¹Hiroshima University, Department of Cellular and Molecular Pharmacology, Graduate School of Biomedical and Health Sciences, Hiroshima, Japan, ²Osaka University, Global Center for Medical Engineering and Informatics, Suita, Japan, ³Hiroshima University, Translational Research Center, Hiroshima, Japan, ⁴Osaka University, Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Suita, Japan, ⁵University of Tsukuba, Division of Biomedical Science, Institute of Medicine, Tsukuba, Japan, ⁶University of Tsukuba, Transborder Medical Research Center, Tsukuba, Japan, ⁷Japan Advanced Institute of Science and Technology, Graduate School of Advanced Science and Technology, Nomi, Japan, ⁸Ichimaru Pharcos Co. Ltd., Research & Development Department, Motosu, Japan

Background: Some patients with schizophrenia do not respond adequately to current treatments. In 2011, a strong association between the vasoactive intestinal peptide (VIP) receptor 2 (VIPR2) gene duplication and schizophrenia was reported. Therefore, VIPR2-selective inhibitors may help in the development of new treatments for schizophrenia. In 2021, we discovered a bicyclic peptide KS-133, Ac-(CPPYLP[KYLC)D]LI-NH₂ (1558.8 g/mol), with a potent and selective VIPR2 antagonist activity and high in vivo stability (Front Pharmacol 12: 751587, 2021). However, the development of KS-133 as an anti-schizophrenia drug has been challenging because KS-133 does not effectively penetrate the blood-brain barrier (BBB) and there is no preclinical proof of concept that VIPR2 inhibition improves symptoms in a rodent model of schizophrenia.

Aims & Objectives: To overcome these challenges, we combined KS-133 with a reliable brain-targeting technology and investigated in vivo efficacy in a relevant mouse model of schizophrenia.

Method: To facilitate drug transport across the BBB into the brain, we focused on the receptor-mediated transcytosis using low-density lipoprotein receptor-related protein 1 (LRP1). We previously identified KS-487 as a novel LRP1-binding cyclic peptide with high affinity, high stability and high BBB permeability (Biochem Biophys Rep 32: 101367, 2022). Therefore, we attempted to develop a novel drug delivery system by combining KS-487 with KS-133 to enhance brain permeability. All animal experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Plasma and tissue concentrations of KS-133 were measured using LC-MS/MS.

Results: We investigated whether the VIPR2 antagonist KS-133 could be transported into the brain when encapsulated in nanoparticles (NPs) displaying KS-487. KS-133 encapsulated in NPs composed of Cremophor EL, a polyethoxylated surfactant, was stably retained at 4 °C but was gradually released from the NPs at 37 °C. Transmission electron microscopy and particle size distribution analysis of KS-133/KS-487 NPs revealed monodisperse particles with diameters of approximately 12 nm. A pharmacokinetic study showed the time-dependent translocation of KS-133 in the brain after subcutaneous administration (3 mg/kg) of KS-133/KS-487 NPs. KS-133 concentrations were consistent in all tissues and peaked 1-2 h after administration. The mean maximum concentration was 8.734 μM in the plasma, 0.054 μmol/kg in the cerebral cortex, 0.058 μmol/kg in the hypothalamus, 0.062 μmol/kg in the hippocampus, 0.044 μmol/kg in the striatum, and 0.103 μmol/kg in the cerebellum. These data suggest that KS-133 is uniformly distributed throughout the brain. The peak concentration of KS-133 in the whole brain was estimated to be approximately 70 nM, which is sufficient for VIPR2 antagonist activity (IC₅₀ = 25 nM). Under these conditions, KS-133/KS-487 or KS-133 NPs (without KS-487) at a dose of 3 mg/kg were administered subcutaneously once daily for 2 weeks to a mouse model of schizophrenia induced by early postnatal treatment with the VIPR2 agonist Ro25-1553. KS-133/KS-487 NPs, but not KS-133 NPs, improved cognitive dysfunction in the novel object recognition test.

Discussion & Conclusions: This is the first study to demonstrate the potential therapeutic efficacy of a multifunctionalized multi-peptide NP with VIPR2 inhibition for the treatment of schizophrenia.