantification of HsTX1[R14A] in plasma was optimised and validated from 2.7 to 66.7 nM. Both Lip1-HsTX1[R14A] and Lip2-HsTX1[R14A] LC-MS/MS assays had a lower limit of quantification of 5 nM. From the pharmacokinetic profiles, both lipidated analogues of HsTX1[R14A] showed a delay in peak plasma concentration compared to the native peptide. Lipidated analogues were also found to have prolonged plasma exposure after subcutaneous injection. Using ultracentrifugation, we found that Cy5-Lip1-HsTX1R14A] showed much stronger association with plasma protein compared to Cy5-HsTX1[R14A], suggesting that albumin hitchhiking could be one of the mechanisms contributing to the prolonged circulation time of the lipidated peptide. Biodistribution studies using Cy5-labelled HsTX1[R14A] analogues demonstrated significantly higher accumulation of lipidated analogues in the skin, muscle and adipose tissues at the injection site at both 4 and 12 h post-dosing, which aligns with the delayed time in reaching the maximum concentration in the pharmacokinetic profile. This could suggest a depot-like effect that slowed the systemic absorption of the lipidated analogues. Cy5-Lip1-HsTX1[R14A] enhanced exposure in disease-relevant organs at 4 and 12 h after subcutaneous administration, including kidney, liver, lungs, heart, pancreas and the gastrointestinal tract. No significant accumulation in the brain was found for either natïve or lipidated peptides. Compared to Cy5-HsTX1[R14A], lipidated analogues were also found to have higher accumulation in the primary draining lymph nodes on the dosing side following subcutaneous administration.

Conclusion: In summary, lipidation of HsTX1[R14A] showed prolonged plasma and tissue exposure and enhanced lymphatic trafficking. This may relate to binding to plasma protein and/or to depot formation at the injection site, leading to delayed and prolonged exposure. These findings suggested that lipidation is a promising approach for optimising the pharmacokinetics of HsTX1[R14A] and provide a basis for improving its dosing regimen in the treatment of autoimmune diseases.

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Acknowledgements: Disclaimers: All animal studies were approved by the Monash Institute of Pharmaceutical Sciences (MIPS) Animal Ethics Committee and were conducted in accordance with the Australian and New Zealand Council for the Care of Animals in Research and Teaching guidelines. Disclosure or conflict of interest statement: The authors declare no conflict of interest. The authors would like to thank Cameron Nowell for his valuable assistance with the imaging analysis. The authors would also like to thank staff from HMST lab for their kind guidance in LC-MS/MS assay development.

Figure 1. Pharmacokinetic profiles of HsTX1[R14A] and two lipidated analogues. N=3-6. Data expressed as Mean+/- SD.

Figure 2. Fluorescence Intensity of Cy5-HsTX1[R14A] and Cy5-Lip1-HsTX1[R14A] at the skin, muscle and adipose tissue at the site of administration of the dosing side and the symmetrical area at the non-dosing side at 4 hr and 12 hr post-dosing in mice. N=5. Data was expressed as mean +/- SD. Statistical analysis was done using Student's t-test. DS: dosing side. NDS: non-dosing side.

Figure 2. Fluorescence Intensity of Cy5-HsTX1[R14A] and Cy5-Lip1-HsTX1[R14A] at the inguinal and axillary lymph node of the dosing side at 4 hr and 12 hr post-dosing in mice. Note that the inguinal lymph node is the one closest to the injection site, and the inguinal lymph node drains to the axillary lymph node following lymphatic drainage direction. N=5. Data was expressed as mean +/- SD. Statistical analysis was done using Student's t-test. DS: dosing side.