Publications by authors named "Laura Lucarini"

38 Publications

NCX 1741, a Novel Nitric Oxide-Donating Phosphodiesterase-5 Inhibitor, Exerts Rapid and Long-Lasting Intraocular Pressure-Lowering in Cynomolgus Monkeys.

J Ocul Pharmacol Ther 2021 Feb 15. Epub 2021 Feb 15.

Nicox Research Institute, Milan, Italy.

We studied the IOP-lowering effects of NCX 1741, a novel nitric oxide (NO)-donating derivative of the phosphodiesterase type-5 inhibitor, avanafil, in Cynomolgus monkey with laser-induced ocular hypertension (OHT-monkeys). NCX 1193 (NO-donating moiety), NCX 1744 (NCX 1741 without ester nitrate moiety), and travoprost (PGF2α analogue) were used for comparison. Ocular exposure after NCX 1741 dosing also was addressed. Vehicle (phosphate buffer pH 6.0, Kolliphor 5%, DMSO 0.3%, benzalkonium chloride 0.02%), NCX 1741, NCX 1193, NCX 1744, or travoprost were instilled (30 μL; single dose) masked and conscious IOPs were measured by pneumatonometry. LC-MS/MS-based methods were employed to monitor ocular exposure of NCX 1741 and main metabolites after ocular dosing in New Zealand White rabbits. NCX 1741 (2.2%, 0.8 μmol/eye) lowered IOP with an E (ΔΔIOP, IOP change vs. pre-dose and vehicle) between 5 and 8 h post-dosing (ΔΔIOP, -5.3 ± 2.0 mmHg and ΔΔIOP, -6.0 ± 2.1 mmHg). Conversely, equimolar (0.47%, 0.8 μmol/eye) NCX 1193 IOP-lowering effects were maximal 3 h post-dosing (ΔΔIOP, -4.7 ± 1.6 mmHg) and declined thereafter (ΔΔIOP, -1.6 ± 1.1 mmHg). In a follow-up study, NCX 1741 (1.5%, 0.5 μmol/eye) was more effective than NCX 1744 despite a similar duration. Further, NCX 1741 was as effective as travoprost (0.1%, 0.06 μmol/eye) at 5 and 8 h post-dosing (travoprost, ΔΔIOP, -3.4 ± 2.2 mmHg and ΔΔIOP, -4.9 ± 1.3 mmHg) but had shorter duration (NCX 1741, ΔΔIOP, -1.5 ± 1.1 mmHg; travoprost, ΔΔIOP, -7.1 ± 2.8 mmHg). NCX 1741 resulted in significant aqueous humor exposure, as determined by the levels of the main metabolite, avanafil. NCX 1741 rapidly and effectively lowers IOP in OHT-monkeys for several hours post-dosing. How these effects translate in humans is still to be defined.
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http://dx.doi.org/10.1089/jop.2020.0126DOI Listing
February 2021

Targeting the RNA-Binding Protein HuR Alleviates Neuroinflammation in Experimental Autoimmune Encephalomyelitis: Potential Therapy for Multiple Sclerosis.

Neurotherapeutics 2020 Nov 16. Epub 2020 Nov 16.

Section of Pharmacology, Department of Neuroscience, Psychology, Drug Research and Child Health (NEUROFARBA), University of Florence, Viale G. Pieraccini 6, 50139, Florence, Italy.

Multiple sclerosis (MS) is a chronic autoimmune inflammatory and neurodegenerative disease of the central nervous system characterized by demyelination, axonal loss, and motor dysfunction. Activated microglia are associated with the destruction of myelin in the CNS. Activated microglia produce cytokines and proinflammatory factors, favoring neuroinflammation, myelin damage, and neuronal loss, and it is thought to be involved in the disease pathogenesis. The present study investigated the role of post-transcriptional regulation of gene expression on the neuroinflammation related to experimental autoimmune encephalomyelitis (EAE) in mice, by focusing on HuR, an RNA-binding protein involved in inflammatory and immune phenomena. Spinal cord sections of EAE mice showed an increased HuR immunostaining that was abundantly detected in the cytoplasm of activated microglia, a pattern associated with its increased activity. Intrathecal administration of an anti-HuR antisense oligonucleotide (ASO) decreased the proinflammatory activated microglia, inflammatory infiltrates, and the expression of the proinflammatory cytokines IL-1β, TNF-α, and IL-17, and inhibited the activation of the NF-κB pathway. The beneficial effect of anti-HuR ASO in EAE mice corresponded also to a decreased permeability of the blood-brain barrier. EAE mice showed a reduced spinal CD206 immunostaining that was restored by anti-HuR ASO, indicating that HuR silencing promotes a shift to the anti-inflammatory and regenerative microglia phenotype. Mice that received anti-HuR ASO exhibited improved EAE-related motor dysfunction, pain hypersensitivity, and body weight loss. Targeting HuR might represent an innovative and promising perspective to control neurological disturbances in MS patients.
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http://dx.doi.org/10.1007/s13311-020-00958-8DOI Listing
November 2020

Effects of New NSAID-CAI Hybrid Compounds in Inflammation and Lung Fibrosis.

Biomolecules 2020 09 10;10(9). Epub 2020 Sep 10.

Department of NEUROFARBA, Pharmaceutical Science Section, University of Florence, Via Ugo Schiff 6, Sesto Fiorentino, 50019 Florence, Italy.

Pulmonary fibrosis is a severe lung disease with progressive worsening of dyspnea, characterized by chronic inflammation and remodeling of lung parenchyma. Carbonic anhydrases are a family of zinc-metallo-enzymes that catalyze the reversible interconversion of carbon-dioxide and water to bicarbonate and protons. Carbonic Anhydrase Inhibitor (CAI) exhibited anti-inflammatory effects in animals with permanent-middle-cerebral artery occlusion, arthritis and neuropathic pain. The pharmacological profile of a new class of hybrid compounds constituted by a CAI connected to a Nonsteroidal-Anti-Inflammatory Drug (NSAID) was studied in the modulation of inflammation and fibrosis. In-vitro tests were performed to assess their effects on cyclo-oxygenase enzyme (COX)-1 and COX-2, namely inhibition of platelet aggregation and thromboxane B2 production in the human-platelet-rich plasma, and reduction of Prostaglandin-E2 production in lipopolysaccharide-treated-RAW-264.7 macrophage cell line. The activity of compound , one of the most active, was studied in a model of bleomycin-induced lung fibrosis in C57BL/6 mice. The hybrid compounds showed a higher potency in inhibiting PGE production, but not in modifying the platelet aggregation and the TXB production in comparison to the reference molecules, indicating an increased activity in COX-2 inhibition. In the in-vivo murine model, the compound was more effective in decreasing inflammation, lung stiffness and oxidative stress in comparison to the reference drugs given alone or in association. In conclusion, these CAI-NSAID hybrid compounds are promising new anti-inflammatory drugs for the treatment of lung chronic inflammatory diseases.
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http://dx.doi.org/10.3390/biom10091307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564963PMC
September 2020

Sulfonamide Inhibitors of Human Carbonic Anhydrases Designed through a Three-Tails Approach: Improving Ligand/Isoform Matching and Selectivity of Action.

J Med Chem 2020 07 22;63(13):7422-7444. Epub 2020 Jun 22.

Department NEUROFARBA - Pharmaceutical and nutraceutical section, University of Firenze, via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence Italy.

The "tail approach" has become a milestone in human carbonic anhydrase inhibitor (hCAI) design for various therapeutics, including antiglaucoma agents. Besides the classical hydrophobic/hydrophilic division of hCAs active site, several subpockets have been identified at the middle/outer active sites rim, which could be targeted to increase the CAI isoform selectivity. This postulate is explored here by three-tailed benzenesulfonamide CAIs () to fully exploit such amino acid differences among hCAs. In this proof-of-concept study, an extensive structure-activity relationship (SAR) study was carried out with 32 such benzenesulfonamides differing in tails combination that were assayed for hCAs I, II, IV, and XII inhibition. A structural study was undertaken by X-ray crystallography and tools to assess the ligand/target interaction mode. The most active and selective inhibitors against isoforms implicated in glaucoma were assessed in a rabbit model of the disease achieving an intraocular pressure-lowering action comparable to the clinically used dorzolamide.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00733DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008423PMC
July 2020

Discovery of Potent Dual-Tailed Benzenesulfonamide Inhibitors of Human Carbonic Anhydrases Implicated in Glaucoma and in Vivo Profiling of Their Intraocular Pressure-Lowering Action.

J Med Chem 2020 03 20;63(6):3317-3326. Epub 2020 Feb 20.

Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, University of Florence, Via Ugo Schiff 6, Sesto Fiorentino, 50019 Florence, Italy.

The design of three dual-tailed sulfonamide series , , and as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors are presented. All compounds were evaluated for inhibitory action against pharmacologically relevant human CA isoforms I, II, IV, and VII. Compounds emerged as potent CA inhibitors against the four tested isoforms with a significant selectivity to CA II, which is implicated in glaucoma ( in the range 0.36-6.9 nM). X-ray crystallographic analysis of three compounds (, , and ) bound to CA II showed the validity of the adopted drug design strategy as specific moieties within the ligand structure interacted directly with the hydrophobic and hydrophilic halves of the CA II active site. Compounds - and were evaluated for their intraocular pressure-lowering effects in a rabbit model of glaucoma. and showed significant efficacy when compared to the clinically used drug dorzolamide.
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http://dx.doi.org/10.1021/acs.jmedchem.9b02090DOI Listing
March 2020

Effect of Mediterranean Diet Enriched in High Quality Extra Virgin Olive Oil on Oxidative Stress, Inflammation and Gut Microbiota in Obese and Normal Weight Adult Subjects.

Front Pharmacol 2019 15;10:1366. Epub 2019 Nov 15.

Department of Agriculture, Food, Environment and Forestry (DAGRI), University of Florence, Florence, Italy.

The Mediterranean Diet (MD) is useful in the prevention of overweight, obesity and metabolic disease. High Quality-Extra Virgin Olive Oil (HQ-EVOO), an essential component of this diet, exerts protective effects against chronic diseases. Gut Microbiota (GM), recognized as a key factor in driving metabolic activities, is involved in the regulation of host immunity. Lactic Acid Bacteria (LAB) and their probio-active cellular substances produce beneficial effects in the gastrointestinal tract. Eighteen overweight/obese subjects (cases, BMI ≥25 kg/m) and 18 normal weight controls (BMI 18.5-24.9 kg/m) were fed with MD enriched with 40 g/die HQ-EVOO for three months. Feces and blood samples were collected at time 0 (T0) and after three months (T1) for LAB composition, oxidative stress, metabolic and inflammation parameter determinations. Myeloperoxidase and 8-hydroxy-2-deoxyguanosine, markers of inflammation and oxidative stress, were significantly decreased after MD rich in HQ-EVOO both in controls and in cases. Proinflammatory cytokines levels were significantly decreased in cases in comparison to controls, while IL-10 and adiponectin were significantly increased in cases. LAB's B copies/ng of DNA increased 55.6 folds in cases compared to their baseline after MD rich in HQ-EVOO. MD rich in HQ-EVOO increased adiponectin and IL-10 concentration in overweight/obese subjects and decreased oxidative stress and inflammation parameters and at the same time, increased LAB number in GM. Our results indicate that MD rich in HQ-EVOO induces an increase of LAB in GM and could have a potential role in the prevention of inflammation. www.ClinicalTrials.gov, identifier NCT03441802.
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http://dx.doi.org/10.3389/fphar.2019.01366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6872540PMC
November 2019

Bioisosteric Development of Multitarget Nonsteroidal Anti-Inflammatory Drug-Carbonic Anhydrases Inhibitor Hybrids for the Management of Rheumatoid Arthritis.

J Med Chem 2020 03 15;63(5):2325-2342. Epub 2019 Nov 15.

Section of Pharmaceutical and Nutraceutical Sciences, Department of NEUROFARBA, University of Florence, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy.

Multitarget nonsteroidal anti-inflammatory drug (NSAID)-carbonic anhydrase inhibitor (CAI) agents for the management of rheumatoid arthritis are reported. The evidence of the plasma stability of the amide-linked hybrids previously reported prompted us to investigate their pain-relieving mechanism of action. A bioisosteric amide to ester substitution yielded a series of derivatives showing potent target CAs inhibition and to undergo cleavage in rat or human plasma depending on the NSAID portion. A selection of derivatives were assayed to indirectly evaluate their effect on COX-1 and COX-2. MD simulations demonstrated that the entire hybrids are also able to efficiently bind the COX active site. In a rat model of RA, the most promising derivative () showed major antihyperalgesic action compared with the equimolar coadministration of the single agents. The gathered data provided new insights on the action mechanism of these multitarget compounds, which induce markedly improved pain relief compared with the parent NSAIDs.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01130DOI Listing
March 2020

Different Antioxidant Efficacy of Two Mn-Containing Superoxide Anion Scavengers on Hypoxia/Reoxygenation-Exposed Cardiac Muscle Cells.

Sci Rep 2019 07 16;9(1):10320. Epub 2019 Jul 16.

Department of Experimental & Clinical Medicine, Research Unit of Histology & Embryology, University of Florence, viale G. Pieraccini 6, 50139, Florence, Italy.

Oxidative stress due to excess superoxide anion ([Formula: see text]) produced by dysfunctional mitochondria is a key pathogenic event of aging and ischemia-reperfusion diseases. Here, a new [Formula: see text]-scavenging Mn complex with a new polyamino-polycarboxylate macrocycle (4,10-dimethyl-1,4,7,10-tetraazacyclododecane-1,7-diacetate) containing 2 quinoline units (MnQ2), designed to improve complex stability and cell permeability, was compared to parental Mn complex with methyls replacing quinolines (MnM2). MnQ2 was more stable than MnM2 (log K = 19.56(8) vs. 14.73(2) for the equilibrium Mn + L, where L = Q2 and M2) due to the involvement of quinoline in metal binding and to the hydrophobic features of the ligand which improve metal desolvation upon complexation. As oxidative stress model, H9c2 rat cardiomyoblasts were subjected to hypoxia-reoxygenation. MnQ2 and MnM2 (10 μmol L) were added at reoxygenation for 1 or 2 h. The more lipophilic MnQ2 showed more rapid cell and mitochondrial penetration than MnM2. Both MnQ2 and MnM2 abated endogenous ROS and mitochondrial [Formula: see text], decreased cell lipid peroxidation, reduced mitochondrial dysfunction, in terms of efficiency of the respiratory chain and preservation of membrane potential (Δψ) and permeability, decreased the activation of pro-apoptotic caspases 9 and 3, and increased cell viability. Of note, MnQ2 was more effective than MnM2 to exert cytoprotective anti-oxidant effects in the short term. Compounds with redox-inert Zn replacing the functional Mn were ineffective. This study provides clues which further our understanding of the structure-activity relationships of Mn-chelates and suggests that Mn-polyamino-polycarboxylate macrocycles could be developed as new anti-oxidant drugs.
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http://dx.doi.org/10.1038/s41598-019-46476-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635543PMC
July 2019

Effects of PARP-1 Deficiency and Histamine H Receptor Inhibition in an Inflammatory Model of Lung Fibrosis in Mice.

Front Pharmacol 2019 16;10:525. Epub 2019 May 16.

Section of Pharmacology, Department of Neurosciences, Psychology, Drug Research and Child Health (NEUROFARBA), University of Florence, Florence, Italy.

Pulmonary fibrosis is the most frequent form of interstitial lung disease. Effective therapies are not yet available; novel therapeutic approaches are needed for counteracting fibrosis. Poly(ADP-ribose) polymerases are enzymes, involved in DNA repair and cell apoptosis. PARP-1 deficient mice exhibited reduced lung fibrosis in response to bleomycin treatment compared to wild-type controls. Histamine H receptors (HRs) have been recognized as a new target for inflammatory and immune diseases, and HR ligands reduced inflammation and oxidative stress in lung tissue. The aim of the study was to evaluate the cross-talk between PARP-1 and HR in a model of bleomycin-induced lung fibrosis in PARP-1 and WT mice. Animals were treated with bleomycin or saline by intra-tracheal injection. JNJ7777120, an HR antagonist, or VUF8430, an HR agonist, were administered i.p for 21 days. Airway resistance to inflation was evaluated, and lung tissues were processed for PARylated protein content, oxidative stress evaluation, and histology of small bronchi. The levels of pro-inflammatory (IL-1β and TNF-α), regulatory (IL-10), and pro-fibrotic (TGF-β) cytokines were evaluated. The deposition of αSMA was determined by immunofluorescence analysis. The results indicate that JNJ7777120 reduces PARylated protein production, decreases oxidative stress damage, and MPO, a marker for leukocyte tissue infiltration, in PARP-1 mice. A significant decrease in the production of both IL-1β and TNF-α and a significant increase in IL-10 levels are observed in mice treated with HR antagonist, suggesting a crucial anti-inflammatory activity of JNJ7777120. The smooth muscle layer thickness, the goblet cell relative number, and collagen deposition decreased following JNJ7777120 administration. The HR antagonist treatment also reduces TGF-β production and αSMA deposition, suggesting an important role of JNJ7777120 in airway remodeling. Our results show that PARylation is essential for the pathogenesis of pulmonary fibrosis and propose that PARP-1 and HRs are both involved in inflammatory and fibrotic responses. JNJ7777120 treatment, in a condition of PARP-1 inhibition, exerts anti-inflammatory and anti-fibrotic effects, reducing airway remodeling and bronchoconstriction. Therefore, selective inhibition of HRs together with non-toxic doses of selective PARP-1 inhibitors could have clinical relevance for the treatment of idiopathic pulmonary fibrosis.
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http://dx.doi.org/10.3389/fphar.2019.00525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6535496PMC
May 2019

Role of Histamine H₃ Receptor Antagonists on Intraocular Pressure Reduction in Rabbit Models of Transient Ocular Hypertension and Glaucoma.

Int J Mol Sci 2019 Feb 24;20(4). Epub 2019 Feb 24.

Department of Neuroscience, Psychiatry, Drug Area and Child Health (NEUROFARBA), Section of Pharmacology and Toxicology, University of Florence, 50130 Florence, Italy.

Intraocular pressure (IOP) has a tendency to fluctuate throughout the day, reaching its peak in the early morning in healthy subjects or glaucoma patients. Likewise, histamine tone also fluctuates over time, being lower at nighttime. Numerous studies have demonstrated a correlation between short-term IOP fluctuation and glaucoma progression; however, it has not yet been determined whether histamine plays a role in IOP fluctuations. The aim of this research was to establish the distribution of the histamine receptor proteins and respective mRNAs in the eye by western blot, immunohistochemistry and RT-PCR in New Zealand rabbits. Furthermore, we used a transient ocular hypertension (OHT) model induced by injection of 50 µL of 5% hypertonic saline into the vitreous and a stable OHT model (100 µL 0.1% carbomer in the anterior chamber) to address the potential IOP-lowering ability of H₃ receptor (H₃R) antagonists (ciproxifan, DL76 and GSK189254). IOPs were performed with a Tono-Pen at baseline and 60, 120 and 240 min post treatment after transient OHT induction and, every day for 12 days in the stable OHT model. All histamine receptor subtypes were localized in the rabbit retina and ciliary body/trabecular meshwork. All the treatments lowered IOP in a dose-dependent fashion between 0.3% and 1%. More specifically, the effects were maximal with ciproxifan at 60 min post-dose (IOP change = -18.84 ± 4.85 mmHg, at 1%), remained stable until 120 min (IOP change = -16.38 ± 3.8 mmHg, at 1%) and decayed thereafter to reach baseline values at 240 min. These effects were highly specific and dependent on histamine release as pre-treatment with imetit (H₃R agonist, 1%) or pyrilamine (H₁R antagonist, 1%) largely blocked ciproxifan-mediated effects. Color Doppler ultrasound examination was performed to evaluate changes in ophtalmic artery resistivity index (RI) before and after repeated dosing with DL 76, GSK189254, ciproxifan and timolol. Chronic treatments with H₃R antagonists and timolol improved the vascular performance of ophthalmic arteries and reduced retinal ganglion cell death. Oxidative stress was also reduced and measured 8-Hydroxy-2'-deoxyguanosine (8OHG) expression, and by dihidroethydium (DHE) staining. These results demonstrated that the histamine system participates in IOP regulation and that H₃R antagonists could represent a future promising therapy for glaucoma. Further studies should be focused on the long-term IOP circadian fluctuations.
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http://dx.doi.org/10.3390/ijms20040981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6412827PMC
February 2019

A Selective Histamine H Receptor Antagonist, JNJ7777120, Is Protective in a Rat Model of Transient Cerebral Ischemia.

Front Pharmacol 2018 29;9:1231. Epub 2018 Oct 29.

Department of Neuroscience, Psychology, Drug Research and Child Health (NEUROFARBA), Division of Pharmacology and Toxicology, University of Florence, Florence, Italy.

Cerebral ischemia is a multifactorial pathology characterized by different events evolving in time. The acute injury, characterized by excitoxicity, is followed by a secondary brain injury that develops from hours to days after ischemia. Extracellular levels of histamine increase in the ischemic area after focal cerebral ischemia induced by occlusion of the middle cerebral artery (MCAo). The histamine H receptor (HR) is predominantly expressed in cell types of immune system where is involved in the regulation of immunological and inflammatory responses, and in numerous area of the Central Nervous System (CNS) including cortex and striatum. Our aim was to assess the putative neuroprotective effects of the potent and selective HR antagonist, JNJ7777120 (JNJ), chronically administered (1 mg/kg, i.p., twice/day for 7 days) on damage parameters in a rat model of focal ischemia induced by transient MCAo (tMCAo). Chronic treatment with the HR antagonist JNJ, significantly protected from the neurological deficit and from body weight loss after tMCAo. Seven days after the ischemic insult, JNJ reduced the volume of the ischemic cortical and striatal damage, the number of activated microglia and astrocytes in the ischemic cortex and striatum and decreased the plasma levels of IL-1β and TNF-α, while increased the levels of IL-10. Two days after ischemia, JNJ has reduced granulocyte infiltration in the ischemic area. Results demonstrate that the selective antagonist of HR, JNJ, systemically and chronically administered after ischemia, reduces the ischemic brain damage, improves the neurological deficit and decreases blood pro-inflammatory cytokines, suggesting that HR is a valuable pharmacological target after focal brain ischemia.
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http://dx.doi.org/10.3389/fphar.2018.01231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6215858PMC
October 2018

Reduced Susceptibility to Sugar-Induced Metabolic Derangements and Impairments of Myocardial Redox Signaling in Mice Chronically Fed with D-Tagatose when Compared to Fructose.

Oxid Med Cell Longev 2018 19;2018:5042428. Epub 2018 Sep 19.

Department of Drug Science and Technology, University of Turin, Italy.

Background: D-tagatose is an isomer of fructose and is ~90% as sweet as sucrose with less caloric value. Nowadays, D-tagatose is used as a nutritive or low-calorie sweetener. Despite clinical findings suggesting that D-tagatose could be beneficial in subjects with type 2 diabetes, there are no experimental data comparing D-tagatose with fructose, in terms of metabolic derangements and related molecular mechanisms evoked by chronic exposure to these two monosaccharides.

Materials And Methods: C57Bl/6j mice were fed with a control diet plus water (CD), a control diet plus 30% fructose syrup (L-Fr), a 30% fructose solid diet plus water (S-Fr), a control diet plus 30% D-tagatose syrup (L-Tg), or a 30% D-tagatose solid diet plus water (S-Tg), during 24 weeks.

Results: Both solid and liquid fructose feeding led to increased body weight, abnormal systemic glucose homeostasis, and an altered lipid profile. These effects were associated with vigorous increase in oxidative markers. None of these metabolic abnormalities were detected when mice were fed with both the solid and liquid D-tagatose diets, either at the systemic or at the local level. Interestingly, both fructose formulations led to significant Advanced Glycation End Products (AGEs) accumulation in mouse hearts, as well as a robust increase in both myocardial AGE receptor (RAGE) expression and NF-B activation. In contrast, no toxicological effects were shown in hearts of mice chronically exposed to liquid or solid D-tagatose.

Conclusion: Our results clearly suggest that chronic overconsumption of D-tagatose in both formulations, liquid or solid, does not exert the same deleterious metabolic derangements evoked by fructose administration, due to differences in carbohydrate interference with selective proinflammatory and oxidative stress cascades.
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http://dx.doi.org/10.1155/2018/5042428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6169220PMC
December 2018

Identification of Bivalent Ligands with Melatonin Receptor Agonist and Fatty Acid Amide Hydrolase (FAAH) Inhibitory Activity That Exhibit Ocular Hypotensive Effect in the Rabbit.

J Med Chem 2018 09 4;61(17):7902-7916. Epub 2018 Sep 4.

Dipartimento di Scienze degli Alimenti e del Farmaco , Università degli Studi di Parma , Parco Area delle Scienze 27/A I-43124 Parma , Italy.

Activation of melatonin receptors and inhibition of fatty acid amide hydrolase (FAAH) have both shown potential benefits for the treatment of glaucoma. To exploit the combination of these biological activities in single therapeutic agents, we designed dual-acting compounds sharing the pharmacophore elements required for the two targets, in search for balanced potencies as MT/MT agonists and FAAH inhibitors. In particular, the N-anilinoethylamide scaffold, previously developed for melatonergic ligands, was decorated at meta position with a polymethylene linker bound to an O-arylcarbamate group, substituted according to known structure-activity relationships for FAAH inhibition. For the most active series, the N-anilinoethylamide portion was also replaced with the indole scaffold of melatonin. O-Biphenyl-3-ylcarbamate derivatives were characterized by remarkable and balanced activity at both targets, in the nanomolar range for compound 29. Topical administration reduced elevated intraocular pressure in rabbits, with a longer action and improved efficacy compared to the reference compounds melatonin and URB597.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00893DOI Listing
September 2018

2-Benzylpiperazine: A new scaffold for potent human carbonic anhydrase inhibitors. Synthesis, enzyme inhibition, enantioselectivity, computational and crystallographic studies and in vivo activity for a new class of intraocular pressure lowering agents.

Eur J Med Chem 2018 May 3;151:363-375. Epub 2018 Apr 3.

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

Two series of 2-benzylpiperazines have been prepared and tested for the inhibition of physiologically relevant isoforms of human carbonic anhydrases (hCA, EC 4.2.1.1). The new compounds carry on one nitrogen atom of the piperazine ring a sulfamoylbenzamide group as zinc-binding moiety, and different alkyl/acyl/sulfonyl groups on the other nitrogen. Regio- and stero-isomers are described. The majority of these compounds showed Ki values in the low-medium nanomolar range against hCA I, II and IV, but not IX. In many instances interaction with the enzyme was enantioselective. The binding mode has been studied by means of X-ray crystallography and molecular modelling. Two compounds, evaluated in rabbit models of glaucoma, were able to significantly reduce intraocular pressure, making them interesting candidates for further studies.
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http://dx.doi.org/10.1016/j.ejmech.2018.04.002DOI Listing
May 2018

ERRATUM.

Oncol Res 2018 Mar;26(2):333-334

Department of Health Sciences, University of Florence, Florence, Italy.

The poly(ADP-ribose) polymerase (PARP) enzymes play a key role in the regulation of cellular processes (e.g., DNA damage repair, genomic stability). It has been shown that PARP inhibitors (PARPIs) are selectively cytotoxic against cells having dysfunctions in genes involved in DNA repair mechanisms (synthetic lethality). Drug-induced PARP inhibition potentiates the activity of anticancer drugs such as 5-fluorouracil in enhancing DNA damage, whose repair involves PARP-1 activity. The aim of this study was to evaluate the inhibitory effects of a novel PARPI, HYDAMTIQ, on growth in human tumor cell lines characterized by different features with regard to DNA damage response pathways (BRCA mutational status, microsatellite status, and ATM expression level) and degree of sensitivity/resistance to 5-fluorouracil. HYDAMTIQ showed a more potent inhibitory effect on cell growth in a BRCA2 mutant cell line (CAPAN-1) compared with wild-type cells (C2-6, C2-12, and C2-14 CAPAN-1 clones, and MCF-7). No statistically significant difference was observed after HYDAMTIQ exposure between cells having a different MS status or a different MRE11 mutational status. HYDAMTIQ induced greater antiproliferative effects in SW620 cells expressing a low level of ATM than in H630 cells expressing a high level of ATM. Finally, the combination of HYDAMTIQ and 5-fluorouracil exerted a synergistic effect on the inhibition of SW620 cell growth and an antagonistic effect on that of H630 cell growth. Our results show that the novel PARP inhibitor HYDAMTIQ potently inhibits the growth of human tumor cells with defective DNA damage response pathways and exerts synergistic cytotoxicity in combination with 5-fluorouracil. These data provide relevant examples of synthetic lethality and evidence for further development of this novel PARPI.
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http://dx.doi.org/10.3727/096504018X15187172557369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7844681PMC
March 2018

Plasmatic carbonic anhydrase IX as a diagnostic marker for clear cell renal cell carcinoma.

J Enzyme Inhib Med Chem 2018 Dec;33(1):234-240

a Department of Neuroscience, Psychiatry and Drug Area and Child Health (NEUROFARBA), Section of Pharmacology and Toxicology and Section of Pharmaceutical Sciences , University of Florence , Florence , Italy.

Carbonic anhydrase (CA, EC 4.2.1.1) IX is regarded as a tumour hypoxia marker and CA inhibitors have been proposed as a new class of antitumor agents, with one such agent in Phase II clinical trials. The expression of some CAs, in particular the isoforms CA IX and CA XII, has been correlated with tumour aggressiveness and progression in several cancers. The aim of this study was to evaluate the possibility that CA IX could represent a marker related to clear cell Renal Cell Carcinoma (ccRCC). Bcl-2 and Bax, and the activity of caspase-3, evaluated in tissue biopsies from patients, were congruent with resistance to apoptosis in ccRCCs with respect to healthy controls, respectively. In the same samples, the CA IX and pro-angiogenic factor VEGF expressions revealed that both these hypoxia responsive proteins were strongly increased in ccRCC with respect to controls. CA IX plasma concentration and CA activity were assessed in healthy volunteers and patients with benign kidney tumours and ccRCCs. CA IX expression levels were found strongly increased only in plasma from ccRCC subjects, whereas, CA activity was found similarly increased both in plasma from ccRCC and benign tumour patients, compared to healthy volunteers. These results show that the plasmatic level of CA IX, but not the CA total activity, can be considered a diagnostic marker of ccRCCs. Furthermore, as many reports exist relating CA IX inhibition to a better outcome to anticancer therapy in ccRCC, plasma levels of CA IX could be also predictive for response to therapy.
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http://dx.doi.org/10.1080/14756366.2017.1411350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7011953PMC
December 2018

-Dinitroalkyl Benzenes: A Novel Class of IOP-Lowering Agents for the Treatment of Ocular Hypertension.

ACS Med Chem Lett 2017 Oct 13;8(10):1054-1059. Epub 2017 Sep 13.

Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino, Via P. Giuria 9, 10125 Turin, Italy.

Primary open angle glaucoma is the second most common cause of blindness worldwide. Nitric oxide has recently received particular attention as a potential antiglaucoma agent. In this work, -dinitroalkyl benzenes are evaluated for their capability to act as a new class of IOP lowering agents. These derivatives have been endowed with a variety of NO-release capacities and found to relax contracted rat aorta strips in a concentration-dependent manner. They have been studied for their IOP-lowering activity in a transient ocular hypertensive rabbit model at 1% dose. The most effective IOP-lowering products were compounds - and , whose activity was similar to that of Molsidomine 120 min after administration. Compounds and were selected for evaluation using carbomer-induced glaucoma as the chronic model of IOP. They cause a significant reduction in IOP in the first 24 h, and their activity is maintained over 5 days, displaying a Molsidomine-like profile.
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http://dx.doi.org/10.1021/acsmedchemlett.7b00264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5641960PMC
October 2017

Role of TGFBR1 and TGFBR2 genetic variants in Marfan syndrome.

J Vasc Surg 2018 07 26;68(1):225-233.e5. Epub 2017 Aug 26.

Department of Experimental and Clinical Medicine, Section of Critical Medical Care and Medical Specialities, University of Florence, Florence, Italy; Marfan Syndrome and Related Disorders Regional (Tuscany) Referral Center, Careggi Hospital, Florence, Italy; Center of Excellence for the Study at Molecular and Clinical Level of Chronic, Degenerative and Neoplastic Diseases to Develop Novel Therapies, DENOTHE Center, University of Florence, Florence, Italy; Atherothrombotic Diseases Center, Careggi Hospital, Florence, Italy. Electronic address:

Objective: Genetic variants in transforming growth factor beta (TGF-β) receptors type 1 (TGFBR1) and type 2 (TGFBR2) genes have been associated with different hereditary connective tissue disorders sharing thoracic aortic aneurysm and dissection (TAA/D). Mutations in both TGFBR1/2 genes have been described in patients with TAA/D and Marfan syndrome (MFS), and they are associated consistently with Loeys-Dietz syndrome. The existing literature shows discordant data resulting from mutational screening of TGFBR1/2 genes in patients with MFS. The aim of the study was to investigate the role of TGFBR1/2 genetic variants in determining and/or modulating MFS clinical phenotype.

Methods: We investigated 75 unrelated patients with MFS referred to the Center for Marfan Syndrome and Related Disorders (Careggi University Hospital, Florence) who were subjected to FBN1 and TGFBR1/2 Sanger mutational screening.

Results: Forty-seven patients with MFS (63%) carried a pathogenetic FBN1 mutation. No pathogenetic mutations were detected in TGFBR1/2 genes. Ten common polymorphisms were identified in TGFBR2 and 6 in TGFBR1. Their association with cardiovascular manifestations was evaluated. Carriers of the A allele of rs11466512, delA allele of c.383delA or delT allele of c.1256-15del1T polymorphisms had a trend toward or significantly reduced z-scores (median [interquartile range (IQR)], 2.2 [1.13-4.77]; 2.1 [1.72-3.48]; 2.5 [1.85-3.86]) with respect to homozygous patients with wild-type MFS (median [IQR], 4.20 [2.39-7.25]; 3.9 [2.19-7.00]; 3.9 [2.14-6.93]). Carriers of the A allele of the rs2276767 polymorphism showed a trend toward increased z-score (median [IQR], 4.9 [2.14-7.16]) with respect to patients with wild-type MFS (median [IQR], 3.3 [1.75-5.45]). The protective effect of TGFBR1/2 genetic score including all the 4 variants was also evaluated. Patients with MFS with two or more protective alleles included in the score had statistically significant reduced aortic z-scores (median [IQR], 2.20 [1.48-3.37]) with respect to patients with 1 or no protective alleles (median [IQR], 4.20 [2.48-7.12]; P = .007). Patients with severe aortic manifestations (aortic z-score ≥ 2 or aortic surgery) showed a significantly lower prevalence of subjects with two or more protective alleles included in the genetic score (29.7%) than patients with no or milder cardiovascular involvement (63.6%; P = .029). The genetic score protective effect on global aortic manifestations severity (aortic z-score ≥ 2 or aortic surgery) was also observed at the logistic regression analysis adjusted for the presence of FBN1 gene mutations (odds ratio, 0.21; 95% CI, 0.05-0.84; P = .028).

Conclusions: In conclusion, our data reappraise the role of TGFBR1 and TGFBR2 as major genes in patients with MFS, and suggest that TGFBR1/2 genetic variants (in particular when evaluated as a burden by score) might play a role in modulating the severity of cardiovascular manifestation in MFS.
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http://dx.doi.org/10.1016/j.jvs.2017.04.071DOI Listing
July 2018

1,3-Oxazole-based selective picomolar inhibitors of cytosolic human carbonic anhydrase II alleviate ocular hypertension in rabbits: Potency is supported by X-ray crystallography of two leads.

Bioorg Med Chem 2017 09 11;25(17):4560-4565. Epub 2017 Jul 11.

Institute of Chemistry, Saint Petersburg State University, Peterhof 198504, Russian Federation. Electronic address:

Two lead 1,3-oxazole-based carbonic anhydrase inhibitors (CAIs) earlier identified as selective, picomolar inhibitors of hCA II (a cytosolic target for treatment of glaucoma) have been investigated further. Firstly, they were found to be conveniently synthesized on multigram scale, which enables further development. These compounds were found to be comparable in efficacy to dorzolamide eye drops when applied in the eye drop form as well. Finally, the reasons for unusually high potency of these compounds became understood from their high-resolution X-ray crystallography structures. These data significantly expand our understanding of heterocycle-based primary sulfonamides, many of which have recently emerged from our labs - particularly, from the corneal permeability standpoint.
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http://dx.doi.org/10.1016/j.bmc.2017.06.054DOI Listing
September 2017

The Inhibitory Effects of HYDAMTIQ, a Novel PARP Inhibitor, on Growth in Human Tumor Cell Lines With Defective DNA Damage Response Pathways.

Oncol Res 2017 Nov 20;25(9):1441-1451. Epub 2017 Apr 20.

The poly(ADP-ribose) polymerase (PARP) enzymes play a key role in the regulation of cellular processes (e.g., DNA damage repair, genomic stability). It has been shown that PARP inhibitors (PARPIs) are selectively cytotoxic against cells having dysfunctions in genes involved in DNA repair mechanisms (synthetic lethality). Drug-induced PARP inhibition potentiates the activity of anticancer drugs such as 5-fluorouracil in enhancing DNA damage, whose repair involves PARP-1 activity. The aim of this study was to evaluate the inhibitory effects of a novel PARPI, HYDAMTIQ, on growth in human tumor cell lines characterized by different features with regard to DNA damage response pathways (BRCA mutational status, microsatellite status, and ATM expression level) and degree of sensitivity/resistance to 5-fluorouracil. HYDAMTIQ showed a more potent inhibitory effect on cell growth in a BRCA2 mutant cell line (CAPAN-1) compared with wild-type cells (C2-6, C2-12, and C2-14 CAPAN-1 clones, and MCF-7). No statistically significant difference was observed after HYDAMTIQ exposure between cells having a different MS status or a different MRE11 mutational status. HYDAMTIQ induced greater antiproliferative effects in SW620 cells expressing a low level of ATM than in H630 cells expressing a high level of ATM. Finally, the combination of HYDAMTIQ and 5-fluorouracil exerted a synergistic effect on the inhibition of SW620 cell growth and an antagonistic effect on that of H630 cell growth. Our results show that the novel PARP inhibitor HYDAMTIQ potently inhibits the growth of human tumor cells with defective DNA damage response pathways and exerts synergistic cytotoxicity in combination with 5-fluorouracil. These data provide relevant examples of synthetic lethality and evidence for further development of this novel PARPI.
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http://dx.doi.org/10.3727/096504017X14926854178616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7841208PMC
November 2017

Histamine H receptor agonist-induced relief from painful peripheral neuropathy is mediated by inhibition of spinal neuroinflammation and oxidative stress.

Br J Pharmacol 2017 01 18;174(1):28-40. Epub 2016 Nov 18.

Department of Neurosciences, Psychology, Drug Research and Child Health (NEUROFARBA), Section of Pharmacology, University of Florence, Florence, Italy.

Background And Purpose: Neuropathic pain is under-treated, with a detrimental effect on quality of life, partly because of low treatment efficacy, but also because pathophysiological mechanisms are not fully elucidated. To clarify the pathobiology of neuropathic pain, we studied the contribution of neuroinflammation and oxidative stress in a model of peripheral neuropathy. We also assessed an innovative treatment for neuropathic pain by investigating the effects of histamine H receptor ligands in this model.

Experimental Approach: A peripheral mononeuropathy was induced in mice, by spared nerve injury (SNI). Neuroinflammation and oxidative stress parameters were evaluated by spectrophotometry. The mechanical (von Frey test) and thermal (plantar test) nociceptive thresholds were evaluated.

Key Results: SNI mice showed increased expression of the pro-inflammatory cytokines IL-1ß and TNF-α, decreased antioxidant enzyme Mn-containing SOD (MnSOD), increased levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), an indicator of oxidative DNA damage, and of PARP, nuclear enzyme activated upon DNA damage. Intrathecal administration of VUF 8430 (H receptor agonist) reversed the mechanical and thermal allodynia and was associated with decreased expression of IL-1ß, TNF-α, 8-OHdG and PARP and with restoration of MnSOD activity in the spinal cord and sciatic nerve. These effects were prevented by JNJ 10191584 (H receptor antagonist).

Conclusion And Implications: In the SNI mouse model of neuropathic pain, neuronal H receptor stimulation counteracts hyperalgesia and reduces neuroinflammation and oxidative stress in the spinal cord and sciatic nerve. Targeting both oxidative stress and pro-neuroinflammatory pathways through H receptor-mediated mechanisms could have promising therapeutic potential for neuropathic pain management.
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http://dx.doi.org/10.1111/bph.13644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341487PMC
January 2017

HYDAMTIQ, a selective PARP-1 inhibitor, improves bleomycin-induced lung fibrosis by dampening the TGF-β/SMAD signalling pathway.

J Cell Mol Med 2017 02 4;21(2):324-335. Epub 2016 Oct 4.

Department of Neuroscience, Psychiatry, Drug Area and Child Health (NEUROFARBA), Section of Pharmacology and Toxicology, University of Florence, Florence, Italy.

Idiopathic pulmonary fibrosis is a severe disease characterized by excessive myofibroblast proliferation, extracellular matrix and fibrils deposition, remodelling of lung parenchyma and pulmonary insufficiency. Drugs able to reduce disease progression are available, but therapeutic results are unsatisfactory; new and safe treatments are urgently needed. Poly(ADP-ribose) polymerases-1 (PARP-1) is an abundant nuclear enzyme involved in key biological processes: DNA repair, gene expression control, and cell survival or death. In liver and heart, PARP-1 activity facilitates oxidative damage, collagen deposition and fibrosis development. In this study, we investigated the effects of HYDAMTIQ, a potent PARP-1 inhibitor, in a murine model of lung fibrosis. We evaluated the role of PARP on transforming growth factor-β (TGF-β) expression and TGF-β/SMAD signalling pathway in lungs. Mice were intratracheally injected with bleomycin and then treated with either vehicle or different doses of HYDAMTIQ for 21 days. Airway resistance to inflation and lung static compliance, markers of lung stiffness, were assayed. Histochemical and biochemical parameters to evaluate TGF-β/SMAD signalling pathway with alpha-smooth muscle actin (αSMA) deposition and the levels of a number of inflammatory markers (tumour necrosis factor-α, interleukin-1β, iNOS and COX-2) were performed. Bleomycin administration increased lung stiffness. It also increased lung PARP activity, TGF-β levels, pSMAD3 expression, αSMA deposition and content of inflammatory markers. HYDAMTIQ attenuated all the above-mentioned physiological, biochemical and histopathological markers. Our findings support the proposal that PARP inhibitors could have a therapeutic potential in reducing the progression of signs and symptoms of the disease by decreasing TGF-β expression and the TGF-β/SMAD transduction pathway.
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http://dx.doi.org/10.1111/jcmm.12967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5264150PMC
February 2017

Role of histamine H4 receptor ligands in bleomycin-induced pulmonary fibrosis.

Pharmacol Res 2016 09 27;111:740-748. Epub 2016 Jul 27.

Departments of NEUROFARBA, Section of Pharmacology, University of Florence, Florence, Italy. Electronic address:

Fibrosis of lung tissue is a disease where a chronic inflammatory process determines a pathological remodelling of lung parenchyma. The animal model obtained by intra-tracheal administration of bleomycin in C57BL/6 mice is one of the most validated murine model. Bleomycin stimulates oxidative stress and the production of pro-inflammatory mediators. Histamine H4R have recently been implicated in inflammation and immune diseases. This study was focused to investigate the effects of H4R ligands in the modulation of inflammation and in the reduction of lung fibrosis in C57BL/6 mice treated with bleomycin. C57BL/6 mice were treated with vehicle, JNJ7777120 (JNJ, selective H4R antagonist) or ST-1006 (partial H4R agonist), ST-994 (H4R neutral antagonist) and ST-1012 (inverse H4R agonist) at equimolar doses, released by micro-osmotic pumps for 21days. Airway resistance to inflation was assayed and lung samples were processed to measure malondialdehyde (TBARS); 8-hydroxy-2'-deoxyguanosine (8OHdG); myeloperoxidase (MPO); COX-2 expression and activity as markers of oxidative stress and inflammation. Fibrosis and airway remodelling were evaluated throughout transforming growth factor-β (TGF-β), percentage of positive Goblet cells, smooth muscle layer thickness determination. Our results indicated that JNJ, ST-994 and ST-1012 decreased inflammation and oxidative stress markers, i.e. the number of infiltrating leukocytes evaluated as lung tissue MPO, COX-2 expression and activity, TBARS and 8OHdG production. They also reduced the level of TGF-β, a pro-fibrotic cytokine, collagen deposition, thickness of smooth muscle layer, Goblet cells hyperplasia; resulting in a decrease of airway functional impairment. The results here reported clearly demonstrated that H4R ligands have a beneficial effect in a model of lung fibrosis in the mouse, thus indicating that H4R antagonists or inverse agonists could be a novel therapeutic strategy for lung inflammatory diseases.
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http://dx.doi.org/10.1016/j.phrs.2016.07.037DOI Listing
September 2016

Protection from Cigarette Smoke-Induced Lung Dysfunction and Damage by H2 Relaxin (Serelaxin).

J Pharmacol Exp Ther 2016 06 5;357(3):451-8. Epub 2016 Apr 5.

Anatomy and Histology Section and Histology and Embryology Research Unit, Department of Experimental and Clinical Medicine (A.P., G.B., S.C., D.G., D.B., S.N.), and Pharmacology Section, Department NEUROFARBA (L.L., E.M.), University of Florence, Florence, Italy

Cigarette smoke (CS) is the major etiologic factor of chronic obstructive pulmonary disease (COPD), which is characterized by airway remodeling, lung inflammation and fibrosis, emphysema, and respiratory failure. The current therapies can improve COPD management but cannot arrest its progression and reduce mortality. Hence, there is a major interest in identifying molecules susceptible of development into new drugs to prevent or reduce CS-induced lung injury. Serelaxin (RLX), or recombinant human relaxin-2, is a promising candidate because of its anti-inflammatory and antifibrotic properties highlighted in lung disease models. Here, we used a guinea pig model of CS-induced lung inflammation, and remodeling reproducing some of the hallmarks of COPD. Animals exposed chronically to CS (8 weeks) were treated with vehicle or RLX, delivered by osmotic pumps (1 or 10 μg/day) or aerosol (10 μg/ml/day) during CS treatment. Controls were nonsmoking animals. RLX maintained airway compliance to a control-like pattern, likely because of its capability to counteract lung inflammation and bronchial remodeling. In fact, treatment of CS-exposed animals with RLX reduced the inflammatory recruitment of leukocytes, accompanied by a significant reduction of the release of proinflammatory cytokines (tumor necrosis factor α and interleukin-1β). Moreover, RLX was able to counteract the adverse bronchial remodeling and emphysema induced by CS exposure by reducing goblet cell hyperplasia, smooth muscle thickening, and fibrosis. Of note, RLX delivered by aerosol has shown a comparable efficacy to systemic administration in reducing CS-induced lung dysfunction and damage. In conclusion, RLX emerges as a new molecule to counteract CS-induced inflammatory lung diseases.
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http://dx.doi.org/10.1124/jpet.116.232215DOI Listing
June 2016

Histamine H4 receptor activation alleviates neuropathic pain through differential regulation of ERK, JNK, and P38 MAPK phosphorylation.

Pain 2015 Dec;156(12):2492-2504

Department of Neuroscience, Psychology, Drug Research and Child Health (NEUROFARBA), Section of Pharmacology, University of Florence, Florence, Italy Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Frankfurt, Germany.

Histamine plays a complex role in pain modulation with opposite roles in nociception for histamine receptor subtypes 1, 2, and 3. The histamine H4 receptor (H4R) is expressed primarily on cells involved in inflammation and immune responses with a proinflammatory activity, but little is known about the role in nociception of neuronal H4R. To investigate the effects of neuronal H4R in pain transmission, the effects produced by the H4R agonist ST-1006 were detected in the spared nerve injury model of neuropathic pain. ST-1006 counteracted mechanical allodynia in neuropathic mice, an effect prevented by the H4R antagonist JNJ 10191584. In spared nerve injury mice, an early over-phosphorylation of ERK1 and ERK2 was observed in the dorsal root ganglia (DRG), spinal cord, and sciatic nerve. A progressive and long-lasting activation of JNK1 was observed in the sciatic nerve and, to a lesser extent, in the spinal cord and DRG. An increased p-P38 content was detected in the spinal cord and DRG, with no modification in the sciatic nerve. Administration of ST-1006 prevented phosphorylation of all 3 MAPK within DRG, and phosphorylation of ERK1, ERK2, and pJNK1 in the sciatic nerve. In the spinal cord, the H4R agonist prevented selectively the pERK2 increase with no effect on pJNK1 and p-P38 levels. Double immunofluorescence experiments showed a neuronal localization and site of action for H4R. These findings suggest a prevalent modulation of ERK activity after H4R stimulation and indicate the DRG as prominent site of action for H4R-mediated antineuropathic activity. Targeting neuronal H4R with selective agonists could have therapeutic potential for neuropathic pain treatment.
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http://dx.doi.org/10.1097/j.pain.0000000000000319DOI Listing
December 2015

Structural insights on carbonic anhydrase inhibitory action, isoform selectivity, and potency of sulfonamides and coumarins incorporating arylsulfonylureido groups.

J Med Chem 2014 Nov 17;57(21):9152-67. Epub 2014 Oct 17.

Laboratorio di Chimica Bioinorganica, Polo Scientifico, Università degli Studi di Firenze , Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy.

Sulfonamides and coumarins incorporating arylsulfonylureido tails were prepared and assayed as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). Some derivatives incorporating 3-pyridinesulfonamide and arylsulfonylureoido fragments were low nanomolar inhibitors of isoforms CA II and XII (upregulated or overexpressed in glaucoma) and showed effective in vivo intraocular pressure lowering effects in an animal model of the disease, which were several times better compared to those of the antiglaucoma drug dorzolamide. By means of X-ray crystallography of adducts of several sulfonamides with CA II, the effective inhibitory properties were rationalized at the molecular level. The coumarins were ineffective as hCA I and II inhibitors but showed low nanomolar activity for the inhibition of the tumor-associated isoforms hCA IX and XII. The presence of arylsulfonylureido tails in these CA inhibitors possessing quite different mechanisms of action led to highly effective and isoform-selective compounds targeting enzymes involved in severe pathologies such as glaucoma or cancer.
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http://dx.doi.org/10.1021/jm501314cDOI Listing
November 2014

Prevention of bleomycin-induced lung inflammation and fibrosis in mice by naproxen and JNJ7777120 treatment.

J Pharmacol Exp Ther 2014 Nov 2;351(2):308-16. Epub 2014 Sep 2.

Departments of Drug Science and Technology, University of Turin, Turin, Italy (A.C.R., E.V.); Department of Neuroscience, Psychiatry, and Drug Research, Section of Pharmacology (L.L., C.L., E.M.), and Experimental and Clinical Medicine (A.P.), University of Florence, Florence, Italy; Janssen Research & Development, L.L.C., San Diego, California (R.L.T.); and Heinrich-Heine Düsseldorf University, Institute of Medicinal Chemistry, Düsseldorf, Germany (H.S.)

Pulmonary fibrosis, a progressive and lethal lung disease characterized by inflammation and accumulation of extracellular matrix components, is a major therapeutic challenge for which new therapeutic strategies are warranted. Cyclooxygenase (COX) inhibitors have been previously utilized to reduce inflammation. Histamine H4 receptor (H4R), largely expressed in hematopoietic cells, has been identified as a novel target for inflammatory and immune disorders. The aim of this study was to evaluate the effect of JNJ7777120 (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine), a selective H4R antagonist, and naproxen, a well known nonsteroidal anti-inflammatory drug, and their combination in a murine model of bleomycin-induced fibrosis. Bleomycin (0.05 IU) was instilled intratracheally to C57BL/6 mice, which were then treated by micro-osmotic pump with vehicle, JNJ7777120 (40 mg/kg b.wt.), naproxen (21 mg/kg b.wt.), or a combination of both. Airway resistance to inflation, an index of lung stiffness, was assessed, and lung specimens were processed for inflammation, oxidative stress, and fibrosis markers. Both drugs alone were able to reduce the airway resistance to inflation induced by bleomycin and the inflammatory response by decreasing COX-2 and myeloperoxidase expression and activity and thiobarbituric acid-reactive substance and 8-hydroxy-2'-deoxyguanosine production. Lung fibrosis was inhibited, as demonstrated by the reduction of tissue levels of transforming growth factor-β, collagen deposition, relative goblet cell number, and smooth muscle layer thickness. Our results demonstrate that both JNJ7777120 and naproxen exert an anti-inflammatory and antifibrotic effect that is increased by their combination, which could be an effective therapeutic strategy in the treatment of pulmonary fibrosis.
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http://dx.doi.org/10.1124/jpet.114.215152DOI Listing
November 2014

Poly(ADP-ribose) polymerase inhibition with HYDAMTIQ reduces allergen-induced asthma-like reaction, bronchial hyper-reactivity and airway remodelling.

J Cell Mol Med 2014 Mar 20;18(3):468-79. Epub 2014 Jan 20.

Department of NEUROFARBA, Section of Pharmacology and Toxicology, University of Florence, Florence, Italy.

Activation of poly(ADP-ribose) polymerases (PARPs) is considered a key event in the molecular and cellular processes leading from acute asthma attacks to bronchial hyper-reactivity, leucocyte recruitment, chronic inflammation, airway remodelling and lung damage. The present investigation has been carried out to investigate the action of hydroxyl-dimethylaminomethyl-thieno[2,3-c]isoquinolin-5(4H)-one (HYDAMTIQ), a new potent PARP inhibitor, in the process leading from asthma-like events to airway damage. Ovalbumin-sensitized guinea pigs exposed two times to allergen inhalation were treated for 8 days with vehicle or HYDAMTIQ. Asthma-like signs, bronchial hyper-reactivity to methacholine, cytokine production, histamine release from mast cells, airway remodelling, collagen deposition and lung damage were evaluated. Repeated HYDAMTIQ administration (1-10 mg/kg/day i.p.) reduced lung PARP activity, delayed the appearance and reduced the severity of allergen-induced cough and dyspnoea and dampened the increased bronchial responses to methacholine. HYDAMTIQ-treated animals presented reduced bronchial or alveolar abnormalities, lower number of eosinophils and other leucocytes in the lung and decreased smooth muscle or goblet cell hyperplasia. The treatment also reduced lung oxidative stress markers, such as malondialdehyde or 8-hydroxy-2'-deoxyguanosine and the lung content of pro-inflammatory cytokines (TNF-α, interleukin (IL)-1β, IL-5, IL-6 and IL-18). Finally, mast cells isolated from the peritoneal or pleural cavities of sensitized, HYDAMTIQ-treated animals had a reduced ability to release histamine when exposed to ovalbumin in vitro. Our findings support the proposal that PARP inhibitors could have a therapeutic potential to reduce chronic lung inflammation, airway damage and remodelling in severe unresponsive asthmatic patients.
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http://dx.doi.org/10.1111/jcmm.12197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3955153PMC
March 2014

Cyclooxygenase-2 and inflammation mediators have a crucial role in reflux-related esophageal histological changes and Barrett's esophagus.

Dig Dis Sci 2014 May 20;59(5):949-57. Epub 2013 Dec 20.

Department of Surgery and Translational Medicine, Section of General Surgery, University of Florence-Careggi General Hospital, AOU Careggi, Largo Brambilla n. 3, 50134, Florence, Italy.

Background: Gastroesophageal reflux (GER) causes injury of the esophageal squamous epithelium, a condition called reflux esophagitis. The sequence reflux-esophagitis-intestinal metaplasia-dysplasia-invasive cancer is widely accepted as the main adenocarcinogenetic pathway in the esophagus; however, the mechanisms of this progression need to be better defined.

Aims: We evaluated COX-2 expression and activity in biopsies from patients affected with GER, and these parameters have been correlated with the stage of the disease, ceramide expression, apoptotic process, and angiogenesis. The effects of celecoxib on bile acid- and EGF-induced mucosal proliferation, apoptosis and angiogenesis have been also investigated.

Methods: Four groups of patients were distinguished: non esophagitis, non erosive esophagitis, erosive esophagitis, and Barrett's esophagus. COX-2 expression, basal PGE2 levels, proliferative activity, VEGF expression and apoptosis were evaluated in esophageal biopsies.

Results: COX-2 expression, basal PGE2 levels, proliferative activity, VEGF expression and apoptosis progressively increase from non esophagitis patients to patients with non erosive and erosive esophagitis, to those with BE. Incubation of the cells with DCA/EGF increases PGE2 production, proliferative activity and VEGF production, effects prevented by celecoxib pretreatment. Ceramide expression increased from non esophagitis patients to patients with non erosive and erosive esophagitis, and decreased in BE; caspase-3 activity progressively decreased from non esophagitis to BE patients, suggesting an impairment of the apoptotic process with disease progression.

Conclusion: These results stand for a close relationship between progression of initial steps of gastroesophageal reflux disease (GERD) and COX-2, proliferative activity and EGF/VEGF expression and could have implications in GERD treatment in order to prevent its neoplastic evolution.
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http://dx.doi.org/10.1007/s10620-013-2975-4DOI Listing
May 2014

Fibroblast autofluorescence in connective tissue disorders: a future tool for clinical and differential diagnosis?

J Biomed Opt 2008 Sep-Oct;13(5):054025

University of Florence, Department of Clinical Physiopathology, ASAcampus, ASA Research Division, Florence, Italy.

Marfan syndrome (MFS) is an inherited disorder of connective tissue due to mutations in FBN1 (90%) and TGFBR1 and TGFBR2 (5 to 10%) genes. Clinical and differential diagnosis is difficult because of the inter- and intrafamiliar marked heterogeneity and the variable onset age of clinical manifestations. Among the disorders, in differential diagnosis, thoracic aortic aneurysm (TAA) and Ullrich scleroatonic muscular dystrophy (UCMD) are reported. We evaluate the possibility of utilizing autofluorescence (AF) analysis as a diagnostic tool in the clinical and/or differential diagnosis of MFS and related disorders and in the investigation of the molecular mechanisms involved. Both multispectral imaging autofluorescence microscopy (MIAM) and autofluorescence microspectroscopy (AMS) have been used to characterize AF emission of fibroblasts from patients affected by inherited connective tissue disorders. Our preliminary results show significant differences in AF emission between normal and pathological fibroblasts, suggesting possible improvement in diagnostics of connective tissue disorders by AF analysis.
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http://dx.doi.org/10.1117/1.2982533DOI Listing
February 2009