Anti-Inflammatory Effects of a Novel Acetonitrile-Water Extract of Lens Culinaris against LPS-Induced Damage in Caco-2 Cells.

Natural compounds like flavonoids preserve intestinal mucosal integrity through their antioxidant, anti-inflammatory, and antimicrobial properties. Additionally, some flavonoids show prebiotic abilities, promoting the growth and activity of beneficial gut bacteria. This study investigates the protective impact of Lens culinaris extract (LE), which is abundant in flavonoids, on intestinal mucosal integrity during LPS-induced inflammation. Using Caco-2 cells as a model for the intestinal barrier, the study found that LE did not affect cell viability but played a cytoprotective role in the presence of LPS. LE improved transepithelial electrical resistance (TEER) and tight junction (TJ) protein levels, which are crucial for barrier integrity. It also countered the upregulation of pro-inflammatory genes TRPA1 and TRPV1 induced by LPS and reduced pro-inflammatory markers like TNF-α, NF-κB, IL-1ß, and IL-8. Moreover, LE reversed the LPS-induced upregulation of AQP8 and TLR-4 expression. These findings emphasize the potential of natural compounds like LE to regulate the intestinal barrier and reduce inflammation's harmful effects on intestinal cells. More research is required to understand their mechanisms and explore therapeutic applications, especially for gastrointestinal inflammatory conditions.

The Role of Ion Channels in Functional Gastrointestinal Disorders (FGID): Evidence of Channelopathies and Potential Avenues for Future Research and Therapeutic Targets.

Several gastrointestinal (GI) tract abnormalities, including visceral hypersensitivity, motility, and intestinal permeability alterations, have been implicated in functional GI disorders (FGIDs). Ion channels play a crucial role in all the functions mentioned above. Hormones and natural molecules modulate these channels and represent targets of drugs and bacterial toxins. Mutations and abnormal functional expression of ion channel subunits can lead to diseases called channelopathies. These channelopathies in gastroenterology are gaining a strong interest, and the evidence of co-relationships is increasing. In this review, we describe the correlation status between channelopathies and FGIDs. Different findings are available. Among others, mutations in the ABCC7/CFTR gene have been described as a cause of constipation and diarrhea. Mutations of the SCN5A gene are instead associated with irritable bowel syndrome. In contrast, mutations of the TRPV1 and TRPA genes of the transient receptor potential (TRP) superfamily manifest hypersensitivity and visceral pain in sensory nerves. Recently, mice and humans affected by Cantu syndrome (CS), which is associated with the mutations of the KCNJ8 and ABCC9 genes encoding for the Kir6.1 and SUR2 subunits, showed dysfunction of contractility throughout the intestine and death in the mice after the weaning on solid food. The discovery of a correlation between channelopathies and FIGD opens new avenues for discovering new direct drug targets for specific channelopathies, leading to significant implications for diagnosing and treating functional GI diseases.

The Importance of Nutraceuticals in COVID-19: What's the Role of Resveratrol?

Since COVID-19 has affected global public health, there has been an urgency to find a solution to limit both the number of infections, and the aggressiveness of the disease once infected. The main characteristic of this infection is represented by a strong alteration of the immune system which, day by day, increases the risk of mortality, and can lead to a multiorgan dysfunction. Because nutritional profile can influence patient's immunity, we focus our interest on resveratrol, a polyphenolic compound known for its immunomodulating and anti-inflammatory properties. We reviewed all the information concerning the different roles of resveratrol in COVID-19 pathophysiology using PubMed and Scopus as the main databases. Interestingly, we find out that resveratrol may exert its role through different mechanisms. In fact, it has antiviral activity inhibiting virus entrance in cells and viral replication. Resveratrol also improves autophagy and decreases pro-inflammatory agents expression acting as an anti-inflammatory agent. It regulates immune cell response and pro-inflammatory cytokines and prevents the onset of thrombotic events that usually occur in COVID-19 patients. Since resveratrol acts through different mechanisms, the effect could be enhanced, making a totally natural agent particularly effective as an adjuvant in anti COVID-19 therapy.

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

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.

Cell Cycle Regulation by Ca2+-Activated K⁺ (BK) Channels Modulators in SH-SY5Y Neuroblastoma Cells.

The effects of Ca2+-activated K⁺ (BK) channel modulation by Paxilline (PAX) (10-7⁻10-4 M), Iberiotoxin (IbTX) (0.1⁻1 × 10-6 M) and Resveratrol (RESV) (1⁻2 × 10-4 M) on cell cycle and proliferation, AKT1pSer473 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 × 10-7 M), PAX (5 × 10-5 M) and RESV (10-4 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 × 10-4 M) (IC50 = 1.50 × 10-4 M), -65 ± 6% for IbTX (10-6 M) (IC50 = 5 × 10-7 M), -97 ± 6% for PAX (1 × 10-4 M) (IC50 = 1.06 × 10-5 M) and AKT1pser473 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, AKT1pser473 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.

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

The large-conductance Ca2+-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 (Ca2+). 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 ß- and γ-subunits. The association of the α-subunit with ß- 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 KCNMA1/slo1 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.

Calcium homeostasis is altered in skeletal muscle of spontaneously hypertensive rats: cytofluorimetric and gene expression analysis.

Hypertension is often associated with skeletal muscle pathological conditions related to function and metabolism. The mechanisms underlying the development of these pathological conditions remain undefined. Because calcium homeostasis is a biomarker of muscle function, we assessed whether it is altered in hypertensive muscles. We measured resting intracellular calcium and store-operated calcium entry (SOCE) in fast- and slow-twitch muscle fibers from normotensive Wistar-Kyoto rats and spontaneously hypertensive rats (SHRs) by cytofluorimetric technique and determined the expression of SOCE gene machinery by real-time PCR. Hypertension caused a phenotype-dependent dysregulation of calcium homeostasis; the resting intracellular calcium of extensor digitorum longus and soleus muscles of SHRs were differently altered with respect to the related muscle of normotensive animals. In addition, soleus muscles of SHR showed reduced activity of the sarcoplasmic reticulum and decreased sarcolemmal calcium permeability at rest and after SOCE activation. Accordingly, we found an alteration of the expression levels of some SOCE components, such as stromal interaction molecule 1, calcium release-activated calcium modulator 1, and transient receptor potential canonical 1. The hypertension-induced alterations of calcium homeostasis in the soleus muscle of SHRs occurred with changes of some functional outcomes as excitability and resting chloride conductance. We provide suitable targets for therapeutic interventions aimed at counterbalancing muscle performance decline in hypertension, and propose the reported calcium-dependent parameters as indexes to predict how the antihypertensive drugs could influence muscle function.

The Community Pharmacy as a Study Center for the Epidemiological Analysis of the Population Vaccination against SARS-CoV-2: Evaluation of Vaccine Safety and Pharmaceutical Service.

We conducted a monocentric observational study aimed at evaluating the vaccine safety and the pharmaceutical service provided at a community pharmacy (C.PHARM) in the Puglia Region in the period from 29 December 2021 to 12 March 2022 using data from 550 patients of various ages and sexes and with concomitant diseases. We collected anamnestic data, the number of hospitalizations, and any post-vaccination adverse reactions. Interviews using the integrated EQ5 method were also performed to evaluate the quality of the service offered and any therapy preference. As expected, the vaccines were reactogenic after the first dose in the patients with mild-moderate reactions, with younger age and female gender as risk factors. Immune-allergic reactions of a moderate-severe degree were observed in adult females. In the elderly, the vaccination was well tolerated. Comirnaty® showed a favorable O.R. < 1 vs. other vaccines. No cardiovascular events or hospitalizations were observed up to May 2023. Regional data indicate that all treatments during May 2023 were correlated with the viremia. PaxlovidTM was prescribed in 3% of the patients in our center and in 1.46% in the region, and distributed/dispensed on behalf of third parties in accordance with a novel distribution/dispensation protocol of the C.PHARM that resulted in a safe vaccination center providing appropriate patient inclusion during vaccination.

Immunohistochemical, pharmacovigilance, and omics analyses reveal the involvement of ATP-sensitive K+ channel subunits in cancers: role in drug-disease interactions.

Background: ATP-sensitive-K+ channels (KATP) are involved in diseases, but their role in cancer is poorly described. Pituitary macroadenoma has been observed in Cantu' syndrome (C.S.), which is associated with the gain-of-function mutations of the ABCC9 and KCNJ8 genes. We tested the role of the ABCC8/Sur1, ABCC9/Sur2A/B, KCNJ11/Kir6.2, and KCNJ8/Kir6.1 genes experimentally in a minoxidil-induced renal tumor in male rats and in the female canine breast cancer, a spontaneous animal model of disease, and in the pharmacovigilance and omics databases. Methods: We performed biopsies from renal tissues of male rats (N = 5) following a sub-chronic high dosing topical administration of minoxidil (0.777-77.7 mg/kg/day) and from breast tissues of female dogs for diagnosis (N = 23) that were analyzed by immunohistochemistry. Pharmacovigilance and omics data were extracted from EudraVigilance and omics databases, respectively. Results: An elevated immunohistochemical reactivity to Sur2A-mAb was detected in the cytosol of the Ki67+/G3 cells other than in the surface membrane in the minoxidil-induced renal tumor and the breast tumor samples. KCNJ11, KCNJ8, and ABCC9 genes are upregulated in cancers but ABCC8 is downregulated. The Kir6.2-Sur2A/B-channel opener minoxidil showed 23 case reports of breast cancer and one case of ovarian cancer in line with omics data reporting, respectively, and the negative and positive prognostic roles of the ABCC9 gene in these cancers. Sulfonylureas and glinides blocking the pancreatic Kir6.2-Sur1 subunits showed a higher risk for pancreatic cancer in line with the positive prognostic role of the ABCC8 gene but low risks for common cancers. Glibenclamide, repaglinide, and glimepiride show a lower cancer risk within the KATP channel blockers. The Kir6.2-Sur1 opener diazoxide shows no cancer reactions. Conclusion: An elevated expression of the Sur2A subunit was found in proliferating cells in two animal models of cancer. Immunohistochemistry/omics/pharmacovigilance data reveal the role of the Kir6.1/2-Sur2A/B subunits as a drug target in breast/renal cancers and in C.S.

Molecular Composition and Biological Activity of a Novel Acetonitrile-Water Extract of Lens Culinaris Medik in Murine Native Cells and Cell Lines Exposed to Different Chemotherapeutics Using Mass Spectrometry.

We evaluated the effects of a new extract (70% acetonitrile, 2E0217022196DIPFARMTDA) of Lens culinaris Medik (Terre di Altamura SRL, Altamura BA) to prevent cytotoxic damage from cisplatin, staurosporine, irinotecan, doxorubicin, and the glucocorticoid dexamethasone. The acetonitrile-water extract (range 0.1-5 mg/mL) was obtained by extracting 10 g of lentil flour with 50 milliliters of the acetonitrile-water extraction mixture in a 70:30 ratio, first for 3 h and then overnight in a shaker at room temperature. The next day, the extract was filtered and passed through a Rotavapor to obtain only the aqueous component and eliminate that with acetonitrile, and then freeze-dried to finally have the powdered extract. In vitro experiments showed that the extract prevented the cytotoxic damage induced by cisplatin, irinotecan, and doxorubicin on HEK293 and SHSY5Y cell lines after 24-96 h. In murine osteoblasts after 24-72 h of incubation time, the extract was cytoprotective against all chemicals. The extract was effective against dexamethasone, leading to synergic cell proliferation in all cell types. In bone marrow cells, the extract is cytoprotective after 72 h against doxorubicin, staurosporine, and dexamethasone. Instead, on muscle fibers, the extract has a synergic effect with chemotherapeutics, increasing cytotoxicity induced by doxorubicin and staurosporine. LC-MS attested to the existence of several phenolic structures in the extract. The most abundant families of compounds were flavonoids (25.7%) and mellitic acid (18%). Thus, the development of this extract could be implemented in the area of research related to the chemoprevention of damage to renal, neuronal, bone marrow cells, and osteoblasts by chemotherapeutics; moreover, it could be used as a reinforcer of cytotoxic action of chemotherapeutics on muscle fibers.

Zoledronic Acid Blocks Overactive Kir6.1/SUR2-Dependent KATP Channels in Skeletal Muscle and Osteoblasts in a Murine Model of Cantú Syndrome.

Cantú syndrome (CS) is caused by the gain of function mutations in the ABCC9 and KCNJ8 genes encoding, respectively, for the sulfonylureas receptor type 2 (SUR2) and the inwardly rectifier potassium channel 6.1 (Kir6.1) of the ATP-sensitive potassium (KATP) channels. CS is a multi-organ condition with a cardiovascular phenotype, neuromuscular symptoms, and skeletal malformations. Glibenclamide has been proposed for use in CS, but even in animals, the drug is incompletely effective against severe mutations, including the Kir6.1wt/V65M. Patch-clamp experiments showed that zoledronic acid (ZOL) fully reduced the whole-cell KATP currents in bone calvaria cells from wild type (WT/WT) and heterozygous Kir6.1wt/V65MCS mice, with IC50 for ZOL block < 1 nM in each case. ZOL fully reduced KATP current in excised patches in skeletal muscle fibers in WT/WT and CS mice, with IC50 of 100 nM in each case. Interestingly, KATP currents in the bone of heterozygous SUR2wt/A478V mice were less sensitive to ZOL inhibition, showing an IC50 of ~500 nM and a slope of ~0.3. In homozygous SUR2A478V/A478V cells, ZOL failed to fully inhibit the KATP currents, causing only ~35% inhibition at 100 µM, but was responsive to glibenclamide. ZOL reduced the KATP currents in Kir6.1wt/VMCS mice in both skeletal muscle and bone cells but was not effective in the SUR2[A478V] mice fibers. These data indicate a subunit specificity of ZOL action that is important for appropriate CS therapies.

Structural nucleotide analogs are potent activators/inhibitors of pancreatic ß cell KATP channels: an emerging mechanism supporting their use as antidiabetic drugs.

The 2H-1,4-benzoxazine derivatives are novel drugs structurally similar to nucleotides; however, their actions on the pancreatic ß cell ATP-sensitive K+ (KATP) channel and on glucose disposal are unknown. Therefore, the effects of the linear/branched alkyl substituents and the aliphatic/aromatic rings at position 2 of the 2H-1,4-benzoxazine nucleus on the activity of these molecules against the pancreatic ß cell KATP channel and the Kir6.2ΔC36 subunit were investigated using a patch-clamp technique. The effects of these compounds on glucose disposal that followed glucose loading by intraperitoneal glucose tolerance test and on fasting glycemia were investigated in normal mice. The 2-n-hexyl analog blocked the KATP (IC50 = 10.1 × 10⁻9 M) and Kir6.2ΔC36 (IC50 = 9.6 × 10⁻9 M) channels, which induced depolarization. In contrast, the 2-phenyl analog was a potent opener (drug concentration needed to enhance the current by 50% = 0.04 × 10⁻9 M), which induced hyperpolarization. The ranked order of the potency/efficacy of the analog openers was 2-phenyl > 2-benzyl > 2-cyclohexylmethyl. The 2-phenylethyl and 2-isopropyl analogs were not effective as blockers/openers. The 2-n-hexyl (2-10 mg/kg) and 2-phenyl analogs (2-30 mg/kg) reduced and enhanced the glucose areas under the curves, respectively, after glucose loading in mice. These compounds did not affect the fasting glycemia as is observed with glibenclamide. The linear alkyl chain and the aromatic ring at position 2 of the 1,4-benzoxazine nucleus are the determinants, which confer the KATP channel blocking action with glucose-lowering effects and the opening action with increased glucose levels, respectively. The opening/blocking actions of these compounds mimic those that were observed with ATP and ADP. The results support the use of these compounds as novel antidiabetic drugs.

Opening/blocking actions of pyruvate kinase antibodies on neuronal and muscular KATP channels.

ATP-sensitive-K(+) (KATP) channels couple metabolism to the electrical activity of the cells. This channel is associated with glycolytic enzymes to form complexes regulating the channel activity in various tissues. The pyruvate-kinase (PK) enzyme is an antigen in the Paediatric Autoimmune Neuropsychiatric Disorders Associated Streptococcal infection known as PANDAS which is characterized by an abnormal production of auto-antibodies against PK. Here, the effects of the anti-pyruvate kinase antibody (anti-PK-ab) on the muscle and neuronal KATP channels were investigated in native rat skeletal muscle fibres and human neuroblastoma cell-line (SH-SY5Y), respectively. Furthermore, the interaction of PK with the inwardly rectifier potassium channel (Kir6.1/Kir6.2) subunits of the KATP channels was investigated by co-immunoprecipitation experiments in mouse brain using the anti-PK-ab. Patch-clamp experiments showed that the short-term incubation (1h) of the fibres with the anti-PK-ab at the dilutions of 1:500 and 1:300 enhanced the KATP current of 19.6% and 33.5%, respectively. As opposite, the long-term incubation (24h) of the fibres with the anti-PK-ab at the dilutions of 1:500 and 1:300 reduced the KATP current of 16% and 24%, respectively, reducing the diameter with atrophy. The direct application of the anti-PK-ab to the excised patches in the absence of intracellular ATP caused channel block, while in the presence of nucleotide channel opened. In neuronal cell line, in the short-term the anti-PK-ab potentiated KATP currents without affecting survival, while in the long-term the anti-PK-ab reduced KATP currents inducing neuronal death. Opening/blocking actions of the anti-PK antibodies on the KATP channels were observed, the blocking action causes fibre atrophy and neuronal death. We demonstrated that PK and Kir subunits are physically/functionally coupled in neurons. The KATP/PK complex can be proposed a novel target in the autoimmune diseases associated with anti-PK production as in PANDAS.

ATP-sensitive Potassium Channel Subunits in Neuroinflammation: Novel Drug Targets in Neurodegenerative Disorders.

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-ß (Aß)-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, are 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 being 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.

Inflammatory Related Reactions in Humans and in Canine Breast Cancers, A Spontaneous Animal Model of Disease.

Inflammatory cells are emerging markers in various cancers in human trials. The relationship between the inflammatory cells response, cancer grade, and progression has been investigated experimentally in a spontaneous canine model of breast cancer and in the unselected population (18-64 years.o.) under anti-HER2 treatments that represent the most prevalent population in this cancer type. The canine data (N samples = 101) were collected retrospectively for diagnosis in our regional area and evaluated by immunohistochemistry and haemato-chemistry. The inflammatory and immune-related adverse reactions (ADR) in humans were evaluated using EudraVigilance. The "Proportional Reporting Ratio" (PRR) of the mabs was calculated for each ADR with values >2 indicative of high risk. In dogs, we found elevated immunostaining of CD68-macrophages in the lymph node of the aggressive cancer G3 and infiltrating CD20+-lymphocyte. A high density of CD20 + lymphocytes was observed in G1 and a decrease in the density was observed with the histological degree of the tumors. The animals with the sample in G1 showed reduced serum platelet and neutrophil count and elevated lymphocytes and the opposite in severely affected animals. Inflammatory reactions with edema, skin reactions, extravasation, loss of effectiveness, and platelet count decrease (PRR > 13) were found with trastuzumab emtansine in humans, in the absence of immune system reactions. Trastuzumab i.v.-s.c. showed immune system reactions, loss of effectiveness, intolerances with drug withdrawal, technological issues (PRR > 7), and neutrophil count decrease reports. These reactions were less frequently reported for pertuzumab i.v. Case reports of platelet and neutrophil count decrease were not associated with disease progression with a better outcome in humans as in canine breast cancer. Therefore, infiltrating CD68-macrophages are associated with G3, while infiltrating CD20+ and elevated serum lymphocytes in parallel with reduced platelet and neutrophil count play a favorable role in human and canine breast cancer.

Counteractions of a Novel Hydroalcoholic Extract from Lens Culinaria against the Dexamethasone-Induced Osteoblast Loss of Native Murine Cells.

The cytoprotective effects of a novel hydroalcoholic extract (0.01-5 mg/mL) from Lens culinaria (Terre di Altamura Srl) were investigated within murine native skeletal muscle fibers, bone marrow cells, and osteoblasts, and in cell lines treated with the apoptotic agent staurosporine (2.14 × 10-6 M), the alkylating drug cisplatin (10-4 M), the topoisomerase I inhibitor irinotecan (10-4 M), the antimitotic pro-oxidant doxorubicin (10-6 M), and the immunosuppressant dexamethasone (2 × 10-6 M). An amount of 10g of plant material was used to obtain a 70% ethanol/water product, following two-step extraction, evaporation, lyophilization, and storage at -20 °C. For the murine osteoblasts, doxorubicin reduced survival by -65%, dexamethasone by -32% and -60% after 24 and 48 h of incubation time, respectively. The extract was effective in preventing the osteoblast count-reduction induced by dexamethasone; it was also effective at preventing the inhibition of mineralization induced by dexamethasone. Doxorubicin and cisplatin caused a significant reduction in cell growth by -77% for bone marrow cells, -43% for irinotecan, and -60% for dexamethasone, but there was no evidence for the cytoprotective effects of the extract in these cells. Staurosporine and doxorubicin caused a fiber death rate of >-40% after 18 and 24 h of incubation, yet the extract was not effective at preventing these effects. The extract was effective in preventing the staurosporine-induced reduction of HEK293 proliferation and colony formation in the crystal violet DNA staining and the clonogenic assays. It was also effective for the cisplatin-induced reduction in HEK293 cell proliferation. The extract, however, failed to protect the SHSY5Y neurons against cisplatin and irinotecan-induced cytotoxicity. A UV/VIS spectroscopy analysis showed three peaks at the wavelengths of 350, 260, and 190 nm, which correspond to flavonoids, proanthocyanins, salicylates, and AA, constituting the extract. These data suggest the possible development of this extract for use against dexamethasone-induced bone loss and renal chemotherapy-induced damage.

Multicenter Observational/Exploratory Study Addressed to the Evaluation of the Effectiveness and Safety of Pharmacological Therapy in Opioid-Dependent Patients in Maintenance Therapy in Southern Italy.

A multicenter-observational study was performed to assess the effectiveness of rac-methadone, levomethadone, and buprenorphine in opioid-dependent patients in polytherapy in Southern Italy. The primary endpoint was the reduction of urinary positivity to the substances and the maintaining doses. Patients (N = 266, age = 44.80 ± 5.65, male = 79.70%, female = 20.30%) have been recruited. At recruitment, 75% of them were on treatment with rac-methadone, levomethadone, and buprenorphine/naloxone. The patients were grouped into three clusters. The levomethadone patients of Cluster A (N patients = 211), after 180 days, showed stability in urinary methadone positivity, with a marked decrease in heroin -53 ± 4%, cannabinol's -48 ± 2%, and cocaine -37 ± 6% positivity, with no differences between treatments. A lower QTcF value of 426 ± 8.4 ms was recorded in the levomethadone patients (delta = -19 ms) vs. rac-methadone, at significantly lower doses of levomethadone (-34%, -50.2% in males) (p < 0.05). The Cluster B data were collected from 37 patients, with a high prevalence of comorbidity infections (HIV/HCV/HPV), monitored for 21 months during COVID-19. High doses of levomethadone (58.33 ± 31.58 mg/day) were needed to stabilize those that were negative for opioids and cannabinoids, in contrast to the rac-methadone and buprenorphine/naloxone patients that showed positive toxicology. Eighteen patients of the Cluster C in double diagnosis (major depressive 38.90%, bipolar 27.78%, and schizophrenia 16.67%) were stabilized with high doses of racemate 97.5 ± 8 mg/day, 51.8 ± 5 mg/day of levomethadone (-46.8% vs. rac-methadone; -71% in men), and 2.5 ± 1 mg/day of buprenorphine/naloxone. Three patients in remission were treated with tapering doses of levomethadone. Significantly reduced QTcF values were recorded with levomethadone (delta -32 ms vs. rac-methadone) in the bipolar patients, as well as the schizophrenia patients in remission (delta -45.19 ms vs. rac-methadone). Our patients were safely stabilized. Levomethadone, compared to the racemate, contributes to reducing the illicit use, especially of opioids and cannabinoids at significantly lower doses with cardiovascular safety, which, in bipolar patients, is clinically significant.

Bisphosphonates Targeting Ion Channels and Musculoskeletal Effects.

Bisphosphonates (BPs) are the most used bone-specific anti-resorptive agents, often chosen as first-line therapy in several bone diseases characterized by an imbalance between osteoblast-mediated bone production and osteoclast-mediated bone resorption. BPs target the farnesyl pyrophosphate synthase (FPPS) in osteoclasts, reducing bone resorption. Lately, there has been an increasing interest in BPs direct pro-survival/pro-mineralizing properties in osteoblasts and their pain-relieving effects. Even so, molecular targets involved in these effects appear now largely elusive. Ion channels are emerging players in bone homeostasis. Nevertheless, the effects of BPs on these proteins have been poorly described. Here we reviewed the actions of BPs on ion channels in musculoskeletal cells. In particular, the TRPV1 channel is essential for osteoblastogenesis. Since it is involved in bone pain sensation, TRPV1 is a possible alternative target of BPs. Ion channels are emerging targets and anti-target for bisphosphonates. Zoledronic acid can be the first selective musculoskeletal and vascular KATP channel blocker targeting with high affinity the inward rectifier channels Kir6.1-SUR2B and Kir6.2-SUR2A. The action of this drug against the overactive mutants of KCNJ9-ABCC9 genes observed in the Cantu' Syndrome (CS) may improve the appropriate prescription in those CS patients affected by musculoskeletal disorders such as bone fracture and bone frailty.

Zoledronic Acid as a Novel Dual Blocker of KIR6.1/2-SUR2 Subunits of ATP-Sensitive K+ Channels: Role in the Adverse Drug Reactions.

Zoledronic acid (ZOL) is used as a bone-specific antiresorptive drug with antimyeloma effects. Adverse drug reactions (A.D.R.) are associated with ZOL-therapy, whose mechanics are unknown. ZOL is a nitrogen-containing molecule whose structure shows similarities with nucleotides, ligands of ATP-sensitive K+ (KATP) channels. We investigated the action of ZOL by performing in vitro patch-clamp experiments on native KATP channels in murine skeletal muscle fibers, bone cells, and recombinant subunits in cell lines, and by in silico docking the nucleotide site on KIR and SUR, as well as the glibenclamide site. ZOL fully inhibited the KATP currents recorded in excised macro-patches from Extensor digitorum longus (EDL) and Soleus (SOL) muscle fibers with an IC50 of 1.2 ± 1.4 × 10-6 and 2.1 ± 3.7 × 10-10 M, respectively, and the KATP currents recorded in cell-attached patches from primary long bone cells with an IC50 of 1.6 ± 2.8 × 10-10 M. ZOL fully inhibited a whole-cell KATP channel current of recombinant KIR6.1-SUR2B and KIR6.2-SUR2A subunits expressed in HEK293 cells with an IC50 of 3.9 ± 2.7 × 10-10 M and 7.1 ± 3.1 × 10-6 M, respectively. The rank order of potency in inhibiting the KATP currents was: KIR6.1-SUR2B/SOL-KATP/osteoblast-KATP > KIR6.2-SUR2A/EDL-KATP >>> KIR6.2-SUR1 and KIR6.1-SUR1. Docking investigation revealed that the drug binds to the ADP/ATP sites on KIR6.1/2 and SUR2A/B and on the sulfonylureas site showing low binding energy <6 Kcal/mol for the KIR6.1/2-SUR2 subunits vs. the <4 Kcal/mol for the KIR6.2-SUR1. The IC50 of ZOL to inhibit the KIR6.1/2-SUR2A/B channels were correlated with its musculoskeletal and cardiovascular risks. We first showed that ZOL blocks at subnanomolar concentration musculoskeletal KATP channels and cardiac and vascular KIR6.2/1-SUR2 channels.

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

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.