Publications by authors named "Katia Varani"

198 Publications

Upregulation of Cortical A2A Adenosine Receptors Is Reflected in Platelets of Patients with Alzheimer's Disease.

J Alzheimers Dis 2021 ;80(3):1105-1117

Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy.

Background: Alzheimer's disease (AD) is a neurodegenerative pathology covering about 70%of all cases of dementia. Adenosine, a ubiquitous nucleoside, plays a key role in neurodegeneration, through interaction with four receptor subtypes. The A2A receptor is upregulated in peripheral blood cells of patients affected by Parkinson's and Huntington's diseases, reflecting the same alteration found in brain tissues. However, whether these changes are also present in AD pathology has not been determined.

Objective: In this study we verified any significant difference between AD cases and controls in both brain and platelets and we evaluated whether peripheral A2A receptors may reflect the status of neuronal A2A receptors.

Methods: We evaluated the expression of A2A receptors in frontal white matter, frontal gray matter, and hippocampus/entorhinal cortex, in postmortem AD patients and control subjects, through [3H]ZM 241385 binding experiments. The same analysis was performed in peripheral platelets from AD patients versus controls.

Results: The expression of A2A receptors in frontal white matter, frontal gray matter, and hippocampus/entorhinal cortex, revealed a density (Bmax) of 174±29, 219±33, and 358±84 fmol/mg of proteins, respectively, in postmortem AD patients in comparison to 104±16, 103±19, and 121±20 fmol/mg of proteins in controls (p < 0.01). The same trend was observed in peripheral platelets from AD patients versus controls (Bmax of 214±17 versus 95±4 fmol/mg of proteins, respectively, p < 0.01).

Conclusion: AD subjects show significantly higher A2A receptor density than controls. Values on platelets seem to correlate with those in the brain supporting a role for A2A receptor as a possible marker of AD pathology and drug target for novel therapies able to modify the progression of dementia.
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http://dx.doi.org/10.3233/JAD-201437DOI Listing
January 2021

Adenosine A receptor inhibition reduces synaptic and cognitive hippocampal alterations in Fmr1 KO mice.

Transl Psychiatry 2021 Feb 5;11(1):112. Epub 2021 Feb 5.

National Center for Drug Research and Evaluation, Istituto Superiore di Sanità, Rome, Italy.

In fragile X syndrome (FXS) the lack of the fragile X mental retardation protein (FMRP) leads to exacerbated signaling through the metabotropic glutamate receptors 5 (mGlu5Rs). The adenosine A receptors (ARs), modulators of neuronal damage, could play a role in FXS. A synaptic colocalization and a strong permissive interaction between A and mGlu5 receptors in the hippocampus have been previously reported, suggesting that blocking ARs might normalize the mGlu5R-mediated effects of FXS. To study the cross-talk between A and mGlu5 receptors in the absence of FMRP, we performed extracellular electrophysiology experiments in hippocampal slices of Fmr1 KO mouse. The depression of field excitatory postsynaptic potential (fEPSPs) slope induced by the mGlu5R agonist CHPG was completely blocked by the AR antagonist ZM241385 and strongly potentiated by the AR agonist CGS21680, suggesting that the functional synergistic coupling between the two receptors could be increased in FXS. To verify if chronic AR blockade could reverse the FXS phenotypes, we treated the Fmr1 KO mice with istradefylline, an AR antagonist. We found that hippocampal DHPG-induced long-term depression (LTD), which is abnormally increased in FXS mice, was restored to the WT level. Furthermore, istradefylline corrected aberrant dendritic spine density, specific behavioral alterations, and overactive mTOR, TrkB, and STEP signaling in Fmr1 KO mice. Finally, we identified AR mRNA as a target of FMRP. Our results show that the pharmacological blockade of ARs partially restores some of the phenotypes of Fmr1 KO mice, both by reducing mGlu5R functioning and by acting on other AR-related downstream targets.
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http://dx.doi.org/10.1038/s41398-021-01238-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864914PMC
February 2021

Pulsed Electromagnetic Field Stimulation in Osteogenesis and Chondrogenesis: Signaling Pathways and Therapeutic Implications.

Int J Mol Sci 2021 Jan 15;22(2). Epub 2021 Jan 15.

Department of Medical Sciences, University of Ferrara, 44121 Ferrara, Italy.

Mesenchymal stem cells (MSCs) are the main cell players in tissue repair and thanks to their self-renewal and multi-lineage differentiation capabilities, they gained significant attention as cell source for tissue engineering (TE) approaches aimed at restoring bone and cartilage defects. Despite significant progress, their therapeutic application remains debated: the TE construct often fails to completely restore the biomechanical properties of the native tissue, leading to poor clinical outcomes in the long term. Pulsed electromagnetic fields (PEMFs) are currently used as a safe and non-invasive treatment to enhance bone healing and to provide joint protection. PEMFs enhance both osteogenic and chondrogenic differentiation of MSCs. Here, we provide extensive review of the signaling pathways modulated by PEMFs during MSCs osteogenic and chondrogenic differentiation. Particular attention has been given to the PEMF-mediated activation of the adenosine signaling and their regulation of the inflammatory response as key player in TE approaches. Overall, the application of PEMFs in tissue repair is foreseen: (1) in vitro: to improve the functional and mechanical properties of the engineered construct; (2) in vivo: (i) to favor graft integration, (ii) to control the local inflammatory response, and (iii) to foster tissue repair from both implanted and resident MSCs cells.
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http://dx.doi.org/10.3390/ijms22020809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7830993PMC
January 2021

Targeting Adenosine Receptors: A Potential Pharmacological Avenue for Acute and Chronic Pain.

Int J Mol Sci 2020 Nov 18;21(22). Epub 2020 Nov 18.

Department of Translational Medicine and for Romagna, Pharmacology Section, University of Ferrara, 44121 Ferrara, Italy.

Adenosine is a purine nucleoside, responsible for the regulation of multiple physiological and pathological cellular and tissue functions by activation of four G protein-coupled receptors (GPCR), namely A, A, A, and A adenosine receptors (ARs). In recent years, extensive progress has been made to elucidate the role of adenosine in pain regulation. Most of the antinociceptive effects of adenosine are dependent upon AAR activation located at peripheral, spinal, and supraspinal sites. The role of AAR and AAR is more controversial since their activation has both pro- and anti-nociceptive effects. AAR agonists are emerging as promising candidates for neuropathic pain. Although their therapeutic potential has been demonstrated in diverse preclinical studies, no AR ligands have so far reached the market. To date, novel pharmacological approaches such as adenosine regulating agents and allosteric modulators have been proposed to improve efficacy and limit side effects enhancing the effect of endogenous adenosine. This review aims to provide an overview of the therapeutic potential of ligands interacting with ARs and the adenosinergic system for the treatment of acute and chronic pain.
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http://dx.doi.org/10.3390/ijms21228710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7698931PMC
November 2020

Pulsed Electromagnetic Fields Stimulate HIF-1α-Independent VEGF Release in 1321N1 Human Astrocytes Protecting Neuron-Like SH-SY5Y Cells from Oxygen-Glucose Deprivation.

Int J Mol Sci 2020 Oct 28;21(21). Epub 2020 Oct 28.

Department of Morphology, Surgery and Experimental Medicine, Section of Pharmacology, University of Ferrara, 44121 Ferrara, Italy.

Pulsed electromagnetic fields (PEMFs) are emerging as an innovative, non-invasive therapeutic option in different pathological conditions of the central nervous system, including cerebral ischemia. This study aimed to investigate the mechanism of action of PEMFs in an in vitro model of human astrocytes, which play a key role in the events that occur following ischemia. 1321N1 cells were exposed to PEMFs or hypoxic conditions and the release of relevant neurotrophic and angiogenic factors, such as VEGF, EPO, and TGF-β1, was evaluated by means of ELISA or AlphaLISA assays. The involvement of the transcription factor HIF-1α was studied by using the specific inhibitor chetomin and its expression was measured by flow cytometry. PEMF exposure induced a time-dependent, HIF-1α-independent release of VEGF from 1321N1 cells. Astrocyte conditioned medium derived from PEMF-exposed astrocytes significantly reduced the oxygen-glucose deprivation-induced cell proliferation and viability decrease in the neuron-like cells SH-SY5Y. These findings contribute to our understanding of PEMFs action in neuropathological conditions and further corroborate their therapeutic potential in cerebral ischemia.
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http://dx.doi.org/10.3390/ijms21218053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7663527PMC
October 2020

A2A adenosine receptors are involved in the reparative response of tendon cells to pulsed electromagnetic fields.

PLoS One 2020 30;15(9):e0239807. Epub 2020 Sep 30.

Orthopaedic Biotechnology Lab, IRCCS Istituto Ortopedico Galeazzi, Milan, Italy.

Tendinopathy is a degenerative disease in which inflammatory mediators have been found to be sometimes present. The interaction between inflammation and matrix remodeling in human tendon cells (TCs) is supported by the secretion of cytokines such as IL-1β, IL-6 and IL-33. In this context, it has been demonstrated that pulsed electromagnetic fields (PEMFs) were able to reduce inflammation and promote tendon marker synthesis. The aim of this study was to evaluate the anabolic and anti-inflammatory PEMF-mediated response on TCs in an in vitro model of inflammation. Moreover, since PEMFs enhance the anti-inflammatory efficacy of adenosine through the adenosine receptors (ARs), the study also focused on the role of A2AARs. Human TCs were exposed to PEMFs for 48 hours. After stimulation, A2AAR saturation binding experiments were performed. Along with 48 hours PEMF stimulation, TCs were treated with IL-1β and A2AAR agonist CGS-21680. IL-1Ra, IL-6, IL-8, IL-10, IL-33, VEGF, TGF-β1, PGE2 release and SCX, COL1A1, COL3A1, ADORA2A expression were quantified. PEMFs exerted A2AAR modulation on TCs and promoted COL3A1 upregulation and IL-33 secretion. In presence of IL-1β, TCs showed an upregulation of ADORA2A, SCX and COL3A1 expression and an increase of IL-6, IL-8, PGE2 and VEGF secretion. After PEMF and IL-1β exposure, IL-33 was upregulated, whereas IL-6, PGE2 and ADORA2A were downregulated. These findings demonstrated that A2AARs have a role in the promotion of the TC anabolic/reparative response to PEMFs and to IL-1β.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0239807PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7527253PMC
December 2020

Piperazine- and Piperidine-Containing Thiazolo[5,4-]pyrimidine Derivatives as New Potent and Selective Adenosine A Receptor Inverse Agonists.

Pharmaceuticals (Basel) 2020 Jul 24;13(8). Epub 2020 Jul 24.

Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino, Sezione di Farmaceutica e Nutraceutica, Universita'degli Studi di Firenze, Via Ugo Schiff 6, 50019 Sesto Fiorentino (FI), Italy.

The therapeutic use of A adenosine receptor (AR) antagonists for the treatment of neurodegenerative disorders, such as Parkinson and Alzheimer diseases, is a very promising approach. Moreover, the potential therapeutic role of A AR antagonists to avoid both immunoescaping of tumor cells and tumor development is well documented. Herein, we report on the synthesis and biological evaluation of a new set of piperazine- and piperidine- containing 7-amino-2-(furan-2-yl)thiazolo[5,4-]pyrimidine derivatives designed as human A AR antagonists/inverse agonists. Binding and potency data indicated that a good number of potent and selective hA AR inverse agonists were found. Amongst them, the 2-(furan-2-yl)--(2-(4-phenylpiperazin-1-yl)ethyl)thiazolo[5,4-]pyrimidine-5,7-diamine exhibited the highest A AR binding affinity (K = 8.62 nM) as well as inverse agonist potency (IC = 7.42 nM). In addition, bioinformatics prediction using the web tool SwissADME revealed that , , and possessed good drug-likeness profiles.
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http://dx.doi.org/10.3390/ph13080161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7465344PMC
July 2020

Pharmacological data of cannabidiol- and cannabigerol-type phytocannabinoids acting on cannabinoid CB, CB and CB/CB heteromer receptors.

Pharmacol Res 2020 09 26;159:104940. Epub 2020 May 26.

Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CiberNed), Spain; Department of Biochemistry and Molecular Biomedicine. Universitat de Barcelona, Spain. Electronic address:

Background: Recent approved medicines whose active principles are ΔTetrahidrocannabinol (Δ-THC) and/or cannabidiol (CBD) open novel perspectives for other phytocannabinoids also present in Cannabis sativa L. varieties. Furthermore, solid data on the potential benefits of acidic and varinic phytocannabinoids in a variety of diseases are already available. Mode of action of cannabigerol (CBG), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabidivarin (CBDV) and cannabigerivarin (CBGV) is, to the very least, partial.

Hypothesis/purpose: Cannabinoid CB or CB receptors, which belong to the G-protein-coupled receptor (GPCR) family, are important mediators of the action of those cannabinoids. Pure CBG, CBDA, CBGA, CBDV and CBGV from Cannabis sativa L. are differentially acting on CB or CB cannabinoid receptors.

Study Design: Determination of the affinity of phytocannabinoids for cannabinoid receptors and functional assessment of effects promoted by these compounds when interacting with cannabinoid receptors.

Methods: A heterologous system expressing the human versions of CB and/or CB receptors was used. Binding to membranes was measured using radioligands and binding to living cells using a homogenous time resolved fluorescence resonance energy transfer (HTRF) assay. Four different functional outputs were assayed: determination of cAMP levels and of extracellular-signal-related-kinase phosphorylation, label-free dynamic mass redistribution (DMR) and ß-arrestin recruitment.

Results: Affinity of cannabinoids depend on the ligand of reference and may be different in membranes and in living cells. All tested phytocannabinoids have agonist-like behavior but behaved as inverse-agonists in the presence of selective receptor agonists. CBGV displayed enhanced potency in many of the functional outputs. However, the most interesting result was a biased signaling that correlated with differential affinity, i.e. the overall results suggest that the binding mode of each ligand leads to specific receptor conformations underlying biased signaling outputs.

Conclusion: Results here reported and the recent elucidation of the three-dimensional structure of CB and CB receptors help understanding the mechanism of action that might be protective and the molecular drug-receptor interactions underlying biased signaling.
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http://dx.doi.org/10.1016/j.phrs.2020.104940DOI Listing
September 2020

The Detrimental Action of Adenosine on Glutamate-Induced Cytotoxicity in PC12 Cells Can Be Shifted towards a Neuroprotective Role through AAR Positive Allosteric Modulation.

Cells 2020 05 18;9(5). Epub 2020 May 18.

Department of Morphology, Surgery and Experimental Medicine, Pharmacology Section, University of Ferrara, 44121 Ferrara, Italy.

Glutamate cytotoxicity is implicated in neuronal death in different neurological disorders including stroke, traumatic brain injury, and neurodegenerative diseases. Adenosine is a nucleoside that plays an important role in modulating neuronal activity and its receptors have been identified as promising therapeutic targets for glutamate cytotoxicity. The purpose of this study is to elucidate the role of adenosine and its receptors on glutamate-induced injury in PC12 cells and to verify the protective effect of the novel A adenosine receptor positive allosteric modulator, TRR469. Flow cytometry experiments to detect apoptosis revealed that adenosine has a dual role in glutamate cytotoxicity, with A and A adenosine receptor (AR) activation exacerbating and A AR activation improving glutamate-induced cell injury. The overall effect of endogenous adenosine in PC12 cells resulted in a facilitating action on glutamate cytotoxicity, as demonstrated by the use of adenosine deaminase and selective antagonists. However, enhancing the action of endogenous adenosine on AARs by TRR469 completely abrogated glutamate-mediated cell death, caspase 3/7 activation, ROS production, and mitochondrial membrane potential loss. Our results indicate a novel potential therapeutic strategy against glutamate cytotoxicity based on the positive allosteric modulation of AARs.
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http://dx.doi.org/10.3390/cells9051242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290574PMC
May 2020

Adenosinergic System Involvement in Ischemic Stroke Patients' Lymphocytes.

Cells 2020 04 25;9(5). Epub 2020 Apr 25.

Department of Morphology, Surgery and Experimental Medicine, Pharmacology Section, University of Ferrara, 44121 Ferrara, Italy.

Adenosine modulates many physiological processes through the interaction with adenosine receptors (ARs) named as A, A, A and AARs. During ischemic stroke, adenosine mediates neuroprotective and anti-inflammatory effects through ARs activation. One of the dominant pathways generating extracellular adenosine involves the dephosphorylation of ATP by ecto-nucleotidases CD39 and CD73, which efficiently hydrolyze extracellular ATP to adenosine. The aim of the study is to assess the presence of ARs in lymphocytes from ischemic stroke patients compared to healthy subjects and to analyze changes in CD39 and CD73 expression in CD4 and CD8 lymphocytes. Saturation binding experiments revealed that AARs affinity and density were significantly increased in ischemic stroke patients whilst no differences were found in A, A and AARs. These results were also confirmed in reverse transcription (RT)-polymerase chain reaction (PCR) assays where AAR mRNA levels of ischemic stroke patients were higher than in control subjects. In flow cytometry experiments, the percentage of CD73 cells was significantly decreased in lymphocytes and in T-lymphocyte subclasses CD4 and CD8 obtained from ischemic stroke patients in comparison with healthy individuals. These data corroborate the importance of the adenosinergic system in ischemic stroke and could open the way to more targeted therapeutic approaches and biomarker development for ischemic stroke.
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http://dx.doi.org/10.3390/cells9051072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290971PMC
April 2020

Structural investigation on thiazolo[5,4-d]pyrimidines to obtain dual-acting blockers of CD73 and adenosine A receptor as potential antitumor agents.

Bioorg Med Chem Lett 2020 05 29;30(9):127067. Epub 2020 Feb 29.

Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino, Sezione di Farmaceutica e Nutraceutica, Università degli Studi di Firenze, via Ugo Schiff, 6, 50019 Sesto Fiorentino, Italy.

Adenosine pathway, including its generating enzyme (CD73) and its receptors represents a key target for cancer immunotherapy. Here we aimed to search for novel compounds able to co-target the CD73 and the A adenosine receptor (A AR) as dual-blockers of adenosine generation and activity. The design project was to combine in the same molecule the thiazolo[5,4-d]pyrimidine core, an essential pharmacophoric feature to block the A AR, with a benzenesulfonamide group which is a characteristic group of CD73 inhibitors. Most of the reported compounds resulted in inverse agonists of the human (h) A AR endowed with high affinity, selectivity and potency. However they were weak inhibitors of CD73 enzyme. Nevertheless, this study can be considered as a starting point to develop more active compounds.
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http://dx.doi.org/10.1016/j.bmcl.2020.127067DOI Listing
May 2020

"" Everything that Could Have Been Avoided If We Had Applied Gender Medicine, Pharmacogenetics and Personalized Medicine in the Gender-Omics and Sex-Omics Era.

Int J Mol Sci 2019 Dec 31;21(1). Epub 2019 Dec 31.

University Center for Studies on Gender Medicine, University of Ferrara, 44121 Ferrara, Italy.

. Gender medicine is the first step of personalized medicine and patient-centred care, an essential development to achieve the standard goal of a holistic approach to patients and diseases. By addressing the interrelation and integration of biological markers (i.e., sex) with indicators of psychological/cultural behaviour (i.e., gender), gender medicine represents the crucial assumption for achieving the personalized health-care required in the third millennium. However, 'sex' and 'gender' are often misused as synonyms, leading to frequent misunderstandings in those who are not deeply involved in the field. Overall, we have to face the evidence that biological, genetic, epigenetic, psycho-social, cultural, and environmental factors mutually interact in defining sex/gender differences, and at the same time in establishing potential unwanted sex/gender disparities. Prioritizing the role of sex/gender in physiological and pathological processes is crucial in terms of efficient prevention, clinical signs' identification, prognosis definition, and therapy optimization. In this regard, the omics-approach has become a powerful tool to identify sex/gender-specific disease markers, with potential benefits also in terms of socio-psychological wellbeing for each individual, and cost-effectiveness for National Healthcare systems. "" is indeed important from a health point of view and it is no longer possible to avoid "" when approaching patients. Accordingly, personalized healthcare must be based on evidence from targeted research studies aimed at understanding how sex and gender influence health across the entire life span. The rapid development of genetic tools in the molecular medicine approaches and their impact in healthcare is an example of highly specialized applications that have moved from specialists to primary care providers (e.g., pharmacogenetic and pharmacogenomic applications in routine medical practice). Gender medicine needs to follow the same path and become an established medical approach. To face the genetic, molecular and pharmacological bases of the existing sex/gender gap by means of omics approaches will pave the way to the discovery and identification of novel drug-targets/therapeutic protocols, personalized laboratory tests and diagnostic procedures (sex/gender-omics). In this scenario, the aim of the present review is not to simply resume the state-of-the-art in the field, rather an opportunity to gain insights into gender medicine, spanning from molecular up to social and psychological stances. The description and critical discussion of some key selected multidisciplinary topics considered as paradigmatic of sex/gender differences and sex/gender inequalities will allow to draft and design strategies useful to fill the existing gap and move forward.
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http://dx.doi.org/10.3390/ijms21010296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6982247PMC
December 2019

Amino-3,5-Dicyanopyridines Targeting the Adenosine Receptors Ranging from Pan Ligands to Combined A/A Partial Agonists.

Pharmaceuticals (Basel) 2019 Oct 22;12(4). Epub 2019 Oct 22.

Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino, Sezione di Farmaceutica e Nutraceutica, Università degli Studi di Firenze, Via Ugo Schiff, 6, 50019 Sesto Fiorentino, Italy.

The amino-3,5-dicyanopyridine derivatives belong to an intriguing series of adenosine receptor (AR) ligands that has been developed by both academic researchers and industry. Indeed, the studies carried out to date underline the versatility of the dicyanopyridine scaffold to obtain AR ligands with not only a wide range of affinities but also with diverse degrees of efficacies at the different ARs. These observations prompted us to investigate on the structure-activity relationships (SARs) of this series leading to important previously reported results. The present SAR study has helped to confirm the 1-imidazol-2-yl group at R position as an important feature for producing potent AR agonists. Moreover, the nature of the R substituent highly affects not only affinity/activity at the hA and hA ARs but also selectivity versus the other subtypes. Potent hA and hA AR ligands were developed, and among them, the 2-amino-6-[(1-imidazol-2-ylmethyl)sulfanyl]-4-[4-(prop-2-en-1-yloxy)phenyl]pyridine-3,5-dicarbonitrile () is active in the low nanomolar range at these subtypes and shows a good trend of selectivity versus both the hA and hA ARs. This combined hAhA partial agonist activity leads to a synergistic effect on glucose homeostasis and could potentially be beneficial in treating diabetes and related complications.
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http://dx.doi.org/10.3390/ph12040159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6958422PMC
October 2019

Signaling pathways involved in anti-inflammatory effects of Pulsed Electromagnetic Field in microglial cells.

Cytokine 2020 01 7;125:154777. Epub 2019 Aug 7.

Department of Medical Sciences, University of Ferrara, Ferrara 44121, Italy; Center for Studies on Gender Medicine, University of Ferrara, Ferrara 44121, Italy.

Literature studies suggest important protective effects of low-frequency, low-energy pulsed electromagnetic fields (PEMFs) on inflammatory pathways affecting joint and cerebral diseases. However, it is not clear on which bases they affect neuroprotection and the mechanism responsible is yet unknown. Therefore the aim of this study was to identify the molecular targets of PEMFs anti-neuroinflammatory action. The effects of PEMF exposure in cytokine production by lipopolysaccharide (LPS)-activated N9 microglial cells as well as the pathways involved, including adenylyl cyclase (AC), phospholipase C (PLC), protein kinase C epsilon (PKC-ε) and delta (PKC-δ), p38, ERK1/2, JNK1/2 mitogen activated protein kinases (MAPK), Akt and caspase 1, were investigated. In addition, the ability of PEMFs to modulate ROS generation, cell invasion and phagocytosis, was addressed. PEMFs reduced the LPS-increased production of TNF-α and IL-1β in N9 cells, through a pathway involving JNK1/2. Furthermore, they decreased the LPS-induced release of IL-6, by a mechanism not dependent on AC, PLC, PKC-ε, PKC-δ, p38, ERK1/2, JNK1/2, Akt and caspase 1. Importantly, a significant effect of PEMFs in the reduction of crucial cell functions specific of microglia like ROS generation, cell invasion and phagocytosis was found. PEMFs inhibit neuroinflammation in N9 cells through a mechanism involving, at least in part, the activation of JNK MAPK signalling pathway and may be relevant to treat a variety of diseases characterized by neuroinflammation.
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http://dx.doi.org/10.1016/j.cyto.2019.154777DOI Listing
January 2020

Modifications on the Amino-3,5-dicyanopyridine Core To Obtain Multifaceted Adenosine Receptor Ligands with Antineuropathic Activity.

J Med Chem 2019 08 26;62(15):6894-6912. Epub 2019 Jul 26.

Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino, Sezione di Farmaceutica e Nutraceutica , Università degli Studi di Firenze , Via Ugo Schiff, 6 , 50019 Sesto Fiorentino , Italy.

A new series of amino-3,5-dicyanopyridines () was synthesized and biologically evaluated in order to further investigate the potential of this scaffold to obtain adenosine receptor (AR) ligands. In general, the modifications performed have led to compounds having high to good human (h) AAR affinity and an inverse agonist profile. While most of the compounds are hAAR-selective, some derivatives behave as mixed hAAR inverse agonists/A and A AR antagonists. The latter compounds ( showed that they reduce oxaliplatin-induced neuropathic pain by a mechanism involving the alpha7 subtype of nAchRs, similar to the nonselective AR antagonist caffeine, taken as the reference compound. Along with the pharmacological evaluation, chemical stability of methyl 3-(((6-amino-3,5-dicyano-4-(furan-2-yl)pyridin-2-yl)sulfanyl)methyl)benzoate was assessed in plasma matrices (rat and human), and molecular modeling studies were carried out to better rationalize the available structure-activity relationships.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00106DOI Listing
August 2019

Targeting A3 and A2A adenosine receptors in the fight against cancer.

Expert Opin Ther Targets 2019 08 12;23(8):669-678. Epub 2019 Jun 12.

a Department of Medical Sciences , University of Ferrara , Ferrara , Italy.

: There is a vicious cycle of tumor hypoxia, high adenosine levels, immune suppression and cancer growth that involves the use of adenosine receptor ligands in tumors. After several years of research, the candidates emerging as promising new anticancer drugs are A adenosine receptor agonists and A receptor antagonists. : The authors give an updated overview of the field related to A receptor agonists and A receptor antagonists in cancer and propose their perspectives on the status of these compounds in oncology. The rationale for the modulation of adenosine receptors in cancer is addressed, starting from the first in vitro evidence of their efficacy up to the animal and clinical studies. : A and A receptors are attractive targets in oncologic therapy due to their involvement in cancer progression and immune-resistance. Of relevance, the A subtype is also a tumor marker to be used in a personalized drug treatment program while the A receptor, playing a non-redundant role in immunomodulation, may be blocked in combination with checkpoint inhibitors to improve their efficacy. The future will reveal how successful this approach is in the fight against cancer.
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http://dx.doi.org/10.1080/14728222.2019.1630380DOI Listing
August 2019

New Rigid Nicotine Analogues, Carrying a Norbornane Moiety, Are Potent Agonists of α7 and α3* Nicotinic Receptors.

J Med Chem 2019 02 8;62(4):1887-1901. Epub 2019 Feb 8.

Department of Neuroscience, Psychology, Drug Research and Child's Health (NEUROFARBA), Section of Pharmaceutical and Nutraceutical Sciences , University of Florence , Via Ugo Schiff 6 , 50019 Sesto Fiorentino , Italy.

A three-dimensional database search has been applied to design a series of endo- and exo-3-(pyridin-3-yl)bicyclo[2.2.1]heptan-2-amines as nicotinic receptor ligands. The synthesized compounds were tested in radioligand binding assay on rat cortex against [H]-cytisine and [H]-methyllycaconitine to measure their affinity for α4β2* and α7* nicotinic receptors. The new derivatives showed some preference for the α4β2* over the α7* subtype, with their affinity being dependent on the endo/exo isomerism and on the methylation degree of the basic nitrogen. The endo primary amines displayed the lowest K values on both receptor subtypes. Selected compounds (1a, 2a, 3a, and 6a) were tested on heterologously expressed α4β2, α7, and α3β2 receptors and on SHSY-5Y cells. Compounds 1a and 2a showed α4β2 antagonistic properties while behaved as full agonists on recombinant α7 and on SHSY5Y cells. On the α3β2 subtype, only the chloro derivative 2a showed full agonist activity and submicromolar potency (EC = 0.43 μM). The primary amines described here represent new chemotypes for the α7 and α3* receptor subtypes.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01372DOI Listing
February 2019

Pulsed electromagnetic field and relief of hypoxia-induced neuronal cell death: The signaling pathway.

J Cell Physiol 2019 Jan 17. Epub 2019 Jan 17.

Department of Medical Sciences, University of Ferrara, Ferrara, Italy.

Low-energy low-frequency pulsed electromagnetic fields (PEMFs) exert several protective effects, such as the regulation of kinases, transcription factors as well as cell viability in both central and peripheral biological systems. However, it is not clear on which bases they affect neuroprotection and the mechanism responsible is yet unknown. In this study, we have characterized in nerve growth factor-differentiated pheochromocytoma PC12 cells injured with hypoxia: (i) the effects of PEMF exposure on cell vitality; (ii) the protective pathways activated by PEMFs to relief neuronal cell death, including adenylyl cyclase, phospholipase C, protein kinase C epsilon and delta, p38, ERK1/2, JNK1/2 mitogen-activated protein kinases, Akt and caspase-3; (iii) the regulation by PEMFs of prosurvival heat-shock proteins of 70 (HSP70), cAMP response element-binding protein (CREB), brain-derived neurotrophic factor (BDNF), and Bcl-2 family proteins. The results obtained in this study show a protective effect of PEMFs that are able to reduce neuronal cell death induced by hypoxia by modulating p38, HSP70, CREB, BDNF, and Bcl-2 family proteins. Specifically, we found a rapid activation (30 min) of p38 kinase cascade, which in turns enrolles HSP70 survival chaperone molecule, resulting in a significant CREB phosphorylation increase (24 hr). In this cascade, later (48 hr), BDNF and the antiapoptotic pathway regulated by the Bcl-2 family of proteins are recruited by PEMFs to enhance neuronal survival. This study paves the way to elucidate the mechanisms triggered by PEMFs to act as a new neuroprotective approach to treat cerebral ischemia by reducing neuronal cell death.
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http://dx.doi.org/10.1002/jcp.28149DOI Listing
January 2019

Cannabigerol Action at Cannabinoid CB and CB Receptors and at CB-CB Heteroreceptor Complexes.

Front Pharmacol 2018 21;9:632. Epub 2018 Jun 21.

Centro de Investigación Biomédica en Red, Enfermedades Neurodegenerativas (CIBERNED), Instituto de Salud Carlos III, Madrid, Spain.

Cannabigerol (CBG) is one of the major phytocannabinoids present in L. that is attracting pharmacological interest because it is non-psychotropic and is abundant in some industrial hemp varieties. The aim of this work was to investigate in parallel the binding properties of CBG to cannabinoid CB (CBR) and CB (CBR) receptors and the effects of the compound on agonist activation of those receptors and of CB-CB heteroreceptor complexes. Using [H]-CP-55940, CBG competed with low micromolar values the binding to CBR and CBR. Homogeneous binding in living cells, which is only technically possible for the CBR, provided a 152 nM value. Also interesting, CBG competed the binding of [H]-WIN-55,212-2 to CBR but not to CBR (: 2.7 versus >30 μM). The phytocannabinoid modulated signaling mediated by receptors and receptor heteromers even at low concentrations of 0.1-1 μM. cAMP, pERK, β-arrestin recruitment and label-free assays in HEK-293T cells expressing the receptors and treated with endocannabinoids or selective agonists proved that CBG is a partial agonist of CBR. The action on cells expressing heteromers was similar to that obtained in cells expressing the CBR. The effect of CBG on CBR was measurable but the underlying molecular mechanisms remain uncertain. The results indicate that CBG is indeed effective as regulator of endocannabinoid signaling.
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http://dx.doi.org/10.3389/fphar.2018.00632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6021502PMC
June 2018

Structure-activity relationship studies and pharmacological characterization of N-heteroarylalkyl-substituted-2-(2-furanyl)thiazolo[5,4-d]pyrimidine-5,7-diamine-based derivatives as inverse agonists at human A adenosine receptor.

Eur J Med Chem 2018 Jul 9;155:552-561. Epub 2018 Jun 9.

Dipartimento di Scienze Mediche, Sezione di Farmacologia, Università degli Studi di Ferrara, Via Fossato di Mortara 17-19, 44121, Ferrara, Italy.

This paper describes the synthesis and characterization of N-(hetero)arylalkyl-substituted-thiazolo [5,4-d]pyrimidine-5,7-diamine derivatives (4-19) as novel human (h) A adenosine receptor (AR) inverse agonists. Competition binding and cyclic AMP assays indicate that the examined compounds behave as hA AR inverse agonists showing binding affinity values in the nanomolar or subnanomolar range. Notably, compounds 4, 5, 6 and 11 showed two affinity values for the hA ARs with the highest (KH) falling in the femtomolar range and the lowest (KL) of the nanomolar order. In addition, in cyclic AMP assays, compounds 4, 5, 6 and 11 exhibited potency (IC) values in the picomolar range. This study has confirmed that 2-(2-furanyl)thiazolo [5,4-d]pyrimidine-5,7-diamine-based derivatives represent a unique new class of hA AR inverse agonists.
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http://dx.doi.org/10.1016/j.ejmech.2018.06.020DOI Listing
July 2018

Identification of novel thiazolo[5,4-d]pyrimidine derivatives as human A and A adenosine receptor antagonists/inverse agonists.

Bioorg Med Chem 2018 07 31;26(12):3688-3695. Epub 2018 May 31.

Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino, Sezione di Farmaceutica e Nutraceutica, Università degli Studi di Firenze, Via Ugo Schiff, 6, 50019 Sesto Fiorentino, Italy.

In this study a new set of thiazolo[5,4-d]pyrimidine derivatives was synthesized. These derivatives bear different substituents at positions 2 and 5 of the thiazolopyrimidine core while maintaining a free amino group at position-7. The new compounds were tested for their affinity and potency at human (h) A, A, A and A adenosine receptors expressed in CHO cells. The results reveal that the higher affinity of these new set of thiazolopyrimidines is toward the hA and hA adenosine receptors subtypes and is tuned by the substitution pattern at both the 2 and 5 positions of the thiazolopyrimidine nucleus. Functional studies evidenced that the compounds behaved as dual A/A antagonists/inverse agonists. Compound 3, bearing a 5-((2-methoxyphenyl) methylamino) group and a phenyl moiety at position 2, displayed the highest affinity (hA K = 10.2 nM; hA K = 4.72 nM) and behaved as a potent A/A antagonist/inverse agonist (hA IC = 13.4 nM; hA IC = 5.34 nM).
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http://dx.doi.org/10.1016/j.bmc.2018.05.048DOI Listing
July 2018

Pharmacology of Adenosine Receptors: The State of the Art.

Physiol Rev 2018 07;98(3):1591-1625

Department of Medical Sciences, University of Ferrara , Ferrara , Italy.

Adenosine is a ubiquitous endogenous autacoid whose effects are triggered through the enrollment of four G protein-coupled receptors: A, A, A, and A. Due to the rapid generation of adenosine from cellular metabolism, and the widespread distribution of its receptor subtypes in almost all organs and tissues, this nucleoside induces a multitude of physiopathological effects, regulating central nervous, cardiovascular, peripheral, and immune systems. It is becoming clear that the expression patterns of adenosine receptors vary among cell types, lending weight to the idea that they may be both markers of pathologies and useful targets for novel drugs. This review offers an overview of current knowledge on adenosine receptors, including their characteristic structural features, molecular interactions and cellular functions, as well as their essential roles in pain, cancer, and neurodegenerative, inflammatory, and autoimmune diseases. Finally, we highlight the latest findings on molecules capable of targeting adenosine receptors and report which stage of drug development they have reached.
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http://dx.doi.org/10.1152/physrev.00049.2017DOI Listing
July 2018

Development of novel pyridazinone-based adenosine receptor ligands.

Bioorg Med Chem Lett 2018 05 30;28(9):1484-1489. Epub 2018 Mar 30.

Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino, Sezione di Farmaceutica e Nutraceutica, Università degli Studi di Firenze, Via Ugo Schiff, 6, 50019 Sesto Fiorentino, Italy.

With the aim of finding new adenosine receptor (AR) ligands, a preliminary investigation focusing on the thieno[2,3-d]pyridazin-5(4H)-one scaffold was undertaken. The synthesized compounds 1-11 were evaluated for their binding at hA, hA and hA ARs and efficacy at hA subtype in order to determine the affinity at the human adenosine receptor subtypes. Small structural changes on this scaffold highly influenced affinity; compound 5 (5-ethyl-7-(thiazol-2-yl)thieno[2,3-d]pyridazin-4(5H)-one) emerged as the best of this series. The simplicity of the synthetic process, the capability of the scaffold to be easily decorated, together with the predicted ADME properties confirm the role of these compounds as promising hits. A molecular docking investigation at the hAAR crystal structure was performed to rationalize the SARs of the herein reported thienopyridazinones.
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http://dx.doi.org/10.1016/j.bmcl.2018.03.086DOI Listing
May 2018

The aminopyridine-3,5-dicarbonitrile core for the design of new non-nucleoside-like agonists of the human adenosine A receptor.

Eur J Med Chem 2018 Apr 6;150:127-139. Epub 2018 Mar 6.

Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino, Sezione di Farmaceutica e Nutraceutica, Università degli Studi di Firenze, Via Ugo Schiff, 6, 50019 Sesto Fiorentino, Italy.

A new series of amino-3,5-dicyanopyridines (3-28) as analogues of the adenosine hA receptor agonist BAY60-6583 (compound 1) was synthesized. All the compounds that interact with the hA adenosine receptor display EC values in the range 9-350 nM behaving as partial agonists, with the only exception being the 2-{[4-(4-acetamidophenyl)-6-amino-3,5-dicyanopyridin-2-yl]thio}acetamide (8) which shows a full agonist profile. Moreover, the 2-[(1H-imidazol-2-yl)methylthio)]-6-amino-4-(4-cyclopropylmethoxy-phenyl)pyridine-3,5-dicarbonitrile (15) turns out to be 3-fold more active than 1 although less selective. This result can be considered a real breakthrough due to the currently limited number of non-adenosine hA AR agonists reported in literature. To simulate the binding mode of nucleoside and non-nucleoside agonists at the hA AR, molecular docking studies were performed at homology models of this AR subtype developed by using two crystal structures of agonist-bound A AR as templates. These investigations allowed us to represent a hypothetical binding mode of hA receptor agonists belonging to the amino-3,5-dicyanopyridine series and to rationalize the observed SAR.
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http://dx.doi.org/10.1016/j.ejmech.2018.02.081DOI Listing
April 2018

Inhibition of A Adenosine Receptor Signaling in Cancer Cells Proliferation by the Novel Antagonist TP455.

Front Pharmacol 2017 1;8:888. Epub 2017 Dec 1.

Department of Medical Sciences, Pharmacology Section, University of Ferrara, Ferrara, Italy.

Several evidences indicate that the ubiquitous nucleoside adenosine, acting through A, A, A, and A receptor (AR) subtypes, plays crucial roles in tumor development. Adenosine has contrasting effects on cell proliferation depending on the engagement of different receptor subtypes in various tumors. The involvement of AARs in human A375 melanoma, as well as in human A549 lung and rat MRMT1 breast carcinoma proliferation has been evaluated in view of the availability of a novel AAR antagonist, with high affinity and selectivity, named as 2-(2-furanyl)-N-(2-methoxybenzyl)[1,3]thiazolo[5,4-d]pyrimidine-5,7-diammine (TP455). Specifically, the signaling pathways triggered in the cancer cells of different origin and the antagonist effect of TP455 were investigated. The AAR protein expression was evaluated through receptor binding assays. Furthermore, the effect of AAR activation on cell proliferation at 24, 48 and 72 hours was studied. The selective AAR agonist 2--(2-carboxyethyl)phenethylamino-5'--ethylcarboxamidoadenosine hydrochloride (CGS21680), concentration-dependently induced cell proliferation in A375, A549, and MRMT1 cancer cells and the effect was potently antagonized by the AAR antagonist TP455, as well as by the reference AAR blocker 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385). As for the signaling pathway recruited in this response we demonstrated that, by using the specific inhibitors of signal transduction pathways, the effect of AAR stimulation was induced through phospholipase C (PLC) and protein kinase C-delta (PKC-δ). In addition, we evaluated, through the AlphaScreen SureFire phospho(p) protein assay, the kinases enrolled by AAR to stimulate cell proliferation and we found the involvement of protein kinase B (AKT), extracellular regulated kinases (ERK1/2), and c-Jun N-terminal kinases (JNKs). Indeed, we demonstrated that the CGS21680 stimulatory effect on kinases was strongly reduced in the presence of the new potent compound TP455, as well as by ZM241385, confirming the role of the AAR. In conclusion, the AAR activation stimulates proliferation of A375, A549, and MRMT1 cancer cells and importantly TP455 reveals its capability to counteract this effect, suggesting selective AAR antagonists as potential new therapeutics.
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http://dx.doi.org/10.3389/fphar.2017.00888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716981PMC
December 2017

Binding and Signaling Studies Disclose a Potential Allosteric Site for Cannabidiol in Cannabinoid CB Receptors.

Front Pharmacol 2017 23;8:744. Epub 2017 Oct 23.

Molecular Neurobiology Laboratory, Department of Biochemistry and Molecular Biomedicine, University of Barcelona, Barcelona, Spain.

The mechanism of action of cannabidiol (CBD), the main non-psychotropic component of L., is not completely understood. First assumed that the compound was acting via cannabinoid CB receptors (CBRs) it is now suggested that it interacts with non-cannabinoid G-protein-coupled receptors (GPCRs); however, CBD does not bind with high affinity to the orthosteric site of any GPCR. To search for alternative explanations, we tested CBD as a potential allosteric ligand of CBR. Radioligand and non-radioactive homogeneous binding, intracellular cAMP determination and ERK1/2 phosphorylation assays were undertaken in heterologous systems expressing the human version of CBR. Using membrane preparations from CBR-expressing HEK-293T (human embryonic kidney 293T) cells, we confirmed that CBD does not bind with high affinity to the orthosteric site of the human CBR where the synthetic cannabinoid, [H]-WIN 55,212-2, binds. CBD was, however, able to produce minor but consistent reduction in the homogeneous binding assays in living cells using the fluorophore-conjugated CBR-selective compound, CM-157. The effect on binding to CBR-expressing living cells was different to that exerted by the orthosteric antagonist, SR144528, which decreased the maximum binding without changing the . CBD at nanomolar concentrations was also able to significantly reduce the effect of the selective CBR agonist, JWH133, on forskolin-induced intracellular cAMP levels and on activation of the MAP kinase pathway. These results may help to understand CBD mode of action and may serve to revisit its therapeutic possibilities.
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http://dx.doi.org/10.3389/fphar.2017.00744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5660261PMC
October 2017

Psychostimulant Effect of the Synthetic Cannabinoid JWH-018 and AKB48: Behavioral, Neurochemical, and Dopamine Transporter Scan Imaging Studies in Mice.

Front Psychiatry 2017 4;8:130. Epub 2017 Aug 4.

Department of Life Sciences and Biotechnology (SVeB), University of Ferrara, Ferrara, Italy.

JWH-018 and AKB48 are two synthetic cannabinoids (SCBs) belonging to different structural classes and illegally marketed as incense, herbal preparations, or chemical supply for theirs psychoactive cannabis-like effects. Clinical reports from emergency room reported psychomotor agitation as one of the most frequent effects in people assuming SCBs. This study aimed to investigate the psychostimulant properties of JWH-018 and AKB48 in male CD-1 mice and to compare their behavioral and biochemical effects with those caused by cocaine and amphetamine. studies showed that JWH-018 and AKB48, as cocaine and amphetamine, facilitated spontaneous locomotion in mice. These effects were prevented by CB receptor blockade and dopamine (DA) D and D receptors inhibition. SPECT-CT studies on dopamine transporter (DAT) revealed that, as cocaine and amphetamine, JWH-018 and AKB48 decreased the [I]-FP-CIT binding in the mouse striatum. Conversely, competition binding studies revealed that, unlike cocaine and amphetamine, JWH-018 and AKB48 did not bind to mouse or human DAT. Moreover, microdialysis studies showed that the systemic administration of JWH-018, AKB48, cocaine, and amphetamine stimulated DA release in the nucleus accumbens (NAc) shell of freely moving mice. Finally, unlike amphetamine and cocaine, JWH-018 and AKB48 did not induce any changes on spontaneous [H]-DA efflux from murine striatal synaptosomes. The present results suggest that SCBs facilitate striatal DA release possibly with different mechanisms than cocaine and amphetamine. Furthermore, they demonstrate, for the first time, that JWH-018 and AKB48 induce a psychostimulant effect in mice possibly by increasing NAc DA release. These data, according to clinical reports, outline the potential psychostimulant action of SCBs highlighting their possible danger to human health.
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http://dx.doi.org/10.3389/fpsyt.2017.00130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5543288PMC
August 2017

Effects of pulsed electromagnetic fields and platelet rich plasma in preventing osteoclastogenesis in an in vitro model of osteolysis.

J Cell Physiol 2018 Mar 4;233(3):2645-2656. Epub 2017 Sep 4.

Laboratory of Preclinical and Surgical Studies, Rizzoli Orthopedic Institute, Bologna, Italy.

Osteolysis is the main limiting cause for the survival of an orthopedic prosthesis and is accompanied by an enhancement in osteoclastogenesis and inflammation, due by wear debris formation. Unfortunately therapeutic treatments, besides revision surgery, are not available. The aim of the present study was to evaluate the effects of Pulsed Electro Magnetic Fields (PEMFs) and platelet rich plasma (PRP), alone or in combination, in an in vitro model of osteolysis. Rats peripheral blood mononuclear cells were cultured on Ultra High Molecular Weight Polyethylene particles and divided into four groups of treatments: (1) PEMF stimulation (12 hr/day, 2.5 mT, 75 Hz, 1.3 ms pulse duration); (2) 10% PRP; (3) combination of PEMFs, and PRP; (4) no treatment. Treatments were performed for 3 days and cell viability, osteoclast number, expression of genes related to osteoclastogenesis and inflammation and production of pro-inflammatory cytokines were assessed up to 14 days. PEMF stimulation exerted best results because it increased cell viability at early time points and counteracted osteoclastogenesis at 14 days. On the contrary, PRP increased osteoclastogenesis and reduced cell viability in comparison to PEMFs alone. The combination of PEMFs and PRP increased cell viability over time and reduced osteoclastogenesis in comparison to PRP alone. However, these positive results did not exceed the level achieved by PEMF alone. At longer time points PEMF could not counteract osteoclastogenesis increased by PRP. Regarding inflammation, all treatments maintained the production of pro-inflammatory cytokines at low level, although PRP increased the level of interleukin 1 beta.
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http://dx.doi.org/10.1002/jcp.26143DOI Listing
March 2018