Citation: La ˙ zewska, D.; Siwek, A.; Olejarz-Maciej, A.; Doroz-Plonka, A.; Wiktorowska-Owczarek, A.; Jó´ zwiak-B˛ ebenista, M.; Reiner-Link, D.; Frank, A.; Sromek-Trzaskowska, W.; Honkisz-Orzechowska, E.; et al. Dual Targeting Ligands—Histamine H 3 Receptor Ligands with Monoamine Oxidase B Inhibitory Activity—In Vitro and In Vivo Evaluation. Pharmaceutics 2022, 14, 2187. https://doi.org/10.3390/ pharmaceutics14102187 Academic Editor: Tatiana B. Tennikova Received: 12 September 2022 Accepted: 9 October 2022 Published: 13 October 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). pharmaceutics Article Dual Targeting Ligands—Histamine H 3 Receptor Ligands with Monoamine Oxidase B Inhibitory Activity—In Vitro and In Vivo Evaluation Dorota La ˙ zewska 1, * , Agata Siwek 2 , Agnieszka Olejarz-Maciej 1 , Agata Doroz-Plonka 1 , Anna Wiktorowska-Owczarek 3 , Marta Jó ´ zwiak-B˛ ebenista 3 , David Reiner-Link 4 , Annika Frank 4 , Wioletta Sromek-Trzaskowska 1 , Ewelina Honkisz-Orzechowska 1 , Ewelina Królicka 1 , Holger Stark 4 , Marek Wieczorek 5 , Waldemar Wagner 6,7 , Katarzyna Kie´ c-Kononowicz 1 and Anna Stasiak 6, * 1 Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian University Medical College in Kraków, Medyczna 9 Str., 30-688 Kraków, Poland 2 Department of Pharmacobiology, Faculty of Pharmacy, Jagiellonian University Medical College in Kraków, Medyczna 9 Str., 30-688 Kraków, Poland 3 Department of Pharmacology and Toxicology, Medical University of Lodz, ˙ Zeligowskiego 7/9 Str., 90-752 Lód´ z, Poland 4 Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Universitaetsstr. 1, 40225 Düsseldorf, Germany 5 Department of Neurobiology, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/143 Str., 90-236 Lód´ z, Poland 6 Department of Hormone Biochemistry, Medical University of Lodz, ˙ Zeligowskiego 7/9 Str., 90-752 Lód´ z, Poland 7 Laboratory of Cellular Immunology, Institute of Medical Biology of Polish Academy of Sciences, 106 Lodowa Str., 93-232 Lód´ z, Poland * Correspondence: dorota.lazewska@uj.edu.pl (D.L.); anna.stasiak@umed.lodz.pl (A.S.) Abstract: The clinical symptoms of Parkinson’s disease (PD) appear when dopamine (DA) con- centrations in the striatum drops to around 20%. Simultaneous inhibitory effects on histamine H 3 receptor (H 3 R) and MAO B can increase DA levels in the brain. A series of compounds was de- signed and tested in vitro for human H 3 R(hH 3 R) affinity and inhibitory activity to human MAO B (hMAO B). Results showed different activity of the compounds towards the two biological targets. Most compounds had poor affinity for hH 3 R(K i > 500 nM), but very good inhibitory potency for hMAO B (IC 50 < 50 nM). After further in vitro testing (modality of MAO B inhibition, permeability in PAMPA assay, cytotoxicity on human astrocyte cell lines), the most promising dual-acting ligand, 1-(3-(4-(tert-butyl)phenoxy)propyl)-2-methylpyrrolidine (13: hH 3 R: K i = 25 nM; hMAO B IC 50 = 4 nM) was selected for in vivo evaluation. Studies in rats of compound 13, in a dose of 3 mg/kg of body mass, confirmed its antagonistic effects for H 3 R (decline in food and a water consumption), decline in MAO B activity (>90%) in rat cerebral cortex (CTX), and an increase in DA content in CTX and striatum. Moreover, compound 13 caused a slight increase in noradrenaline, but a reduction in sero- tonin concentration in CTX. Thus, compound 13 is a promising dual-active ligand for the potential treatment of PD although further studies are needed to confirm this. Keywords: histamine H 3 receptor; histamine H 3 receptor ligand; monoamine oxidase B (MAO B); MAO B inhibitor; dual-target ligands; pitolisant; in vivo studies 1. Introduction Parkinson’s disease (PD) is characterized by a progressive loss of dopaminergic neu- rons in the substantia nigra and the accumulation of misfolded and aggregated α-synuclein named Lewy bodies. All of this leads to a decrease in the level of dopamine (DA) in the striatum causing memory deficits and also problems with moving. However, it should be remembered that a decline in DA levels is a normal process of ageing and it could Pharmaceutics 2022, 14, 2187. https://doi.org/10.3390/pharmaceutics14102187 https://www.mdpi.com/journal/pharmaceutics