Highly Selective Cyclooxygenase-1 Inhibitors P6 and Mofezolac Counteract Inflammatory State Both in Vitro and in Vivo Models of Neuroinflammation

ORIGINAL RESEARCH published: 09 June 2017 doi: 10.3389/fneur.2017.00251 Highly Selective Cyclooxygenase-1 Inhibitors P6 and Mofezolac counteract inflammatory state both In Vitro and In Vivo Models of Neuroinflammation Rosa Calvello1†, Dario Domenico Lofrumento2†, Maria Grazia Perrone3†, Antonia Cianciulli1, Rosaria Salvatore1, Paola Vitale3, Francesco De Nuccio2, Laura Giannotti2, Giuseppe Nicolardi2*, Maria Antonietta Panaro1* and Antonio Scilimati3* 1 Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari “A. Moro”, Bari, Italy, 2 Department Edited by: of Biological and Environmental Sciences and Technologies, Section of Human Anatomy, University of Salento, Lecce, Italy, Vittorio Maglione, 3 Department of Pharmacy – Pharmaceutical Sciences, University of Bari “A. Moro”, Bari, Italy Istituto Neurologico Mediterraneo (IRCCS), Italy Activated microglia secrete an array of pro-inflammatory factors, such as prostaglandins, Reviewed by: Ilaria Bellezza, whose accumulation contributes to neuronal damages. Prostaglandin endoperoxide University of Perugia, Italy synthases or cyclooxygenases (COX-1 and COX-2), which play a critical role in the inflam- Stefania Filosa, Istituto di Bioscienze mation, are the pharmacological targets of non-steroidal anti-inflammatory drugs, used to e Biorisorse (IBBR), Italy treat pain and inflammation. Since it was reported that COX-1 is the major player in medi- *Correspondence: ating the brain inflammatory response, the aim of this study was to evaluate the effects of Giuseppe Nicolardi highly selective COX-1 inhibitors, such as P6 and mofezolac, in neuroinflammation models. [email protected]; Maria Antonietta Panaro Lipopolysaccharide (LPS)-activated mouse BV-2 microglial cells and LPS intracerebroven- [email protected]; tricular-injected mice as in vitro and in vivo neuroinflammation models, respectively, were Antonio Scilimati [email protected] used to probe the antiinflammatory efficacy of P6 and mofezolac. Both P6 and mofezolac †These authors have equally reduce COX-1 expression in LPS-activated BV-2 cells. This reduction was accompanied contributed to this work. with PGE2 release reduction and NF-kB activation downregulation. Coextensively, in the in vivo model, both glial fibrillary acidic protein and ionized calcium-binding adapter mol- Specialty section: ecule-1 expression, two markers of inflammation, were reduced by mofezolac to a rank This article was submitted to Neurodegeneration, depending on the encephalon area analyzed. The increase of COX-1 expression observed a section of the journal in all the brain sections of LPS-treated mice was selectively downregulated by the in vivo Frontiers in Neurology treatment with mofezolac as well as PGE2 release and Ikβα phosphorylation amount Received: 24 March 2017 Accepted: 19 May 2017 assayed in the brain areas tested. These results indicate the capability of P6 and mofezolac Published: 09 June 2017 to modulate the NF-kB signaling pathway, emphasizing the neuroprotective effect and Citation: therapeutic potential of COX-1 inhibitors in the control of neuroinflammatory diseases. Calvello R, Lofrumento DD, Perrone MG, Cianciulli A, Salvatore R, Keywords: cyclooxygenase-1, P6 and mofezolac, cyclooxygenase-1 inhibitors, neuroinflammation, in vitro and Vitale P, De Nuccio F, Giannotti L, in vivo experiments, lipopolysaccharide-treated BV2 microglial cells Nicolardi G, Panaro MA and Scilimati A (2017) Highly Selective Cyclooxygenase-1 Inhibitors P6 and INTRODUCTION Mofezolac Counteract Inflammatory State both In Vitro and In Vivo Models Neuroinflammation is widely recognized as an inflammatory response originated in the cen- of Neuroinflammation. tral nervous system (CNS). It is a pathological condition mainly caused by the nervous tissue Front. Neurol. 8:251. infiltration of host defense cells and molecules from the bloodstream. In addition, it implies a doi: 10.3389/fneur.2017.00251 complex interplay of glia, in particular microglia, typically associated with neurological and Frontiers in Neurology | www.frontiersin.org 1 June 2017 | Volume 8 | Article 251 Calvello et al. COX-1 Inhibition in Neuroinflammation neurodegenerative diseases, triggering several concerns from a nosological viewpoint (1). Microglia are recognized as the innate immune cells of the CNS, where they mediate a number of tissue homeostatic func- tions, including immune surveillance (2), synaptic regulation (3, 4), and neurogenesis (5, 6). Microglial activation and chronic inflammation thereafter is the starting point for elevated levels of a wide array of potentially neurotoxic molecules including pro- inflammatory cytokines, proteinases, and reactive oxygen species (ROS), which are believed to contribute to neurodegenerative processes (7–9). Epidemiological data-based link between neuroinflammation and neurodegenerative diseases increased the worldwide scien- FIGURE 1 | Chemical structure of P6 and mofezolac. IC50 values refer to the tific interest aimed to determine whether reducing inflamma- human whole blood assay. tion would reverse neurodegeneration. Such data also indicate an inverse relationship between the use of traditional non-steroidal anti-inflammatory drugs (tNSAIDs) and Alzheimer’s disease All the other reagents and solvents were purchased from Sigma- risk. tNSAIDs pharmacological action is due to their ability to Aldrich (Milan, Italy) and used without any further purification. inhibit the cyclooxygenase (COX) and, hence, the biosynthesis Lipopolysaccharide from Escherichia coli serotype 0127: of the prostaglandins (PG) involved in neuroinflammation (10). B8 was purchased from Sigma-Aldrich (Milan, Italy). The goat Two COXs are known, COX-1 and COX-2. Upon inflammatory polyclonal a p-IκB (sc-7977) antibody (Ab) was purchased from stimuli, COX-1, being constitutive in microglia, is responsible of Santa Cruz Biotechnology (DBA, Milan, Italy); COX-1 (Ab 695) the primary inflammatory response by inducing the production and COX-2 (Ab 15191) Abs were obtained from Abcam of PG, mainly PGE2. COX-2 is responsible, upon its induction, of (Cambridge, UK). Goat anti-rabbit IgG (sc-2004), goat anti- a later and secondary response, with the exception of conditions mouse IgG (sc-2005), and donkey anti-goat IgG (sc-2020) were in which the neurons are directly challenged (excitotoxicity and purchased from Santa Cruz Biotechnology; mouse primary ischemia) (11). In the latter case, the primary response is COX-2 monoclonal antibody (mAb) anti-glial fibrillary acidic protein mediated, and microglia derived COX-1 succeeds as a secondary (GFAP) (Merck Millipore, Milan, Italy), mouse mAb anti-ionized response upon perpetuating stimuli. In the first circumstances, calcium-binding adapter molecule-1 (Iba-1) (Merck Millipore). selective COX-1 catalytic activity inhibition may result beneficial. Elisa kit for PGE2 evaluation was purchased from Cayman tNSAIDs inhibit COXs not selectively. Then, drugs useful to treat Chemical (Ann Arbor, MI, USA). MTT [3-(4,5-dimethylthiazol- the different neuroinflammatory conditions are needed (12, 13). 2-yl)-2,5-diphenyltetrazolium bromide], 3,3′-diaminobenzi- Preliminarily, we accomplished an in vitro study using some dine, and tribromoethanol were obtained from Sigma-Aldrich, selective COX-1 inhibitors (P6, P10, and SC-560) compared Milan, Italy. to aspirin and two selective COX-2 inhibitors (celecoxib and etoricoxib) in a in vitro model of neuroinflammation, represented Cell Cultures and Treatment by lipopolysaccharide (LPS)-stimulated N13 microglial cell line BV2 microglia cells (ICLC HTL 03001-Interlab Cell Line (14). In such a context, the treatment of LPS-stimulated micro- Collection) were grown in high glucose Dulbecco’s modified glial cells by selective COX-1 inhibitors P6 and P10 were able Eagle’s medium supplemented with 10% fetal bovine serum, to downregulate COX-1 protein expression without affecting 100 U/mL penicillin, and 100 µg/mL streptomycin. They were COX-2 levels; interfering with NF-kB activation (14). maintained at 37°C in a humidified 5% CO2/95% environ- In continuation of our investigations, herein, we report the mental air. validation of the study above mentioned by using both an in vitro Then, microglial cells were plated, at a density of 25 × 104/ model of neuroinflammation, represented by LPS-activated BV2 well in 6-well plates (Falcon) and treated with the chosen COX cell line, and an in vivo animal model, constituted by LPS-treated inhibitors (P6 and mofezolac) when they reached 80% conflu- mice. ence. Preliminary experiments were conducted to establish the In particular, in this study, COX-1 role in neuroinflamma- optimal concentration and exposure times necessary for LPS tion was explored by using P6 and mofezolac (Figure 1). Both (1 µg/mL) treatment, which were found to be in accordance with compounds belong to the diarylisoxazole class, and mofezolac other reports (17, 18), as well as to establish the optimal dose of is the only clinically used diarylisoxazole approved in Japan as the COX inhibitors and exposure times to detect their effects on Disopain®. LPS-stimulated BV2 microglial cell function. In this regard, a set of experiments was carried out in which microglial cells were MATERIALS AND METHODS 1 h pretreated with the selective COX-1 inhibitors P6 (0.5 and 1.0 µM) or mofezolac (0.1 and 0.5 µM). Cells were then incu- Reagents bated for different times at 37°C with LPS, as pro-inflammatory P6 was synthesized according to Di Nunno et al. (15),

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