LC–MS–MS Method for the Analysis of New Non-Imidazole Histamine H3 Receptor Antagonist 1-[3-(4-tert-Butylphenoxy)propyl]piperidine in Rat Serum—Application to Pharmacokinetic Studies

A sensitive and specific liquid chromatography electrospray ionisation–tandem mass spectrometry method for determination of new non-imidazole histamine H3 receptor antagonist 1-[3-(4-tert-butylphenoxy)propyl]piperidine (DL76) in rat serum has been developed and validated. Chromatography was performed on a XBridge™ C18 analytical column (2.1 × 30 mm, 3.5 µm, Waters, Ireland) with gradient elution using a mobile phase containing acetonitrile and water with an addition of 0.1% of formic acid. Detection was achieved by an Applied Biosystems MDS Sciex (Concord, Ontario, Canada) API 2000 triple quadrupole mass spectrometer. Electrospray ionization (ESI) was used for ion production. The limit of detection in the SRM mode was found to be 0.5 ng mL−1. The limit of quantification was 1 ng mL−1. The precision and accuracy for both intra- and inter-day determination of DL76 ranged from 1.65 to 15.09% and from 88.74 to 113.43%. The results of this analytical method validation allow to carry out pharmacokinetic studies in rats. The method was used for the pilot study of the pharmacokinetic behavior of DL76 in rats after intravenous administration

Eosinophils adhesion assay as a tool for phenotypic drug screening - The pharmacology of 1,3,5 – Triazine and 1H-indole like derivatives against the human histamine H4 receptor

Histamine is a pleiotropic biogenic amine, having affinity towards four distinct histamine receptors. The existing pharmacological studies suggest the usefulness of histamine H4 receptor ligands in the treatment of many inflammatory and immunomodulatory diseases, including allergic rhinitis, asthma, atopic dermatitis, colitis or pruritus. Up to date, several potent histamine H4 receptor ligands were developed, none of which was registered as a drug yet. In this study, a series of potent indole-like and triazine derivatives were tested, in radioligand displacement and functional assays at histamine H4 receptor, as well as in human eosinophils adhesion assay to endothelium. For selected compounds permeability, cytotoxicity, metabolic and in vivo studies were conducted. Adhesion assay differentiated the activity of different groups of compounds with a known affinity towards the histamine H4 receptor. Most of the tested compounds downregulated the number of adherent cells. However, adhesion assay revealed additional properties of tested compounds that had not been detected in radioligand displacement and aequorin-based functional assays. Furthermore, for some tested compounds, these abnormal effects were confirmed during the in vivo studies. In conclusion, eosinophils adhesion assay uncovered pharmacological activity of histamine H4 receptor ligands that has been later confirmed in vivo, underscoring the value of well-suited cell-based phenotypic screening approach in drug discovery

Molecular and functional interaction between GPR18 and cannabinoid CB2 G-protein-coupled receptors. Relevance in neurodegenerative diseases

GPR18, still considered an orphan receptor, may respond to endocannabinoids, whose canonical receptors are CB1 and CB2. GPR18 and CB2 receptors share a role in peripheral immune response regulation and are co-expressed in microglia, which are immunocompetent cells in the central nervous system (CNS). We aimed at identifying heteroreceptor complexes formed by GPR18 and CB1R or CB2R in resting and activated microglia. Receptor-receptor interaction was assessed using energy-transfer approaches, and receptor function by determining cAMP levels and ERK1/2 phosphorylation in heterologous cells and primary cultures of microglia. Heteroreceptor identification in primary cultures of microglia was achieved by in situ proximity ligation assays. Energy transfer results showed interaction of GPR18 with CB2R but not with CB1R. CB2-GPR18 heteroreceptor complexes displayed particular functional properties (heteromer prints) often consisting of negative cross-talk (activation of one receptor reduces signaling arising from the partner receptor) and cross-antagonism (the response of one of the receptors is blocked by a selective antagonist of the partner receptor). Activated microglia showed the heteromer print (negative cross-talk and bidirectional cross-antagonism) and increased expression of CB2R and GPR18. Due to the important role of CB2R in neuroprotection, we further investigated heteroreceptor occurrence in primary cultures of microglia from transgenic mice overexpressing human APPSw,Ind, an Alzheimer's disease model. Microglial cells from transgenic mice showed the heteromer print and functional interactions that were similar to those found in cells from wild-type animals that were activated by treatment with lipopolysaccharide and interferon-ɤ. Our results show that GPR18 and its heteromers may play important roles in neurodegenerative processes

In vitro and in silico ADME-Tox profiling and safety significance of multifunctional monoamine oxidase inhibitors targeting neurodegenerative deseases

Herein we report in vitro metabolic stability in human liver microsomes (HLMs), interactions with cytochrome P450 isoenzymes (CYP3A4, CYP2D6, and CYP2C9), and cytotoxicity analyses on HEK-293, HepG2, Huh7, and WTIIB cell lines of our most recent multitarget directed ligands PF9601N, ASS234, and contilisant. Based on these results, we conclude that (1) PF9601N and contilisant are metabolically stable in the HLM assay, in contrast to the very unstable ASS234; (2) CYP3A4 activity was decreased by PF9601N at all the tested concentrations and by ASS234 and contilisant only at the highest concentration; CYP2D6 activity was reduced by ASS234 at 1, 10, and 25 μM and by PF9601N at 10 and 25 μM, whereas contilisant increased its activity at the same concentrations; CYP2C9 was inhibited by the three compounds; (3) contilisant did not affect cell viability in the widest range of concentrations: up to 10 μM on HEK-293 cells, up to 30 μM on Huh7 cells, up to 50 μM on HepG2 cells, and up to 30 or 100 μM on WTIIB cells. Based on these results, we selected contilisant as a metabolically stable and nontoxic lead compound for further studies in Alzheimer's disease therapy.This study received financial support from the National Science Centre Poland (Grant No. 2016/23/D/NZ7/01328). J.M.-C. thanks AEI (Government of Spain) for grants PDI- 2019-105813RB-C21 and SAF2015-65586-R. J.M.-C. and F.L.- M. thank UCJC (Grants UCJC 2020-33 UCJC 2020-03) for support

Selenocompounds as Novel Antibacterial Agents and Bacterial Efflux Pump Inhibitors

Bacterial multidrug resistance is becoming a growing problem for public health, due to the development and spreading of bacterial strains resistant to antimicrobials. In this study, the antibacterial and multidrug resistance reversing activity of a series of seleno-carbonyl compounds has been evaluated. The effects of eleven selenocompounds on bacterial growth were evaluated in Staphylococcus aureus, methicillin resistant S. aureus (MRSA), Enterococcus faecalis, Escherichia coli, and Chlamydia trachomatis D. The combination effect of compounds with antibiotics was examined by the minimum inhibitory concentration reduction assay. Their efflux pump (EP) inhibitory properties were assessed using real-time fluorimetry. Relative expressions of EP and quorum-sensing genes were studied by quantitative PCR. Results showed that a methylketone selenoester had remarkable antibacterial activity against Gram-positive bacteria and potentiated the activity of oxacillin in MRSA. Most of the selenocompounds showed significant anti-chlamydial effects. The selenoanhydride and the diselenodiester were active inhibitors of the AcrAB-TolC system. Based on these results it can be concluded that this group of selenocompounds can be attractive potential antibacterials and EP inhibitors. The discovery of new derivatives with a significant antibacterial activity as novel selenocompounds, is of high impact in the fight against resistant pathogen

Probing Substituents in the 1- and 3-Position: Tetrahydropyrazino-Annelated Water-Soluble Xanthine Derivatives as Multi-Target Drugs With Potent Adenosine Receptor Antagonistic Activity

Tetrahydropyrazino-annelated theophylline (1,3-dimethylxanthine) derivatives have previously been shown to display increased water-solubility as compared to the parent xanthines due to their basic character. In the present study, we modified this promising scaffold by replacing the 1,3-dimethyl residues by a variety of alkyl groups including combinations of different substituents in both positions. Substituted benzyl or phenethyl residues were attached to the N8 of the resulting 1,3-dialkyl-tetrahydropyrazino[2,1-f ]purinediones with the aim to obtain multi-target drugs that block human A1 and A2A adenosine receptors (ARs) and monoaminoxidase B (MAO-B). 1,3-Diethyl-substituted derivatives showed high affinity for A1 ARs, e.g., 15d (PSB-18339, 8-m-bromobenzyl-substituted) displayed a Ki value of 13.6 nM combined with high selectivity. 1-Ethyl-3-propargyl-substituted derivatives exhibited increased A2A AR affinity. The 8-phenethyl derivative 20h was selective for the A2A AR (Ki 149 nM), while the corresponding 8-benzyl-substituted compound 20e (PSB-1869) blocked A1 and A2A ARs with equal potency (Ki A1, 180 nM; A2A, 282 nM). The 1-ethyl-3-methyl-substituted derivative 16a (PSB-18405) bearing a m,p-dichlorobenzyl residue at N8 blocked all three targets, A1 ARs (Ki 396 nM), A2A ARs (Ki 1,620 nM), and MAO-B (IC50 106 nM) with high selectivity vs. the other subtypes (A2B and A3 ARs, MAO-A), and can thus be considered as a multi-target drug. Our findings were rationalized by molecular docking studies based on previously published X-ray structures of the protein targets. The new drugs have potential for the treatment of neurodegenerative diseases, in particular Parkinson's disease