Research paperAntidepressant-like effects exerted by the intranasal administration of a glucagon-like peptide-2 derivative containing cell-penetrating peptides and a penetration-accelerating sequence in mice
Introduction
Proglucagon-derived glucagon-like peptide-2 (GLP-2) is a 33-amino acid peptide produced in the gut and central nervous system (CNS) [1], [2]. GLP-2 acts by binding to G protein-coupled GLP-2 receptors (GLP-2R) [3]. The GLP-2R gene is expressed not only in distinct gastrointestinal cells [3], [4], but also in specific regions of the CNS including the dorsomedial hypothalamic nucleus (DMH), amygdala, thalamus, cerebellum, hippocampus, and cerebral cortex [5], [6]. The intracerebroventricular (i.c.v.) administration of GLP-2 to rodents was previously reported to suppress food intake [7] and decrease blood pressure [8]. We also reported that the i.c.v. administration of GLP-2 exerted antidepressant-like effects in naïve mice [9] and adrenocorticotropic hormone (ACTH)-treated mice [10] through the modulation of the hypothalamic-pituitary-adrenal (HPA)-axis and the restoration of neurogenesis in the subgranular zone (SGZ) of the hippocampal dentate gyrus [11] in addition to activation of monoamine receptors and the changes in monoamine levels in the brain [9], [10]. These findings raise the possibility that GLP-2 may become a novel treatment tool for major depression with different mechanisms of action from existing drugs. However, the i.c.v. administration is invasive, costly, and impractical for the delivery of drugs into human brains, and thus we need to develop a non-invasive and effective way to deliver GLP-2 in the brain.
A difficulty associated with the development of peptides as CNS therapeutic agents is their limited ability to cross the blood-brain barrier (BBB) in order to reach their targets in the CNS at the pharmacological level following their systemic administration. Intranasal (i.n.) delivery offers a non-invasive alternative for getting protein and peptide drugs into the brain by utilizing olfactory neuronal distribution pathway in the cribriform plate, which may lead to direct nose-to-brain drug distribution [12].
Cell-penetrating peptides (CPPs), including arginine-rich peptides, have been shown to deliver various bioactive molecules with low membrane permeability into cells in order to regulate cell functions [13], [14], [15]. Macropinocytosis also plays an important role in the cellular uptake of arginine-rich CPPs, resulting in highly efficient intracellular delivery [16], [17]. Macropinocytosis is transient, actin-driven fluid-phase endocytosis that involves membrane ruffling and the formation of large vacuoles called macropinosomes [18], [19]. The addition of a penetration-accelerating sequence (PAS) to CPPs has been reported to enhance the efficiency of the intracellular delivery of bioactive peptides by promoting endosomal escape [20]. Although some CPP-peptide chimeras have reached clinical trials for the alleviation of pain, hearing loss, or stroke [21], it currently remains unknown whether the addition of a PAS to CPPs increases the effectiveness of peptide medicines.
In the present study, we synthesized several GLP-2 derivatives containing CPPs with or without a PAS, and examined antidepressant-like effects after the i.n. administration and distribution patterns in the CNS.
Section snippets
Preparation of GLP-2 derivatives
PAS-CPPs-GLP-2 (FFLIPKG-RRRRRRRR-GG-HADGSFSDEMNTILDNLAARDFINWLIQTKITD) (JPN Patent pending: No. 2014-184436, PCT applied for: WO 2016/035820 A1), CPPs-GLP-2 (RRRRRRRR-GG-HADGSFSDEMNTILDNLAARDFINWLIQTKITD), fluorescein isothiocyanate (FITC)-labeled PAS-CPPs-GLP-2, and FITC-labeled CPPs-GLP-2 were synthesized by the SCRUM Inc (Tokyo, Japan) with a peptide synthesizer (433A: Applied Biosystems) following the standard 9-fluorenylmethoxycarbonyl (Fmoc) method.
Animals
All experimental protocols were approved
Effects of PAS-CPPs-GLP-2 in the TST, and FST in naïve mice
We previously reported that the i.c.v. administration of native GLP-2 (3 or 6 μg/mouse) once a day for 2 days significantly decreased the immobility time in the FST [9]. The experimental schedule is shown in Fig. 1A. No abnormal changes in body weight were observed after the 2-day treatment (data not shown). In the present study, the i.n. administration of PAS-CPPs-GLP-2 (3.6 μg/mouse) for 2 days significantly decreased the immobility time in the TST (n = 9) (U = 10, P = 0.0056, Mann-Whitney test) and
Discussion
We herein showed for the first time that the GLP-2 derivative containing CPPs and a PAS exerted antidepressant-like effects in vivo, even though the addition of a PAS to CPPs has been shown to enhance the efficiency of the intracellular delivery of bioactive peptides in vitro [20]. Furthermore, we demonstrated that PAS-CPPs-GLP-2 was effectively delivered into the brain by the i.n. administration route.
A recent study demonstrated that modified CPPs promoted brain delivery of coumarin-containing
Conclusions
The present study provided first evidence that PAS-CPPs-GLP-2 (i.n.) exerted antidepressant-like effects on naïve and ACTH-treated mice, and was effectively delivered into the brain by the i.n. administration route. We also demonstrated for the first time the efficiency of the addition of a PAS to CPPs in vivo. These results suggest that PAS-CPPs-GLP-2 is effective for i.n. delivery to the brain, and may be useful in the clinical treatment of major depression.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
This study was partially supported by JSPS KAKENHI (Grant numbers 24590126, 15K07974 to J.-I.O.); the Mochida Memorial Foundation for Medical and Pharmaceutical Research (2015 to S.S.-H.); MEXT-Supported Program for the Strategic Research Foundation at Private Universities (2014-2018 to J.-I.O.); and the Senshin Medical Research Foundation (2015 to J.-I.O.).
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