Publications by authors named "Domenico Tricarico"

55 Publications

Oxtr/TRPV1 expression and acclimation of skeletal muscle to cold-stress in male mice.

J Endocrinol 2021 May;249(2):135-148

Department of Pharmacy-Pharmaceutical Sciences, University of Bari Aldo Moro, Bari, Italy.

We explored the involvement of oxytocin receptor (Oxtr)/transient-receptor-potential-vanilloid-1 (TRPV1) genes and oxytocin (Oxt) on the adaptation of skeletal muscle to cold stress challenge in mice. Oxtr expression in hypothalamic paraventricular (PVN), supraoptic nuclei (SON), and hippocampus (HIPP) were evaluated by immunohistochemistry in parallel with the measurement of circulating Oxt. The Oxtr and TRPV1 gene expressions in soleus (SOL) and tibialis anterior (TA) muscles were investigated by RT-PCR. Histological studies of the cardiac muscle after cold stress were also performed. Male mice (n = 15) were divided into controls maintained at room temperature (RT = 24°C), exposed to cold stress (CS) at T = 4°C for 6 h , and 5 days. Immunohistochemical studies showed that Oxtr protein expression increased by two-fold (P = 0.01) in PVN and by 1.5-fold (P = 0.0001) in HIPP after 6 h- and 5 days of CS but decreased by 2-fold (P = 0.026) in SON in 5 days. Both Oxtr and TRPV1 gene expression increased after 6 h and 5 days of CS in SOL and TA muscles. Oxtr vs TRPV1 gene expression in SOL and TA muscles evaluated by regression analysis was linearly correlated following CS at 6 h and 5 days but not at control temperature of 24 ± 1°C, supporting the hypothesis of coupling between these genes. The circulating levels of Oxt are unaffected after 6 h of CS but decreased by 0.2-fold (P = 0.0141) after 5 days-CS. This is the first report that Oxtr and TRPV1 expressions are upregulated in response to cold acclimation in skeletal muscle. The up-regulation of Oxtr in PVN and HIPP balances the decrease of circulating Oxt.
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http://dx.doi.org/10.1530/JOE-20-0346DOI Listing
May 2021

Peripheral Neuropathy under Oncologic Therapies: A Literature Review on Pathogenetic Mechanisms.

Int J Mol Sci 2021 Feb 17;22(4). Epub 2021 Feb 17.

Interventional and Medical Oncology Unit, IRCCS Istituto Tumori "Giovanni Paolo II", Viale Orazio Flacco65, 70124 Bari, Italy.

Peripheral neurologic complications are frequent adverse events during oncologic treatments and often lead to dose reduction, administration delays with time elongation of the therapeutic plan and, not least, worsening of patients' quality of life. Experience skills are required to recognize symptoms and clinical evidences and the collaboration between different health professionals, in particular oncologists and hospital pharmacists, grants a correct management of this undesirable occurrence. Some classes of drugs (platinates, vinca alkaloids, taxanes) typically develop this kind of side effect, but the genesis of chemotherapy-induced peripheral neuropathy is not linked to a single mechanism. This paper aims from one side at summarizing and explaining all the scattering mechanisms of chemotherapy-induced peripheral neuropathy through a detailed literature revision, on the other side at finding new approaches to possible treatments, in order to facilitate the collaboration between oncologists, hematologists and hospital pharmacists.
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http://dx.doi.org/10.3390/ijms22041980DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7922628PMC
February 2021

Analysis of the Introduction in Clinical Practice of New Oral Anticoagulants in Local Health Agency BT: Translation of the Clinical Trial Data to a Local Health Care Area.

Pharmaceutics 2021 Feb 11;13(2). Epub 2021 Feb 11.

Local Health Agency BT, via Fornaci 201, 76123 Andria, Italy.

The commercial release of the New Oral Anticoagulants (NOACs) has been the most significant change in anticoagulant therapy in recent years. The work aimed to evaluate the economic and health impact for the Local Health Agency Barletta-Andria-Trani (BT). Through the Regional Information System data about naïve patients on NOAC treatment and patients on anti-vitamin-k (VKA), treatments were extrapolated. We assessed therapeutic continuity, pharmaceutical expenditure, hospitalizations, and deaths in 2017 and 2018. Therapeutic continuity was similar in the two groups. The number and the average cost of hospitalizations for a patient treated with VKAs were almost constant, while those of patients treated with NOACs decreased. The treatment of adult-aged naïve patients with NOACs, compared to VKAs therapy, involves an increase in expenditure of about 100€ for a patient, but the reduced hospitalizations could generate, in the long term, saving for the Health System. Clinical data, according to the Real-World Data, confirmed the safety and effectiveness of these drugs. However, attention to the special population is necessary to improve the safety and effectiveness of NOACs. Innovative formulations for pediatric patients are being developed. The challenge for Health Systems is the appropriate use of available resources through health interventions with transversal competences.
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http://dx.doi.org/10.3390/pharmaceutics13020252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918324PMC
February 2021

ATP-sensitive potassium channel subunits in the neuroinflammation: novel drug targets in neurodegenerative disorders.

CNS Neurol Disord Drug Targets 2021 Jan 18. Epub 2021 Jan 18.

Department of Pharmacy-Pharmaceutical Science, University of Bari Aldo Moro, via Orabona 4, 70125-I. Italy.

Arachidonic acids and its metabolites modulate plenty of ligand-gated, voltage-dependent ion channels, and metabolically regulated potassium channels including ATP-sensitive potassium channels (KATP). KATP channels are hetero-multimeric complexes of sulfonylureas receptors (SUR1, SUR2A or SUR2B) and the pore-forming subunits (Kir6.1 and Kir6.2) likewise expressed in the pre-post synapsis of neurons and inflammatory cells, thereby affecting their proliferation and activity. KATP channels are involved in amyloid-β (Aβ)-induced pathology, therefore emerging as therapeutic targets against Alzheimer's and related diseases. The modulation of these channels can represent an innovative strategy for the treatment of neurodegenerative disorders; nevertheless, the currently available drugs are not selective for brain KATP channels and show contrasting effects. This phenomenon can be a consequence of the multiple physiological roles of the different varieties of KATP channels. Openings of cardiac and muscular KATP channel subunits, is protective against caspase-dependent atrophy in these tissues and some neurodegenerative disorders, whereas in some neuroinflammatory diseases benefits can be obtained through the inhibition of neuronal KATP channel subunits. For example, glibenclamide exerts an anti-inflammatory effect in respiratory, digestive, urological, and central nervous system (CNS) diseases, as well as in ischemia-reperfusion injury associated with abnormal SUR1-Trpm4/TNF-α or SUR1-Trpm4/ Nos2/ROS signaling. Despite this strategy is promising, glibenclamide may have limited clinical efficacy due to its unselective blocking action of SUR2A/B subunits also expressed in cardiovascular apparatus with pro-arrhythmic effects and SUR1 expressed in pancreatic beta cells with hypoglycemic risk. Alternatively, neuronal selective dual modulators showing agonist/antagonist actions on KATP channels can be an option.
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http://dx.doi.org/10.2174/1871527320666210119095626DOI Listing
January 2021

Pathophysiological Consequences of KATP Channel Overactivity and Pharmacological Response to Glibenclamide in Skeletal Muscle of a Murine Model of Cantù Syndrome.

Front Pharmacol 2020 30;11:604885. Epub 2020 Nov 30.

Section of Pharmacology, Department of Pharmacy-Pharmaceutical Sciences, University of Bari "Aldo Moro", Bari, Italy.

Cantù syndrome (CS) arises from mutations in and genes that lead to gain of function (GOF) of ATP-sensitive potassium (KATP) channels containing SUR2A and Kir6.1 subunits, respectively, of KATP channels. Pathological consequences of CS have been reported for cardiac and smooth muscle cells but consequences in skeletal muscle are unknown. Children with CS show muscle hypotonia and adult manifest fatigability. We analyzed muscle properties of Kir6.1[V65M] CS mice, by measurements of forelimb strength and ultrasonography of hind-limb muscles, as well as assessing KATP channel properties in native (FDB) and (SOL) fibers by the patch-clamp technique in parallel with histopathological, immunohistochemical and Polymerase Chain Reaction (PCR) analysis. Forelimb strength was lower in Kir6.1 mice than in WT mice. Also, a significant enhancement of echodensity was observed in hind-limb muscles of Kir6.1 mice relative to WT, suggesting the presence of fibrous tissue. There was a higher KATP channel current amplitude in Kir6.1 FDB fibers relative to WT and a reduced response to glibenclamide. The IC of glibenclamide to block KATP channels in FDB fibers was 1.3 ± 0.2 × 10 M in WT and 1.2 ± 0.1 × 10 M in Kir6.1 mice, respectively; and it was 1.2 ± 0.4 × 10 M in SOL WT fibers but not measurable in Kir6.1 fibers. The sensitivity of the KATP channel to MgATP was not modified in Kir6.1 fibers. Histopathological/immunohistochemical analysis of SOL revealed degeneration plus regressive-necrotic lesions with regeneration, and up-regulation of Atrogin-1, MuRF1, and BNIP3 mRNA/proteins in Kir6.1 mice. Kir6.1 mutation in skeletal muscle leads to changes of the KATP channel response to glibenclamide in FDB and SOL fibers, and it is associated with histopathological and gene expression changes in slow-twitch muscle, suggesting marked atrophy and autophagy.
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http://dx.doi.org/10.3389/fphar.2020.604885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7734337PMC
November 2020

The hydroxypropyl-β-cyclodextrin-minoxidil inclusion complex improves the cardiovascular and proliferative adverse effects of minoxidil in male rats: Implications in the treatment of alopecia.

Pharmacol Res Perspect 2020 06;8(3):e00585

Section of Pharmacology, Department of Pharmacy - Pharmaceutical Sciences, University of Bari, Bari, Italy.

The efficacy of minoxidil (MXD) ethanolic solutions (1%-5% w/v) in the treatment of androgenetic alopecia is limited by adverse reactions. The toxicological effects of repeated topical applications of escalating dose (0.035%-3.5% w/v) and of single and twice daily doses (3.5% w/v) of a novel hydroxypropyl-β-cyclodextrin MXD GEL formulation (MXD/HP-β-CD) and a MXD solution were investigated in male rats. The cardiovascular effects were evaluated by telemetric monitoring of ECG and arterial pressure in free-moving rats. Ultrasonographic evaluation of cardiac morphology and function, and histopathological and biochemical analysis of the tissues, were performed. A pharmacovigilance investigation was undertaken using the EudraVigilance database for the evaluation of the potential cancer-related effects of topical MXD. Following the application of repeated escalating doses of MXD solution, cardiac hypertrophy, hypotension, enhanced serum natriuretic peptides and K -ion levels, serum liver biomarkers, and histological lesions including renal cancer were observed. In addition, the administration of a twice daily dose of MXD solution, at SF rat vs human = 311, caused reductions in the systolic, diastolic, and mean blood pressure of the rats (-30.76 ± 3%, -28.84 ± 4%, and -30.66 ± 5%, respectively, vs the baseline; t test P < .05). These effects were not reversible following washout of the MXD solution. Retrospective investigation showed 32 cases of cancer associated with the use of topical MXD in humans. The rats treated with MXD HP-β-CD were less severely affected. MXD causes proliferative adverse effects. The MXD HP-β-CD inclusion complex reduces these adverse effects.
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http://dx.doi.org/10.1002/prp2.585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7203570PMC
June 2020

Bridging repair of the abdominal wall in a rat experimental model. Comparison between uncoated and polyethylene oxide-coated equine pericardium meshes.

Sci Rep 2020 04 24;10(1):6959. Epub 2020 Apr 24.

University of Bari "A. Moro", Department of Biomedical Sciences and Human Oncology, Unit of Academic General Surgery "V. Bonomo", Bari, Italy.

Biological meshes improve the outcome of incisional hernia repairs in infected fields but often lead to recurrence after bridging techniques. Sixty male Wistar rats undergoing the excision of an abdominal wall portion and bridging mesh repair were randomised in two groups: Group A (N = 30) using the uncoated equine pericardium mesh; Group B (N = 30) using the polyethylene oxide (PEO)-coated one. No deaths were observed during treatment. Shrinkage was significantly less common in A than in B (3% vs 53%, P < 0.001). Adhesions were the most common complication and resulted significantly higher after 90 days in B than in A (90% vs 30%, P < 0.01). Microscopic examination revealed significantly (P < 0.05) higher mesh integrity, fibrosis and calcification in B compared to A. The enzymatic degradation, as assessed with Raman spectroscopy and enzyme stability test, affected A more than B. The PEO-coated equine pericardium mesh showed higher resistance to biodegradation compared to the uncoated one. Understanding the changes of these prostheses in a surgical setting may help to optimize the PEO-coating in designing new biomaterials for the bridging repair of the abdominal wall.
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http://dx.doi.org/10.1038/s41598-020-63886-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7181852PMC
April 2020

Oxytocin/Osteocalcin/IL-6 and NGF/BDNF mRNA Levels in Response to Cold Stress Challenge in Mice: Possible Oxytonic Brain-Bone-Muscle-Interaction.

Front Physiol 2019 27;10:1437. Epub 2019 Nov 27.

Department of Pharmacy-Pharmaceutical Sciences, University of Bari Aldo Moro, Bari, Italy.

Oxytocin (Oxt), osteocalcin (Ost), and NGF/BDNF have a role in bone homeostasis, reproduction, and cognition. Oxt/Ost is required for muscle repair. We investigated gene response of muscle and the inter-organ communication following cold stress (CS). The mRNA quantity of , , , , , , , , , , and genes in bone, brain, soleus (SOL), and (TA) muscles from adult mice following CS were investigated. The myosin heavy-chain , , , and gene expression were investigated. Mice were maintained at = 23°C or 4°C for 6 h and 5-days (5d). CS mice did not show signs of muscle degeneration. An upregulation of and genes by 2 and 1.5 folds, respectively, in TA after 6 h CS and by 4 and 22 folds in SOL muscle after 6 h and 5d CS, respectively, was observed; while after 6 h CS was downregulated in either muscle. was unaffected, while after 5d CS was upregulated in TA. was downregulated in SOL by 0.9-folds at 5d. Following 5d CS, and genes were upregulated, respectively, by 1 and 1.5 folds in SOL. A downregulation of , respectively, by 0.96 and 0.88-folds after 6 h and 5d CS in SOL and was also downregulated by 0.88-fold after 5d CS in TA. and were not affected. Changes in the expression levels of genes in TA and SOL muscles, bone, and brain following CS were regulated by IL6 and Oxt. CS potentiates the slow-twitch phenotype of SOL which is in line with the metabolic need of this muscle, and the potentiation of the slow-twitch phenotype in TA. Oxt and IL6 coordinate a phenotype-dependent tonic effect of slow-twitch muscle and Oxt regulates the inter-organ interaction between brain and SOL muscle. Muscle tropism is maintained by NGF signaling following CS.
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http://dx.doi.org/10.3389/fphys.2019.01437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6890828PMC
November 2019

Thymidine Phosphorylase Expression and Microvascular Density Correlation Analysis in Canine Mammary Tumor: Possible Prognostic Factor in Breast Cancer.

Front Vet Sci 2019 25;6:368. Epub 2019 Oct 25.

Interventional and Medical Oncology Unit, Department of Pathology National Cancer Research Centre, IRCCS Istituto Tumori Giovanni Paolo II, Bari, Italy.

The thymidine phosphorylase (TP) is a key enzyme involved in the metabolism of pyrimidines. Inhibition or downregulation of this enzyme causes accumulation of metabolites with consequences in DNA replication. TP regulates angiogenesis and chemotactic activity of endothelial cells. Different studies showed the presence of TP upregulation in human cancer but the correlation between TP expression and the microvascular density (MVD) in canine mammary tumors is unknown. The aim of this study was to investigate a possible correlation between the MVD and TP expression in tumor cells of canine mammary tumors of different degree of severity (G1-G3) by immunohistochemical analysis. Sixty-eight samples of spontaneous mammary neoplasia of 5-12 cm in diameter were collected from purebred and mixed-breed dogs (mean aged = 9.5 ± 7), not subject to chemotherapy treatments in veterinary clinics. Histopathological analysis and immunostaining were performed. Carcinoma simple samples have been classified as 72.06% of tubule-papillary, 20.59% cysto-papillary, and 7.35% tubular carcinomas. Immunostainings revealed a marked cytoplasmic expression of TP in 30.88% of samples, mild in 32.35%, weaker in 22.07%, and negative in 14.70%. The correlation analysis and two-way ANOVA showed a linear correlation between MVD and TP with a coefficient of correlation () > 0.5 ( < 0.05) in G2 and G3. No correlation between variables was found in G1. These findings suggest that cytoplasmic TP overexpression is correlated with microvascular density in canine mammary tumors, in severe grade, and it can be a potential prognostic factor in breast cancer.
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http://dx.doi.org/10.3389/fvets.2019.00368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6823610PMC
October 2019

Elucidating the Contribution of Skeletal Muscle Ion Channels to Amyotrophic Lateral Sclerosis in search of new therapeutic options.

Sci Rep 2019 02 28;9(1):3185. Epub 2019 Feb 28.

Department of Pharmacy-Drug Sciences, University of Bari Aldo Moro, 70125, Bari, Italy.

The discovery of pathogenetic mechanisms is essential to identify new therapeutic approaches in Amyotrophic Lateral Sclerosis (ALS). Here we investigated the role of the most important ion channels in skeletal muscle of an ALS animal model (MLC/SOD1) carrying a mutated SOD1 exclusively in this tissue, avoiding motor-neuron involvement. Ion channels are fundamental proteins for muscle function, and also to sustain neuromuscular junction and nerve integrity. By a multivariate statistical analysis, using machine learning algorithms, we identified the discriminant genes in MLC/SOD1 mice. Surprisingly, the expression of ClC-1 chloride channel, present only in skeletal muscle, was reduced. Also, the expression of Protein Kinase-C, known to control ClC-1 activity, was increased, causing its inhibition. The functional characterization confirmed the reduction of ClC-1 activity, leading to hyperexcitability and impaired relaxation. The increased expression of ion channel coupled AMPA-receptor may contribute to sustained depolarization and functional impairment. Also, the decreased expression of irisin, a muscle-secreted peptide protecting brain function, may disturb muscle-nerve connection. Interestingly, the in-vitro application of chelerythrine or acetazolamide, restored ClC-1 activity and sarcolemma hyperexcitability in these mice. These findings show that ion channel function impairment in skeletal muscle may lead to motor-neuron increased vulnerability, and opens the possibility to investigate on new compounds as promising therapy.
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http://dx.doi.org/10.1038/s41598-019-39676-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395744PMC
February 2019

Zoledronic Acid Modulation of TRPV1 Channel Currents in Osteoblast Cell Line and Native Rat and Mouse Bone Marrow-Derived Osteoblasts: Cell Proliferation and Mineralization Effect.

Cancers (Basel) 2019 Feb 11;11(2). Epub 2019 Feb 11.

Section of Pharmacology, Department of Pharmacy-Pharmaceutical Sciences, University of Bari, Via Orabona 4, I-70125 Bari, Italy.

Bisphosphonates (BPs) reduce bone pain and fractures by balancing the osteoblast/osteoclast ratio. The behavior of ion channels in the presence of BPs is not known. To investigate this, the effect of zoledronic acid BP (ZOL) (3 × 10 to 5 × 10 M) treatment, on ion channels, cell proliferation, and mineralization, has been investigated on preosteoclast-like cells, RAW264.7, preosteoblast-like cells MC3T3-E1, and rat/mouse native bone marrow-derived osteoblasts. In whole-cell patch clamp on cell line- and bone marrow-derived osteoblasts, ZOL potentiated outward currents. On RAW264.7, ZOL (10 M)-evoked current was reduced by the Kv channel blocker tetraethylammonium hydrochloride (TEA), but not by the selective TRPV1-channel antagonist capsazepine. On MC3T3-E1 cells and bone marrow-derived osteoblasts, ZOL-evoked current (5 × 10 to 10 M) was reduced by capsazepine, whereas the selective TRPV1-channel agonist capsaicin potentiated the control current. In the cell proliferation assay, 72 h incubation of RAW264.7 and MC3T3-E1 cells with ZOL reduced proliferation, with IC values of 2.62 × 10 M and 2.02 × 10 M, respectively. Mineralization of MC3T3-E1 cells and bone marrow-derived osteoblasts was observed in the presence of capsaicin and ZOL (5 × 10⁻10 M); ZOL effects were antagonized by capsazepine. In summary, the ZOL-induced activation of TRPV1 channel mediates the mineralization of osteoblasts and counterbalances the antiproliferative effects, increasing the IC. This mechanism is not operative in osteoclasts lacking the TRPV1 channel.
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http://dx.doi.org/10.3390/cancers11020206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6406412PMC
February 2019

Novel bisphosphonates with antiresorptive effect in bone mineralization and osteoclastogenesis.

Eur J Med Chem 2018 Oct 18;158:184-200. Epub 2018 Aug 18.

Department of Pharmacy - Pharmaceutical Sciences, University of Bari "A. Moro", via E. Orabona 4, 70125, Bari, Italy. Electronic address:

Bisphosphonates such as zoledronic, alendronic and risedronic acids are a class of drugs clinically used to prevent bone density loss and osteoporosis. Novel P-C-P bisphosphonates were synthesized for targeting human farnesyl pyrophosphate synthase (hFPPS) and human geranylgeranyl pyrophosphate synthase (hGGPPS), key enzymes of the mevalonate pathway, and capable of anti-proliferative action on a number of cell lines (PC3, MG63, MC3T3, RAW 264.7, J774A.1, bone marrow cells and their co-colture with PC3) involved in bone homeostasis, bone formation and death. Among sixteen compounds, [1-hydroxy-2-(pyrimidin-2-ylamino)ethane-1,1-diyl]bis(phosphonic acid) (10) was effective in reducing PC3 and RAW 264.7 cell number in crystal-violet and cell-dehydrogenase activity assays at 100 μM concentration. 10 reduced differentiated osteoclasts number similarly with zoledronic acid in osteoclastogenesis assay. At nanomolar concentrations, 10 was more effective than zoledronic acid in inducing mineralization in MC3T3 and murine bone marrow cells. Further, 10 significantly inhibited the activity of hFPPS showing an IC of 0.31 μM and a remarkable hydroxyapatite binding of 90%. Docking calculations were performed identifying putative interactions between some representative novel bisphosphonates and both hFPPS and hGGPPS. Then, 10 was found to behave similarly or even better than zoledronic acid as a anti-resorptive agent.
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http://dx.doi.org/10.1016/j.ejmech.2018.08.044DOI Listing
October 2018

Cell Cycle Regulation by Ca-Activated K⁺ (BK) Channels Modulators in SH-SY5Y Neuroblastoma Cells.

Int J Mol Sci 2018 Aug 18;19(8). Epub 2018 Aug 18.

Section of Pharmacology, Department of Pharmacy-Pharmaceutical Sciences, University of Bari, Via Orabona 4, 70125 Bari, Italy.

The effects of Ca-activated K⁺ (BK) channel modulation by Paxilline (PAX) (10⁻10 M), Iberiotoxin (IbTX) (0.1⁻1 × 10 M) and Resveratrol (RESV) (1⁻2 × 10 M) on cell cycle and proliferation, AKT1p phosphorylation, cell diameter, and BK currents were investigated in SH-SY5Y cells using Operetta-high-content-Imaging-System, ELISA-assay, impedentiometric counting method and patch-clamp technique, respectively. IbTX (4 × 10 M), PAX (5 × 10 M) and RESV (10 M) caused a maximal decrease of the outward K⁺ current at +30 mV (Vm) of -38.3 ± 10%, -31.9 ± 9% and -43 ± 8%, respectively, which was not reversible following washout and cell depolarization. After 6h of incubation, the drugs concentration dependently reduced proliferation. A maximal reduction of cell proliferation, respectively of -60 ± 8% for RESV (2 × 10 M) (IC50 = 1.50 × 10 M), -65 ± 6% for IbTX (10 M) (IC50 = 5 × 10 M), -97 ± 6% for PAX (1 × 10 M) (IC50 = 1.06 × 10 M) and AKT1p dephosphorylation was observed. PAX induced a G1/G2 accumulation and contraction of the S-phase, reducing the nuclear area and cell diameter. IbTX induced G1 contraction and G2 accumulation reducing diameter. RESV induced G2 accumulation and S contraction reducing diameter. These drugs share common actions leading to a block of the surface membrane BK channels with cell depolarization and calcium influx, AKT1p dephosphorylation by calcium-dependent phosphatase, accumulation in the G2 phase, and a reduction of diameter and proliferation. In addition, the PAX action against nuclear membrane BK channels potentiates its antiproliferative effects with early apoptosis.
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http://dx.doi.org/10.3390/ijms19082442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6121591PMC
August 2018

Evaluation of Short and Long Term Cold Stress Challenge of Nerve Grow Factor, Brain-Derived Neurotrophic Factor, Osteocalcin and Oxytocin mRNA Expression in BAT, Brain, Bone and Reproductive Tissue of Male Mice Using Real-Time PCR and Linear Correlation Analysis.

Front Physiol 2017 11;8:1101. Epub 2018 Jan 11.

Department of Pharmacy-Drug Sciences, University of Bari Aldo Moro, Bari, Italy.

The correlation between the and , Osteocalcin-/ and Oxytocin- genes, was challenged investigating their mRNA levels in 3 months-old mice after cold-stress (CS). Uncoupling protein-1 ( was used as positive control. Control mice were maintained at room temperature T = 25°C, CS mice were maintained at T = 4°C for 6 h and 5-days ( = 15 mice). RT-PCR experiments showed that and genes were up-regulated after 6 h CS in brown adipose tissues (BAT), respectively, by 2 and 1.5-folds; was upregulated also after 5-days, while and genes were downregulated after 6 h and 5-days CS in BAT. and were upregulated in bone and testis following 5-days, and in testis after 6 h CS. was instead up-regulated in bone following 5-days CS and down-regulated in testis. was upregulated by 16 and 3-fold in bone and BAT, respectively, following 5-days CS. was upregulated after 6 h in brain, while ( gene was downregulated. gene was upregulated by 5-fold following 5-days CS in bone. was upregulated by 0.5 and 0.3-fold, respectively, following 6 h and 5-days CS in brain. and were downregulated in testis and in BAT. The changes in the expression levels of control genes vs. genes following 6 h and 5-days CS were correlated in all tissues, but not in BAT. Correlation in BAT was improved eliminating data. The correlation in brain was lost eliminating data. In sum, potentiation in BAT after cold stress is associated with early -response in the same tissue and trophic action in bone and testis. In contrast, BDNF exerts bone and neuroprotective effects. Similarly to ( signaling is enhanced in bone and BAT while it may exert local neuroprotective effects thought its receptor. regulates the adaptation to CS through a feed-back loop in BAT. regulates the gene-response to CS through a feed-forward loop in brain. Overall these results expand the understanding of the physiology of these molecules under metabolic thermogenesis.
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http://dx.doi.org/10.3389/fphys.2017.01101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5768886PMC
January 2018

Alginate-Based Hydrogel Containing Minoxidil/Hydroxypropyl-β-Cyclodextrin Inclusion Complex for Topical Alopecia Treatment.

J Pharm Sci 2018 04 26;107(4):1046-1054. Epub 2017 Nov 26.

Department of Pharmacy-Drug Sciences, University of Bari "Aldo Moro", Bari, Italy.

Cutaneous minoxidil (MXD) formulations were developed with the intent to reduce the side effects of the cosolvents propylene glycol and ethanol, frequently used in commercial MXD solutions. Completely aqueous alginate-based hydrogels were investigated and MXD aqueous solubility was improved using inclusion complexes with hydroxypropyl-β-cyclodextrin (HP-β-CD) at 2 different molar substitution degree (MS), namely 0.65 and 0.85. HP-β-CD MS 0.65 was selected for its improved solubilizing ability toward MXD. At concentration of 39% w/v, this cyclodextrin increased the intrinsic aqueous solubility of MXD of about 22-fold. The calculated complexation constant was 2309 ± 20 M, and the inclusion process was spontaneous and enthalpically driven. Nuclear magnetic resonance studies (Job plot, H, 2D correlations spectroscopy, nuclear overhauser effect spectroscopy, and rotating-frame overhauser enhancement spectroscopy) confirmed the stoichiometry 1:1 between MXD and HP-β-CD providing information about the exact geometry of the inclusion complex. Rheological and in vitro release studies performed on the formulation loaded with MXD 3.5% w/w proved that the inclusion complex increased the viscosity of the hydrogel modulating the release of the free drug. Furthermore, the hydrogel formulation facilitate MXD to permeate into the skin and did not damage epidermis, suggesting that these completely aqueous MXD delivery systems can be proposed as alternative formulations to commercial solutions.
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http://dx.doi.org/10.1016/j.xphs.2017.11.016DOI Listing
April 2018

Characterization of minoxidil/hydroxypropyl-β-cyclodextrin inclusion complex in aqueous alginate gel useful for alopecia management: Efficacy evaluation in male rat.

Eur J Pharm Biopharm 2018 Jan 24;122:146-157. Epub 2017 Oct 24.

Pharmaceutical Technology, Department of Pharmacy - Pharmaceutical Sciences, University of Bari, Italy.

Solid inclusion complex between hydroxypropyl-β-cyclodextrin (HP-β-CD) and minoxidil (MXD) was prepared by freeze-drying and characterized by yield, drug loading and dissolution rate. Moreover, the complex was formulated as alginate gel (GEL HP-β-CD)/MXD 3.5% w/w). The efficacy of the novel GEL HP-β-CD)/MXD 3.5% w/w and of MXD 3.5% w/w ethanolic/propylene-glycol solution (MXD solution) were evaluated by monitoring the hair growth of dorsal skin 1-4 weeks after depilation followed by histological analysis and gene expression in skin biopsies in male rat. Patch-clamp experiments and cell-dehydrogenase activity (CDA) were performed to evaluate the capability of the formulations to activate "in vitro" the ATP-sensitive K-channels (KATP) and their effects on cell viability in skin fibroblasts. After 3 weeks, the MXD solution and MXD/HP-β-CD GEL enhanced the hair growth, respectively, of 80.1 ± 2% and 84.3 ± 4% vs controls. After 4 weeks, the MXD/HP-β-CD GEL significantly enhanced the hair length and bulb diameter vs others groups. The MXD/HP-β-CD GEL significantly enhanced the mRNA levels of the SUR2 and Kir6.1 subunits of the KATP channels and AKT2 vs other groups. The AR gene was down-regulated vs controls following the treatment with either MXD formulations. Either MXD (10 M) formulations were effective in potentiating the KATP currents. The MXD solution and its vehicle after 9 h of incubation time, but not MXD/HP-β-CD, reduced CDA in fibroblasts. In sum, the MXD/HP-β-CD GEL shows a favorable profile following topical long-term use.
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http://dx.doi.org/10.1016/j.ejpb.2017.10.015DOI Listing
January 2018

Risk of Myopathy in Patients in Therapy with Statins: Identification of Biological Markers in a Pilot Study.

Front Pharmacol 2017 27;8:500. Epub 2017 Jul 27.

Section of Pharmacology, Department of Pharmacy and Drug Sciences, University of Bari Aldo MoroBari, Italy.

Statin therapy may induce skeletal muscle damage ranging from myalgia to severe rhabdomyolysis. Our previous preclinical studies showed that statin treatment in rats involves the reduction of skeletal muscle ClC-1 chloride channel expression and related chloride conductance (gCl). An increase of the activity of protein kinase C theta (PKC theta) isoform, able to inactivate ClC-1, may contribute to destabilize sarcolemma excitability. These effects can be detrimental for muscle function leading to drug-induced myopathy. Our goal is to study the causes of statin-induced muscle side effects in patients at the aim to identify biological markers useful to prevent and counteract statin-induced muscle damage. We examined 10 patients, who experienced myalgia and hyper-CK-emia after starting statin therapy compared to 9 non-myopathic subjects not using lipid-lowering drugs. Western Blot (WB) analysis showed a 40% reduction of ClC-1 protein and increased expression of phosphorylated PKC in muscle biopsies of statin-treated patients with respect to untreated subjects, independently from their age and statin type. Real-time PCR analysis showed that despite reduction of the protein, the ClC-1 mRNA was not significantly changed, suggesting post-transcriptional modification. The mRNA expression of a series of genes was also evaluated. MuRF-1 was increased in accord with muscle atrophy, MEF-2, calcineurin (CN) and GLUT-4 transporter were reduced, suggesting altered transcription, alteration of glucose homeostasis and energy deficit. Accordingly, the phosphorylated form of AMPK, measured by WB, was increased, suggesting cytoprotective process activation. In parallel, mRNA expression of Notch-1, involved in muscle cell proliferation, was highly expressed in statin-treated patients, indicating active regeneration. Also, PGC-1-alpha and isocitrate-dehydrogenase increased expression together with increased activity of mitochondrial citrate-synthase, measured by spectrophotometric assay, suggests mitochondrial biogenesis. Thus, the reduction of ClC-1 protein and consequent sarcolemma hyperexcitability together with energy deficiency appear to be among the most important alterations to be associated with statin-related risk of myopathy in humans. Thus, it may be important to avoid statin treatment in pathologies characterized by energy deficit and chloride channel malfunction. This study validates the measure of ClC-1 expression as a reliable clinical test for assessing statin-dependent risk of myopathy.
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http://dx.doi.org/10.3389/fphar.2017.00500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5529355PMC
July 2017

Molecular structure and function of big calcium-activated potassium channels in skeletal muscle: pharmacological perspectives.

Physiol Genomics 2017 Jun 28;49(6):306-317. Epub 2017 Apr 28.

Department of Pharmacy-Drug Science, University of Bari, Bari, Italy;

The large-conductance Ca-activated K (BK) channel is broadly expressed in various mammalian cells and tissues such as neurons, skeletal muscles (sarco-BK), and smooth muscles. These channels are activated by changes in membrane electrical potential and by increases in the concentration of intracellular calcium ion (Ca). The BK channel is subjected to many mechanisms that add diversity to the BK channel α-subunit gene. These channels are indeed subject to alternative splicing, auxiliary subunits modulation, posttranslational modifications, and protein-protein interactions. BK channels can be modulated by diverse molecules that may induce either an increase or decrease in channel activity. The linkage of these channels to many intracellular metabolites and pathways, as well as their modulation by extracellular natural agents, have been found to be relevant in many physiological processes. BK channel diversity is obtained by means of alternative splicing and modulatory β- and γ-subunits. The association of the α-subunit with β- or with γ-subunits can change the BK channel phenotype, functional diversity, and pharmacological properties in different tissues. In the case of the skeletal muscle BK channel (sarco-BK channel), we established that the main mechanism regulating BK channel diversity is the alternative splicing of the gene encoding for the α-subunit generating different splicing isoform in the muscle phenotypes. This finding helps to design molecules selectively targeting the skeletal muscle subtypes. The use of drugs selectively targeting the skeletal muscle BK channels is a promising strategy in the treatment of familial disorders affecting muscular skeletal apparatus including hyperkalemia and hypokalemia periodic paralysis.
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http://dx.doi.org/10.1152/physiolgenomics.00121.2016DOI Listing
June 2017

Commentary: A BK (Slo1) channel journey from molecule to physiology.

Front Pharmacol 2017 5;8:188. Epub 2017 Apr 5.

Department of Pharmacy-Drug Science, University of BariBari, Italy.

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http://dx.doi.org/10.3389/fphar.2017.00188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5380717PMC
April 2017

Pharmacovigilance database search discloses ClC-K channels as a novel target of the AT receptor blockers valsartan and olmesartan.

Br J Pharmacol 2017 07 26;174(13):1972-1983. Epub 2017 Apr 26.

Department of Pharmacy - Drug Sciences, University of Bari 'Aldo Moro', Bari, Italy.

Background And Purpose: Human ClC-K chloride channels are highly attractive targets for drug discovery as they have a variety of important physiological functions and are associated with genetic disorders. These channels are crucial in the kidney as they control chloride reabsorption and water diuresis. In addition, loss-of-function mutations of CLCNKB and BSND genes cause Bartter's syndrome (BS), whereas CLCNKA and CLCNKB gain-of-function polymorphisms predispose to a rare form of salt sensitive hypertension. Both disorders lack a personalized therapy that is in most cases only symptomatic. The aim of this study was to identify novel ClC-K ligands from drugs already on the market, by exploiting the pharmacological side activity of drug molecules available from the FDA Adverse Effects Reporting System database.

Experimental Approach: We searched for drugs having a Bartter-like syndrome as a reported side effect, with the assumption that BS could be causatively related to the block of ClC-K channels. The ability of the selected BS-causing drugs to bind and block ClC-K channels was then validated through an integrated experimental and computational approach based on patch clamp electrophysiology in HEK293 cells and molecular docking simulations.

Key Results: Valsartan and olmesartan were able to block ClC-Ka channels and the molecular requirements for effective inhibition of these channels have been identified.

Conclusion And Implications: These results suggest additional mechanisms of action for these sartans further to their primary AT receptor antagonism and propose these compounds as leads for designing new potent ClC-K ligands.
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http://dx.doi.org/10.1111/bph.13794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5466521PMC
July 2017

Growth hormone secretagogues prevent dysregulation of skeletal muscle calcium homeostasis in a rat model of cisplatin-induced cachexia.

J Cachexia Sarcopenia Muscle 2017 Jun 10;8(3):386-404. Epub 2017 Mar 10.

Department of Pharmacy - Drug Sciences, University of Bari, Via Orabona 4, 70125, Bari, Italy.

Background: Cachexia is a wasting condition associated with cancer types and, at the same time, is a serious and dose-limiting side effect of cancer chemotherapy. Skeletal muscle loss is one of the main characteristics of cachexia that significantly contributes to the functional muscle impairment. Calcium-dependent signaling pathways are believed to play an important role in skeletal muscle decline observed in cachexia, but whether intracellular calcium homeostasis is affected in this situation remains uncertain. Growth hormone secretagogues (GHS), a family of synthetic agonists of ghrelin receptor (GHS-R1a), are being developed as a therapeutic option for cancer cachexia syndrome; however, the exact mechanism by which GHS interfere with skeletal muscle is not fully understood.

Methods: By a multidisciplinary approach ranging from cytofluorometry and electrophysiology to gene expression and histology, we characterized the calcium homeostasis in fast-twitch extensor digitorum longus (EDL) muscle of adult rats with cisplatin-induced cachexia and established the potential beneficial effects of two GHS (hexarelin and JMV2894) at this level. Additionally, in vivo measures of grip strength and of ultrasonography recordings allowed us to evaluate the functional impact of GHS therapeutic intervention.

Results: Cisplatin-treated EDL muscle fibres were characterized by a ~18% significant reduction of the muscle weight and fibre diameter together with an up-regulation of atrogin1/Murf-1 genes and a down-regulation of Pgc1-a gene, all indexes of muscle atrophy, and by a two-fold increase in resting intracellular calcium, [Ca ] , compared with control rats. Moreover, the amplitude of the calcium transient induced by caffeine or depolarizing high potassium solution as well as the store-operated calcium entry were ~50% significantly reduced in cisplatin-treated rats. Calcium homeostasis dysregulation parallels with changes of functional ex vivo (excitability and resting macroscopic conductance) and in vivo (forelimb force and muscle volume) outcomes in cachectic animals. Administration of hexarelin or JMV2894 markedly reduced the cisplatin-induced alteration of calcium homeostasis by both common as well as drug-specific mechanisms of action. This effect correlated with muscle function preservation as well as amelioration of various atrophic indexes, thus supporting the functional impact of GHS activity on calcium homeostasis.

Conclusions: Our findings provide a direct evidence that a dysregulation of calcium homeostasis plays a key role in cisplatin-induced model of cachexia gaining insight into the etiopathogenesis of this form of muscle wasting. Furthermore, our demonstration that GHS administration efficaciously prevents cisplatin-induced calcium homeostasis alteration contributes to elucidate the mechanism of action through which GHS could potentially ameliorate chemotherapy-associated cachexia.
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http://dx.doi.org/10.1002/jcsm.12185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5703021PMC
June 2017

A novel injectable formulation of 6-fluoro-l-DOPA imaging agent for diagnosis of neuroendocrine tumors and Parkinson's disease.

Int J Pharm 2017 Mar 21;519(1-2):304-313. Epub 2017 Jan 21.

Dipartimento di Farmacia - Scienze del Farmaco-Università degli Studi di Bari "Aldo Moro", Via E. Orabona 4, 70125, Bari, Italy, Italy. Electronic address:

Two [19F]F-l-DOPA (F-DOPA) new β-cyclodextrin (CD)-based dosage forms (FA and FB, respectively) have been studied and their physico-chemical and pharmacological features determined to overcome the administration site reactions showed by the currently used [18F]F-l-DOPA formulation (IASOdopa) to perform PET-CT diagnosis in oncology (neuroendocrine tumors) and neurological (Parkinson's disease) field. Chemical stability of FA and FB was found to be longer than IASOdopa by adding the thiol-antioxidant agent, L-Cysteine. H and F NMR investigations suggest the formation of an inclusion complex of F-DOPA with β-CD. In vitro experiments on the effects of FA and FB on mouse skeletal muscle fibers and on the human neuroblastoma SH-SY5Y and embryonal kidney tsA201 cell lines viability showed that FA was the most performant formulation compared to F-DOPA solutions. In vivo tolerability tests of FA on adult male rat showed no significant effects on body weight and no change in their dried organs weight. In addition, their metabolic and physiological parameters were not affected. In conclusion, [18F]F-l-DOPA, formulated as FA, constitutes a promising dosage form for PET-CT diagnosis of both neuroendocrine tumors and Parkinson's disease.
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http://dx.doi.org/10.1016/j.ijpharm.2017.01.038DOI Listing
March 2017

Nerve Growth Factor, Brain-Derived Neurotrophic Factor and Osteocalcin Gene Relationship in Energy Regulation, Bone Homeostasis and Reproductive Organs Analyzed by mRNA Quantitative Evaluation and Linear Correlation Analysis.

Front Physiol 2016 13;7:456. Epub 2016 Oct 13.

Department of Pharmacy - Drug Sciences, University of Bari Bari, Italy.

Nerve Growth Factor (NGF)/Brain-derived Neurotrophic Factor (BDNF) and osteocalcin share common effects regulating energy, bone mass, reproduction and neuronal functions. To investigate on the gene-relationship between NGF, BDNF, and Osteocalcin we compared by RT-PCR the transcript levels of and as well as of their receptors , and in brain, bone, white/brown adipose tissue (WAT/BAT) and reproductive organs of 3 months old female and male mice. Brain and bone were used as positive controls for NGF/BDNF and Osteocalcin respectively. The role of oxitocin() and its receptor() was also investigated. expression shows an opposite trend compared to . / expression is 50% higher in BAT than brain, in both genders, but lower in bone. In contrast, expression in bone is higher than in brain, but low in BAT/WAT. We found gene expressed in brain in both genders, but expression is low in brain and BAT/WAT. As expected, gene is expressed in bone. gene was markedly expressed in brain, in the ovaries and in fat and bone in both genders. is highly expressed in reproductive tissues and mRNA levels are respectively 300, 100, and 50% higher in testis/ovaries/uterus than in brain. In contrast, BDNF genes are not expressed in reproductive tissues. As expected, is expressed in testis but not in the ovaries/uterus. A significant correlation was found between the expression levels of the gene ligands and their receptors in brain, BAT and testis suggesting a common pathway of different genes in these tissues in either male and female. Changes in the expression levels of , or genes may mutually affect the expression levels of the others. Moreover, it may be possible that different ligands may operate through different receptor subtypes. and failed to show significant correlation. The up-regulation of / in BAT is consistent with NGF as an energy regulator and with BDNF regulating bone.
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http://dx.doi.org/10.3389/fphys.2016.00456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5061807PMC
October 2016

Pharmaceutical development of novel lactate-based 6-fluoro-l-DOPA formulations.

Eur J Pharm Sci 2017 Mar 4;99:361-368. Epub 2016 Oct 4.

Dipartimento di Farmacia - Scienze del Farmaco, Università degli Studi di Bari "Aldo Moro", Via E. Orabona 4, 70125 Bari, Italy. Electronic address:

6-[F]fluoro-l-dihydroxyphenylalanine (F-DOPA) is a diagnostic positron emission tomography (PET) agent, which has been used for decades in imaging the loss of dopaminergic neurons in Parkinson's disease, and more recently to detect, stage and restage neuroendocrine tumors (NETs) and to search for recurrence of viable glioma tissue. The commercially available F-DOPA PET radiopharmaceutical for diagnostic use in European Union countries, is formulated in an aqueous solution of acetic acid (1.05mg/mL) and has the disadvantages that, immediately before injection, the pH must be adjusted to 4.0-5.0 by the addition of a sterile solution of sodium bicarbonate (84mg/mL) causing a light and transient burning sensation at the injection site. To overcome these drawbacks, preformulation studies were accomplished to confirm that F-DOPA degradation was affected by pH. Hence, two formulations of F-DOPA, namely ND1 and ND2, were prepared maintaining the pH=5.0 using 1mM l-(+)-lactate buffer, excluding oxygen, and incorporating in the formula the chelating agent NaEDTA (1mM). F-DOPA oxygen exposure, the presence of free metal cations in formulation and high pH values seem to promote F-DOPA degradation. The resulting formulations proved to guarantee the chemical stability of F-DOPA in solution at pH5.0, value also compatible with the direct infusion. In vitro cell viability tests on mouse skeletal muscle fibers, renal tsa201 and neuronal SH-SY5Y cell lines, and in vivo studies in rats reported elsewhere, showed cell tolerability to the new F-DOPA formulations providing the basis for their further in vivo evaluation.
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http://dx.doi.org/10.1016/j.ejps.2016.10.001DOI Listing
March 2017

New azepino[4,3-b]indole derivatives as nanomolar selective inhibitors of human butyrylcholinesterase showing protective effects against NMDA-induced neurotoxicity.

Eur J Med Chem 2017 Jan 13;125:288-298. Epub 2016 Sep 13.

Department of Pharmacy - Drug Sciences, University of Bari "A. Moro", Via E. Orabona 4, 70125 Bari, Italy. Electronic address:

Several 6-substituted 3,4,5,6-tetrahydroazepino[4,3-b]indol-1(2H)-one (THAI) derivatives were synthesized and evaluated for their activity as cholinesterase (ChE) inhibitors. The most potent inhibitors were identified among 6-(2-phenylethyl)-THAI derivatives, and in particular compounds 12b and 12d proved to be very active against human BChE (IC = 13 and 1.8 nM, respectively), with 1000-fold selectivity over AChE. Structure-activity relationships highlighted critical features (e.g., ring fusion [4,3-b], integrity of the lactam CONH function) and favorable physicochemical properties of the 6-(2-phenylethyl) group (i.e., optimal position, size and lipophilicity of phenyl substituents). The effects of a number of compounds against NMDA-induced SH-SY5Y neuronal cell injury were also evaluated. Treatment with 12b increased cell viability in SH-SY5Y cells pretreated with 250 μM NMDA, with significant effects (P < 0.05) at concentrations between 0.5 and 5 μM. These findings suggest that THAI can be used as a scaffold for developing new drug leads for the treatment of Alzheimer-type neurodegeneration syndrome.
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http://dx.doi.org/10.1016/j.ejmech.2016.09.037DOI Listing
January 2017

ATP Sensitive Potassium Channels in the Skeletal Muscle Function: Involvement of the KCNJ11(Kir6.2) Gene in the Determination of Mechanical Warner Bratzer Shear Force.

Front Physiol 2016 10;7:167. Epub 2016 May 10.

Department of Veterinary Medicine, University of Bari Aldo Moro Bari, Italy.

The ATP-sensitive K(+)-channels (KATP) are distributed in the tissues coupling metabolism with K(+) ions efflux. KATP subunits are encoded by KCNJ8 (Kir6.1), KCNJ11 (Kir6.2), ABCC8 (SUR1), and ABCC9 (SUR2) genes, alternative RNA splicing give rise to SUR variants that confer distinct physiological properties on the channel. An high expression/activity of the sarco-KATP channel is observed in various rat fast-twitch muscles, characterized by elevated muscle strength, while a low expression/activity is observed in the slow-twitch muscles characterized by reduced strength and frailty. Down-regulation of the KATP subunits of fast-twitch fibers is found in conditions characterized by weakness and frailty. KCNJ11 gene knockout mice have reduced glycogen, lean phenotype, lower body fat, and weakness. KATP channel is also a sensor of muscle atrophy. The KCNJ11 gene is located on BTA15, close to a QTL for meat tenderness, it has also a role in glycogen storage, a key mechanism of the postmortem transformation of muscle into meat. The role of KCNJ11 gene in muscle function may underlie an effect of KCNJ11 genotypes on meat tenderness, as recently reported. The fiber phenotype and genotype are important in livestock production science. Quantitative traits including meat production and quality are influenced both by environment and genes. Molecular markers can play an important role in the genetic improvement of animals through breeding strategies. Many factors influence the muscle Warner-Bratzler shear force including breed, age, feeding, the biochemical, and functional parameters. The role of KCNJ11gene and related genes on muscle tenderness will be discussed in the present review.
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http://dx.doi.org/10.3389/fphys.2016.00167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4862255PMC
May 2016

Therapeutic Approaches to Genetic Ion Channelopathies and Perspectives in Drug Discovery.

Front Pharmacol 2016 10;7:121. Epub 2016 May 10.

Department of Pharmacy - Drug Sciences, University of Bari "Aldo Moro" Bari, Italy.

In the human genome more than 400 genes encode ion channels, which are transmembrane proteins mediating ion fluxes across membranes. Being expressed in all cell types, they are involved in almost all physiological processes, including sense perception, neurotransmission, muscle contraction, secretion, immune response, cell proliferation, and differentiation. Due to the widespread tissue distribution of ion channels and their physiological functions, mutations in genes encoding ion channel subunits, or their interacting proteins, are responsible for inherited ion channelopathies. These diseases can range from common to very rare disorders and their severity can be mild, disabling, or life-threatening. In spite of this, ion channels are the primary target of only about 5% of the marketed drugs suggesting their potential in drug discovery. The current review summarizes the therapeutic management of the principal ion channelopathies of central and peripheral nervous system, heart, kidney, bone, skeletal muscle and pancreas, resulting from mutations in calcium, sodium, potassium, and chloride ion channels. For most channelopathies the therapy is mainly empirical and symptomatic, often limited by lack of efficacy and tolerability for a significant number of patients. Other channelopathies can exploit ion channel targeted drugs, such as marketed sodium channel blockers. Developing new and more specific therapeutic approaches is therefore required. To this aim, a major advancement in the pharmacotherapy of channelopathies has been the discovery that ion channel mutations lead to change in biophysics that can in turn specifically modify the sensitivity to drugs: this opens the way to a pharmacogenetics strategy, allowing the development of a personalized therapy with increased efficacy and reduced side effects. In addition, the identification of disease modifiers in ion channelopathies appears an alternative strategy to discover novel druggable targets.
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http://dx.doi.org/10.3389/fphar.2016.00121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4861771PMC
May 2016

Kidney CLC-K chloride channels inhibitors: structure-based studies and efficacy in hypertension and associated CLC-K polymorphisms.

J Hypertens 2016 May;34(5):981-92

aDipartimento di Farmacia - Scienze del Farmaco, Università degli Studi di Bari, Bari bIstituto di Biofisica, CNR, Genova, Italy *Antonella Liantonio and Paola Imbrici contributed equally to the writing of this article.

Objective: Alterations in the handling of renal salt reabsorption may contribute to interindividual differences in blood pressure regulation and susceptibility to hypertension. CLC-K chloride channels and their accessory subunit barttin play a pivotal role in kidney by controlling chloride and water absorption. Compounds selective for CLC-Ks, such as the benzofuran derivative MT-189, may have a significant therapeutic potential. Here, we assessed the feasibility of using CLC-K blockers in hypertension and aimed at enhancing drug inhibitory affinity.

Methods And Results: We demonstrated that acute in-vivo administration of MT-189 to spontaneously hypertensive rats (SHR) caused a reduction of blood pressure and defined the CLC-K/barttin gene expression pattern in kidney of SHR in comparison with normotensive Wistar-Kyoto rats. Based on MT-189, we designed and tested a new series of benzofuran derivatives on CLC-K chloride channels heterologously expressed in HEK293 cells. These studies enabled us to elucidate the causative molecular relationship for obtaining the most potent and selective inhibitor (SRA-36) described so far, with an IC50 of 6.6 ± 1 μmol/l. The biophysical and pharmacological characterization of A447T CLC-Ka and Y315F CLC-Ka, both polymorphisms associated with hypertension, showed that SRA-36 is an efficacious inhibitor of the chloride currents sustained by these polymorphisms. Molecular docking studies allowed hypothesizing an inhibition mechanism for the considered ligands, laying the foundations for the rational design of new and more effective CLC-K inhibitors.

Conclusion: The SRA-36 molecule represents a new potential therapeutic option for hypertension.
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http://dx.doi.org/10.1097/HJH.0000000000000876DOI Listing
May 2016

Molecular modeling of antibodies for the treatment of TNFα-related immunological diseases.

Pharmacol Res Perspect 2016 Feb 15;4(1):e00197. Epub 2016 Jan 15.

Department of Pharmacy - Drug Science University of Bari Bari Italy.

Therapeutic monoclonal antibodies (mAbs) have high efficacy in treating TNF α-related immunological diseases. Other than neutralizing TNF α, these IgG1 antibodies exert Fc receptor-mediated effector functions such as the complement-dependent cytotoxicity (CDC) and antibody-dependent cell cytotoxicity (ADCC). The crystallizable fragment (Fc) of these IgG1 contains a single glycosylation site at Asn 297/300 that is essential for the CDC and ADCC. Glycosylated antibodies lacking core fucosylation showed an improved ADCC. However, no structural data are available concerning the ligand-binding interaction of these mAbs used in TNF α-related diseases and the role of the fucosylation. We therefore used comparative modeling for generating complete 3D mAb models that include the antigen-binding fragment (Fab) portions of infliximab, complexed with TNF α (4G3Y.pdb), the Fc region of the human IGHG1 fucosylated (3SGJ) and afucosylated (3SGK) complexed with the Fc receptor subtype Fcγ RIIIA, and the Fc region of a murine immunoglobulin (1IGT). After few thousand steps of energy minimization on the resulting 3D mAb models, minimized final models were used to quantify interactions occurring between Fcγ RIIIA and the fucosylated/afucosylated Fc fragments. While fucosylation does not affect Fab-TNF α interactions, we found that in the absence of fucosylation the Fc-mAb domain and Fcγ RIIIA are closer and new strong interactions are established between G129 of the receptor and S301 of the Chimera 2 Fc mAb; new polar interactions are also established between the Chimera 2 Fc residues Y299, N300, and S301 and the Fcγ RIIIA residues K128, G129, R130, and R155. These data help to explain the reduced ADCC observed in the fucosylated mAbs suggesting the specific AA residues involved in binding interactions.
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http://dx.doi.org/10.1002/prp2.197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4777268PMC
February 2016

The large conductance Ca(2+) -activated K(+) (BKCa) channel regulates cell proliferation in SH-SY5Y neuroblastoma cells by activating the staurosporine-sensitive protein kinases.

Front Physiol 2014 9;5:476. Epub 2014 Dec 9.

Department of Pharmacy-Drug Science, University of Bari "Aldo Moro" Bari, Italy.

Here we investigated on the role of the calcium activated K(+)-channels(BKCa) on the regulation of the neuronal viability. Recordings of the K(+)-channel current were performed using patch-clamp technique in human neuroblastoma cells (SH-SY5Y) in parallel with measurements of the cell viability in the absence or presence of the BKCa channel blockers iberiotoxin(IbTX) and tetraethylammonium (TEA) and the BKCa channel opener NS1619. Protein kinase C/A (PKC, PKA) activities in the cell lysate were investigated in the presence/absence of drugs. The whole-cell K(+)-current showed a slope conductance calculated at negative membrane potentials of 126.3 pS and 1.717 nS(n = 46) following depolarization. The intercept of the I/V curve was -33 mV. IbTX(10(-8) - 4 × 10(-7) M) reduced the K(+)-current at +30 mV with an IC50 of 1.85 × 10(-7) M and an Imax of -46% (slope = 2.198) (n = 21). NS1619(10-100 × 10(-6) M) enhanced the K(+)-current of +141% (n = 6), at -10 mV(Vm). TEA(10(-5)-10(-3) M) reduced the K(+)-current with an IC50 of 3.54 × 10(-5) M and an Imax of -90% (slope = 0.95) (n = 5). A concentration-dependent increase of cell proliferation was observed with TEA showing a maximal proliferative effect(MPE) of +38% (10(-4) M). IbTX showed an MPE of +42% at 10(-8) M concentration, reducing it at higher concentrations. The MPE of the NS1619(100 × 10(-6) M) was +42%. The PKC inhibitor staurosporine (0.2-2 × 10(-6) M) antagonized the proliferative actions of IbTX and TEA. IbTX (10 × 10(-9) M), TEA (100 × 10(-6) M), and the NS1619 significantly enhanced the PKC and PKA activities in the cell lysate with respect to the controls. These results suggest that BKCa channel regulates proliferation of the SH-SY5Y cells through PKC and PKA protein kinases.
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http://dx.doi.org/10.3389/fphys.2014.00476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4260485PMC
December 2014