Targeting glucosylceramide synthase induces antiproliferative and proapoptotic effects in osimertinib-resistant NSCLC cell models.

The EGFR tyrosine kinase inhibitor osimertinib has been approved for the first-line treatment of EGFR-mutated Non-Small Cell Lung Cancer (NSCLC) patients. Despite its efficacy, patients develop resistance. Mechanisms of resistance are heterogeneous and not fully understood, and their characterization is essential to find new strategies to overcome resistance. Ceramides are well-known regulators of apoptosis and are converted into glucosylceramides (GlcCer) by glucosylceramide synthase (GCS). A higher content of GlcCers was observed in lung pleural effusions from NSCLC patients and their role in osimertinib-resistance has not been documented. The aim of this study was to determine the therapeutic potential of inhibiting GCS in NSCLC EGFR-mutant models resistant to osimertinib in vitro and in vivo. Lipidomic analysis showed a significant increase in the intracellular levels of glycosylceramides, including GlcCers in osimertinib resistant clones compared to sensitive cells. In resistant cells, the GCS inhibitor PDMP caused cell cycle arrest, inhibition of 2D and 3D cell proliferation, colony formation and migration capability, and apoptosis induction. The intratumoral injection of PDMP completely suppressed the growth of OR xenograft models. This study demonstrated that dysregulation of ceramide metabolism is involved in osimertinib-resistance and targeting GCS may be a promising therapeutic strategy for patients progressed to osimertinib.

Development of inhalation powders containing lactic acid bacteria with antimicrobial activity against Pseudomonas aeruginosa.

OBJECTIVES: The aim of the project was to develop and characterise powders containing a probiotic (Lactiplantibacillus plantarum [Lpb. plantarum], Lacticaseibacillus rhamnosus, or Lactobacillus acidophilus) to be administered to the lung for the containment of pathogen growth in patients with lung infections. METHODS: The optimised spray drying process for the powder manufacturing was able to preserve viability of the bacteria, which decreased of only one log unit and was maintained up to 30 days. RESULTS: Probiotic powders showed a high respirability (42%-50% of particles had a size < 5 µm) suitable for lung deposition and were proven safe on A549 and Calu-3 cells up to a concentration of 107 colony-forming units/mL. The Lpb. plantarum adhesion to both cell lines tested was at least 10%. Surprisingly, Lpb. plantarum powder was bactericidal at a concentration of 106 colony-forming units/mL on P. aeruginosa, whereas the other two strains were bacteriostatic. CONCLUSION: This work represents a promising starting point to consider a probiotic inhalation powder a value in keeping the growth of pathogenic microflora in check during the antibiotic inhalation therapy suspension in cystic fibrosis treatment regimen. This approach could also be advantageous for interfering competitively with pathogenic bacteria and promoting the restoration of the healthy microbiota.

An Enhanced Dissolving Cyclosporin-A Inhalable Powder Efficiently Reduces SARS-CoV-2 Infection In Vitro.

This work illustrates the development of a dry inhalation powder of cyclosporine-A for the prevention of rejection after lung transplantation and for the treatment of COVID-19. The influence of excipients on the spray-dried powder's critical quality attributes was explored. The best-performing powder in terms of dissolution time and respirability was obtained starting from a concentration of ethanol of 45% (v/v) in the feedstock solution and 20% (w/w) of mannitol. This powder showed a faster dissolution profile (Weibull dissolution time of 59.5 min) than the poorly soluble raw material (169.0 min). The powder exhibited a fine particle fraction of 66.5% and an MMAD of 2.97 µm. The inhalable powder, when tested on A549 and THP-1, did not show cytotoxic effects up to a concentration of 10 µg/mL. Furthermore, the CsA inhalation powder showed efficiency in reducing IL-6 when tested on A549/THP-1 co-culture. A reduction in the replication of SARS-CoV-2 on Vero E6 cells was observed when the CsA powder was tested adopting the post-infection or simultaneous treatment. This formulation could represent a therapeutic strategy for the prevention of lung rejection, but is also a viable approach for the inhibition of SARS-CoV-2 replication and the COVID-19 pulmonary inflammatory process.

Micro-fragmented Fat Inhibits the Progression of Human Mesothelioma Xenografts in Mice.

BACKGROUND: Malignant pleural mesothelioma is a pathology with no effective therapy and a poor prognosis. Our previous study demonstrated an in vitro inhibitory effect on mesothelioma cell lines of both the lysate and secretome of adipose tissue-derived Mesenchymal Stromal Cells. The inhibitory activity on tumor growth has been demonstrated also in vivo: five million Mesenchymal Stromal Cells, injected "in situ", produced a significant therapeutic efficacy against MSTO-211H xenograft equivalent to that observed after the systemic administration of paclitaxel. OBJECTIVE: The objective of this study is to evaluate the efficacy of low amount (half a million) Mesenchymal Stromal Cells and micro-fragmented adipose tissues (the biological tissue from which the Mesenchymal Stromal Cells were isolated) on mesothelioma cells growth. METHODS: Tumor cells growth inhibition was evaluated in vitro and in a xenograft model of mesothelioma. RESULTS: The inhibitory effect of micro-fragmented fat from adipose-tissue has been firstly confirmed in vitro on MSTO-211H cell growth. Then the efficacy against the growth of mesothelioma xenografts in mice of both micro-fragmented fat and low amount of Mesenchymal Stromal Cells has been evaluated. Our results confirmed that both Mesenchymal Stromal Cells and micro-fragmented fat, injected "in situ", did not stimulate mesothelioma cell growth. By contrast, micro-fragmented fat produced a significant inhibition of tumor growth and progression, comparable to that observed by the treatment with paclitaxel. Low amount of Mesenchymal Stromal Cells exerted only a little anticancer activity. CONCLUSION: Micro-fragmented fat inhibited mesothelioma cell proliferation in vitro and exerted a significant control of the mesothelioma xenograft growth in vivo.

Probing the effects of MR120 in preclinical chronic colitis: A first-in-class anti-IBD agent targeting the CCL20/CCR6 axis.

Concerning the growing interest in the role played by the CCL20/CCR6 axis in IBD pathogenesis and in the search for novel anti-IBD small molecules, we have recently discovered the first small-molecule (MR120) endowed with protective action against TNBS-induced colitis and zymosan-induced peritonitis. This protective action occurs through interference with the CCL20/CCR6 signaling. The aim of the present work is to expand the preclinical investigation of MR120, evaluating its beneficial anti-inflammatory effect on a model of chronic colitis obtained by cyclically exposing C57BL/6 mice to 3% DSS. Subcutaneous administration of MR120 at 1 mg/kg, the same dose effective against acute inflammation, helped attenuate several systemic and local inflammatory responses induced by DSS. Besides significantly improving murine health conditions, MR120 counteracted mucosal macroscopic injury, the increase of colonic edema and neutrophils oxidative activity, and mitigated spleen enlargement, while not significantly lowering intestinal IL-6 concentration. Overall, repeated daily treatment with MR120 for approximately 30 days was well tolerated and showed moderate protection in a relevant model of chronic colitis, in line with the beneficial effect previously observed in acute models of intestinal inflammation. Although more potent analogues of MR120 will be needed to more fully evaluate their clinical translatability, the present work provides a valuable example of in vivo efficacy of CCL20/CCR6 modulators in a chronic model of IBD.

Inhibition of Human Malignant Pleural Mesothelioma Growth by Mesenchymal Stromal Cells.

BACKGROUND: Malignant Pleural Mesothelioma (MPM) is an aggressive tumor that has a significant incidence related to asbestos exposure with no effective therapy and poor prognosis. The role of mesenchymal stromal cells (MSCs) in cancer is controversial due to their opposite effects on tumor growth and in particular, only a few data are reported on MSCs and MPM. METHODS: We investigated the in vitro efficacy of adipose tissue-derived MSCs, their lysates and secretome against different MPM cell lines. After large-scale production of MSCs in a bioreactor, their efficacy was also evaluated on a human MPM xenograft in mice. RESULTS: MSCs, their lysate and secretome inhibited MPM cell proliferation in vitro with S or G0/G1 arrest of the cell cycle, respectively. MSC lysate induced cell death by apoptosis. The efficacy of MSC was confirmed in vivo by a significant inhibition of tumor growth, similar to that produced by systemic administration of paclitaxel. Interestingly, no tumor progression was observed after the last MSC treatment, while tumors started to grow again after stopping chemotherapeutic treatment. CONCLUSIONS: These data demonstrated for the first time that MSCs, both through paracrine and cell-to-cell interaction mechanisms, induced a significant inhibition of human mesothelioma growth. Since the prognosis for MPM patients is poor and the options of care are limited to chemotherapy, MSCs could provide a potential new therapeutic approach for this malignancy.

UniPR1331: Small Eph/Ephrin Antagonist Beneficial in Intestinal Inflammation by Interfering with Type-B Signaling.

Eph receptors, comprising A and B classes, interact with cell-bound ephrins generating bidirectional signaling. Although mainly related to carcinogenesis and organogenesis, the role of Eph/ephrin system in inflammation is growingly acknowledged. Recently, we showed that EphA/ephrin-A proteins can modulate the acute inflammatory responses induced by mesenteric ischemia/reperfusion, while beneficial effects were granted by EphB4, acting as EphB/ephrin-B antagonist, in a murine model of Crohn's disease (CD). Accordingly, we now aim to evaluate the effects of UniPR1331, a pan-Eph/ephrin antagonist, in TNBS-induced colitis and to ascertain whether UniPR1331 effects can be attributed to A- or B-type signaling interference. The potential anti-inflammatory action of UniPR1331 was compared to those of the recombinant proteins EphA2, a purported EphA/ephrin-A antagonist, and of ephrin-A1-Fc and EphA2-Fc, supposedly activating forward and reverse EphA/ephrin-A signaling, in murine TNBS-induced colitis and in stimulated cultured mononuclear splenocytes. UniPR1331 antagonized the inflammatory responses both in vivo, mimicking EphB4 protection, and in vitro; EphA/ephrin-A proteins were inactive or only weakly effective. Our findings represent a further proof-of-concept that blockade of EphB/ephrin-B signaling is a promising pharmacological strategy for CD management and highlight UniPR1331 as a novel drug candidate, seemingly working through the modulation of immune responses.

Excipient-free pulmonary insulin dry powder: Pharmacokinetic and pharmacodynamics profiles in rats.

A novel pure insulin spray-dried powder for DPI product (Ins_SD) was studied with respect to physico-chemical stability, in vitro respirability, bioavailability, activity and tolerability. Ins_SD powder exhibited a very high in vitro respirability, independently of the DPI product preparation (manual or semi-automatic). Physico-chemical characteristics of Ins_SD powder remained within the pharmacopoeia limits during 6 months of storage at room temperature. PK/PD profiles were measured in rats that received the pulmonary powders by intratracheal insufflation and compared with Afrezza inhalation insulin. Due to the low drug powder mass to deliver, both insulin powders were diluted with mannitol. Insulin from Ins_SD was promptly absorbed (tmax 15 min and Cmaxx4.9 ±â€¯1.5 mU/ml). Afrezza had a slower absorption (tmax 30 min and Cmax of 1.8 ±â€¯0.37 mU/ml). After glucose injection, Ins_SD determined a rapid reduction of glucose level, similar to Afrezza. As reference, insulin subcutaneous injection showed a long-lasting hypoglycemic effect due to the slow absorption that prolonged insulin plasma level. In summary, Ins_SD product is suitable for post-prandial glucose control, providing a convenient and compliant product, in particular in the event of using a disposable device. Albeit the product has to be stored in fridge, its stability at room temperature allows the diabetic individual to carry the daily dose in normal conditions.

Novel analgesic agents obtained by molecular hybridization of orthosteric and allosteric ligands.

Despite the high incidence of acute and chronic pain in the general population, the efficacy of currently available medications is unsatisfactory. Insufficient management of pain has a profound impact on the quality of life and can have serious physical, psychological, social, and economic consequences. This unmet need reflects a failure to develop novel classes of analgesic drugs with superior clinical properties and lower risk of abuse. Nevertheless, recent advances in our understanding of the neurobiology of pain are offering new opportunities for developing different therapeutic approaches. Among those, the activation of M2 muscarinic acetylcholine receptors, which play a key role in the cholinergic regulation of the nociceptive transmission, constitutes one of the most promising strategies. We have recently developed a small library of novel pharmacological agents by merging the structures of known orthosteric and allosteric muscarinic ligands through their molecular hybridization, an emerging approach in medicinal chemistry based on the combination of pharmacophoric moieties of different bioactive substances to produce a new compound with improved pharmacological properties. Herein we report the functional characterization of the new ligands in vitro and the assessment of their efficacy as analgesic agents and tolerability in mice. This work provides new insights for the development and optimization of novel muscarinic hybrid compounds for the management of pain.

Targeting the Eph/Ephrin System as Anti-Inflammatory Strategy in IBD.

Besides their long-known critical role in embryonic growth and in cancer development and progression, erythropoietin-producing hepatocellular carcinoma type B (EphB) receptor tyrosine kinases and their ephrin-B ligands are involved in the modulation of immune responses and in remodeling and maintaining the integrity of the intestinal epithelial layer. These processes are critically involved in the pathogenesis of inflammatory-based disorders of the gut, like inflammatory bowel diseases (IBDs). Accordingly, our aim was to investigate the role of the EphB/ephrin-B system in intestinal inflammation by assessing the local and systemic effects produced by its pharmacological manipulation in 2,4,6-trinitrobenzenesulfonic acid (TNBS)- (Th1-dependent model) and dextran sulphate sodium (DSS)- (innate response model) induced colitis in mice. To this purpose, we administered chimeric Fc-conjugated proteins, allegedly able to uni-directionally activate either forward (ephrin-B1-Fc) or reverse (EphB1-Fc) signaling, and the soluble monomeric EphB4 extracellular domain protein, that, simultaneously interfering with both signaling pathways, acts as EphB/ephrin-B antagonist.The blockade of the EphB/ephrin-B forward signaling by EphB4 and EphB1-Fc was ineffective against DSS-induced colitis while it evoked remarkable beneficial effects against TNBS colitis: it counteracted all the evaluated inflammatory responses and the changes elicited on splenic T lymphocytes subpopulations, without preventing the appearance of a splice variant of ephrin-B2 gene elicited by the haptenating agent in the colon. Interestingly, EphB4, preferentially displacing EphB4/ephrin-B2 interaction over EphB1/ephrin-B1 binding, was able to promote Tumor Necrosis Factor alpha (TNFα) release by splenic mononuclear cells in vitro. On the whole, the collected results point to a potential role of the EphB/ephrin-B system as a pharmacological target in intestinal inflammatory disorders and suggest that the therapeutic efficacy of its blockade seemingly works through the modulation of immune responses, independent of the changes at the transcriptional and translational level of EphB4 and ephrin-B2 genes.

Third generation EGFR inhibitor osimertinib combined with pemetrexed or cisplatin exerts long-lasting anti-tumor effect in EGFR-mutated pre-clinical models of NSCLC.

BACKGROUND: The third generation Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor (TKI) osimertinib has been initially approved for T790M positive Non-Small Cell Lung Cancer (NSCLC) and more recently for first-line treatment of EGFR-mutant T790M negative NSCLC patients. Similarly to previous generation TKIs, despite the high response rate, disease progression eventually occurs and current clinical research is focused on novel strategies to delay the emergence of osimertinib resistance. In this study we investigated the combination of osimertinib with pemetrexed or cisplatin in EGFR-mutated NSCLC cell lines and xenografts. METHODS: Tumor growth was evaluated in a PC9T790M xenograft model and tissue composition was morphometrically determined. PC9, PC9T790M and HCC827 cell lines were employed to test the efficacy of osimertinib and chemotherapy combination in vitro. Cell viability and cell death were evaluated by MTT assay and fluorescence microscopy. Protein expression and gene status were analysed by Western blotting, fluorescence in situ hybridization analysis, next-generation sequencing and digital droplet PCR. RESULTS: In xenograft models, osimertinib significantly inhibited tumor growth, however, as expected, in 50% of mice drug-resistance developed. A combination of osimertinib with pemetrexed or cisplatin prevented or at least delayed the onset of resistance. Interestingly, such combinations increased the fraction of fibrotic tissue and exerted a long-lasting activity after stopping therapy. In vitro studies demonstrated the stronger efficacy of the combination over the single treatments in inhibiting cell proliferation and inducing cell death in PC9T790M cells as well as in T790M negative PC9 and HCC827 cell lines, suggesting the potential role of this strategy also as first-line treatment. Finally, we demonstrated that osimertinib resistant clones, either derived from resistant tumors or generated in vitro, were less sensitive to pemetrexed prompting to use a chemotherapy regimen non-containing pemetrexed in patients after progression to osimertinib treatment. CONCLUSIONS: Our results identify a combination between osimertinib and pemetrexed or cisplatin potentially useful in the treatment of EGFR-mutated NSCLC patients, which might delay the appearance of osimertinib resistance with long-lasting effects.

3D-printed chitosan-based scaffolds: An in vitro study of human skin cell growth and an in-vivo wound healing evaluation in experimental diabetes in rats.

The fabrication of porous 3D printed chitosan (CH) scaffolds for skin tissue regeneration and their behavior in terms of biocompatibility, cytocompatibility and toxicity toward human fibroblasts (Nhdf) and keratinocytes (HaCaT), are presented and discussed. 3D cell cultures achieved after 20 and 35 days of incubation showed significant in vitro qualitative and quantitative cell growth as measured by neutral red staining and MTT assays and confirmed by scanning electron microphotographs. The best cell growth was obtained after 35 days on 3D scaffolds when the Nhdf and HaCaT cells, seeded together, filled the pores in the scaffolds. An early skin-like layer consisting of a mass of fibroblast and keratinocyte cells growing together was observed. The tests of 3D printed scaffolds in wound healing carried out on streptozotocin-induced diabetic rats demonstrate that 3D printed scaffolds improve the quality of the restored tissue with respect to both commercial patch and spontaneous healing.

Rifaximin anti-inflammatory activity on bovine endometrium primary cell cultures: a preliminary study.

Rifaximin is an unabsorbed oral antibiotic showing anti-inflammatory properties in human pathologies like irritable bowel syndrome and inflammatory bowel disease. In veterinary medicine, rifaximin is primarily used in the treatment of dermatological diseases in all animal species, in therapy and prophylaxis of mastitis in cows and in the treatment of endometritis in cattle and horses. The aim of this preliminary study was to evaluate the anti-inflammatory properties of rifaximin on primary cell cultures from bovine endometrium in which inflammatory response was induced by Lipopolysaccaride (LPS) treatment. Epithelial and stromal cells were isolated from bovine endometrium and separately incubated for 24 h with 1 µg mL-1 LPS after rifaximin (10, 50 and 100 µmol L-1 ) or dexamethasone (10 µmol L-1 ) pre-treatment for 24 h. Supernatants were collected 24 h after LPS treatment and interleukin (IL)-6 and IL-8 accumulation was measured by ELISA. Rifaximin (10, 50 and 100 µmol L-1 ) dose dependently inhibited the LPS-induced increase in IL-6 and IL-8 in stromal cells, whereas in epithelial cells it was not possible to detect any accumulation of these interleukins. Rifaximin reduced IL-6 and IL-8 production, showing a potential anti-inflammatory effect that opens up to new possibilities for the use of this drug in uterine inflammatory diseases.

Trastuzumab emtansine delays and overcomes resistance to the third-generation EGFR-TKI osimertinib in NSCLC EGFR mutated cell lines.

BACKGROUND: Osimertinib is a third-generation EGFR-TKI with a high selective potency against T790M-mutant NSCLC patients. Considering that osimertinib can lead to enhanced HER-2 expression on cell surface and HER-2 overexpression is a mechanism of resistance to osimertinib, this study was addressed to investigate the potential of combining osimertinib with trastuzumab emtansine (T-DM1) in order to improve the efficacy of osimertinib and delay or overcome resistance in NSCLC cell lines with EGFR activating mutation and with T790M mutation or HER-2 amplification. METHODS: The effects of osimertinib combined with T-DM1 on cell proliferation, cell cycle, cell death, antibody-dependent cell-mediated cytotoxicity (ADCC), and acquisition of osimertinib resistance was investigated in PC9, PC9-T790M and H1975 cell lines. The potential of overcoming osimertinib resistance with T-DM1 was tested in a PC9/HER2c1 xenograft model. RESULTS: T-DM1 exerted an additive effect when combined with osimertinib in terms of inhibition of cell proliferation, cell death and ADCC induction in PC9, PC9-T790M and H1975 cell lines. Combining osimertinib and T-DM1 using different schedules in long-term growth experiments revealed that the appearance of osimertinib-resistance was prevented in PC9-T790M and delayed in H1975 cells when the two drugs were given together. By contrast, when osimertinib was followed by T-DM1 an antagonistic effect was observed on cell proliferation, cell death and resistance acquisition. In xenograft models, we demonstrated that HER-2 amplification was associated with osimertinib-resistance and that T-DM1 co-administration is a potential strategy to overcome this resistance. CONCLUSIONS: Our data suggest that concomitant treatment with osimertinib and T-DM1 may be a promising therapeutic strategy for EGFR-mutant NSCLC.

α7 Nicotinic Agonist AR-R17779 Protects Mice against 2,4,6-Trinitrobenzene Sulfonic Acid-Induced Colitis in a Spleen-Dependent Way.

The existence of a cholinergic anti-inflammatory pathway negatively modulating the inflammatory and immune responses in various clinical conditions and experimental models has long been postulated. In particular, the protective involvement of the vagus nerve and of nicotinic Ach receptors (nAChRs) has been proposed in intestinal inflammation and repeatedly investigated in DSS- and TNBS-induced colitis. However, the role of α7 nAChRs stimulation is still controversial and the potential contribution of α4ß2 nAChRs has never been explored in this experimental condition. Our aims were therefore to pharmacologically investigate the role played by both α7 and α4ß2 nAChRs in the modulation of the local and systemic inflammatory responses activated in TNBS-induced colitis in mice and to assess the involvement of the spleen in nicotinic responses. To this end, TNBS-exposed mice were sub-acutely treated with various subcutaneous doses of highly selective agonists (AR-R17779 and TC-2403) and antagonists (methyllycaconitine and dihydro-ß-erythroidine) of α7 and α4ß2 nAChRs, respectively, or with sulfasalazine 50 mg/kg per os and clinical and inflammatory responses were evaluated by means of biochemical, histological and flow cytometry assays. α4ß2 ligands evoked weak and contradictory effects, while α7 nAChR agonist AR-R17779 emerged as the most beneficial treatment, able to attenuate several local markers of colitis severity and to revert the rise in splenic T-cells and in colonic inflammatory cytokines levels induced by haptenization. After splenectomy, AR-R17779 lost its protective effects, demonstrating for the first time that, in TNBS-model of experimental colitis, the anti-inflammatory effect of exogenous α7 nAChR stimulation is strictly spleen-dependent. Our findings showed that the selective α7 nAChRs agonist AR-R17779 exerted beneficial effects in a model of intestinal inflammation characterized by activation of the adaptive immune system and that the spleen is essential to mediate this cholinergic protection.

Novel bipharmacophoric inhibitors of the cholinesterases with affinity to the muscarinic receptors M1 and M2.

A set of hybrid compounds composed of the fragment of allosteric modulators of the muscarinic receptor, i.e. W84 and naphmethonium, and the well-known AChE inhibitor tacrine on the one hand, and the skeletons of the orthosteric muscarinic agonists, iperoxo and isox, on the other hand, were synthesized. The two molecular moieties were connected via a polymethylene linker of varying length. These bipharmacophoric compounds were investigated for inhibition of AChE (from electric eel) and BChE (from equine serum) as well as human ChEs in vitro and compared to previously synthesized dimeric inhibitors. Among the studied hybrids, compound 10-C10, characterized by a 10 carbon alkylene linker connecting tacrine and iperoxo, proved to be the most potent inhibitor with the highest pIC50 values of 9.81 (AChE from electric eel) and 8.75 (BChE from equine serum). Docking experiments with compounds 10-C10, 7b-C10, and 7a-C10 helped to interpret the experimental inhibitory power against AChE, which is affected by the nature of the allosteric molecular moiety, with the tacrine-containing hybrid being much more active than the naphthalimido- and phthalimido-containing analogs. Furthermore, the most active AChE inhibitors were found to have affinity to M1 and M2 muscarinic receptors. Since 10-C10 showed almost no cytotoxicity, it emerged as a promising lead structure for the development of an anti-Alzheimer drug.

Esomeprazole immediate release tablets: Gastric mucosa ex vivo permeation, absorption and antisecretory activity in conscious rats.

The aim of this work was to study the esomeprazole activity on the control of gastric secretion after administration of a novel immediate release tablet. The ex vivo permeation of esomeprazole across porcine gastric mucosa from immediate release tablets, containing sodium carbonate or magnesium oxide as alkalinizing agents, was firstly assessed. Pharmacokinetics and pharmacodynamics studies in conscious rats following the administration of immediate release tablets with sodium carbonate, in comparison with delayed-release tablets having the same formula, were also conducted. The results showed an important effect of sodium carbonate and magnesium oxide on the drug release, on the ex vivo trans-mucosal transport and the stability in acid environment. In particular, the presence of sodium carbonate in esomeprazole tablet formulation provided the maximum increase of the drug in vitro transport across the mucosa. Then, the absorption and the antisecretory activity of this proton pump inhibitor orally administered in rats as immediate release tablets containing Na2CO3, was superior but not significantly different compared to delayed-release tablets having the same formula. In the adopted animal model, an activity of esomeprazole from immediate release alkaline formulation was seen also in presence of partial gastric absorption allowing inhibition of proton pumps reached via systemic circulation. This esomeprazole immediate release formulation could be used for the on-demand treatment of acid-related disorders such as gastro-esophageal reflux disease.

Hake fish bone as a calcium source for efficient bone mineralization.

Calcium is recognized as an essential nutritional factor for bone health. An adequate intake is important to achieve or maintain optimal bone mass in particular during growth and old age. The aim of the present study was to evaluate the efficiency of hake fish bone (HBF) as a calcium source for bone mineralization: in vitro on osteosarcoma SaOS-2 cells, cultured in Ca-free osteogenic medium (OM) and in vivo on young growing rats fed a low-calcium diet. Lithotame (L), a Ca supplement derived from Lithothamnium calcareum, was used as control. In vitro experiments showed that HBF supplementation provided bone mineralization similar to standard OM, whereas L supplementation showed lower activity. In vivo low-Ca HBF-added and L-added diet similarly affected bone deposition. Physico-chemical parameters concerning bone mineralization, such as femur breaking force, tibia density and calcium/phosphorus mineral content, had beneficial effects from both Ca supplementations, in the absence of any evident adverse effect. We conclude HBF derived from by-product from the fish industry is a good calcium supplier with comparable efficacy to L.

Development and validation of a DESI-HRMS/MS method for the fast profiling of esomeprazole and its metabolites in rat plasma: a pharmacokinetic study.

The advances in pharmaceutical development and drug discovery impose the availability of reliable high-throughput screening methods for the rapid evaluation of drug metabolism and pharmacokinetic (PK) in biological samples. Here, a desorption electrospray mass spectrometry (DESI-MS) method has been developed and validated for the PK profiling of esomeprazole and its metabolites (5-hydroxyomeprazole and omeprazole sulfone) in rat plasma. Rats were treated with an esomeprazole solution (2.5 mg/mL) for endovenous administration and the analyte levels were profiled over 2 h after liquid-liquid extraction from plasma. MS and tandem mass spectrometry (MS/MS) experiments were performed by using a DESI-LTQ-Orbitrap XL instrument and an on-spot fixed time analysis on PMMA surfaces. Validation was performed for the esomeprazole. The DESI-MS/MS method exhibited for the esomepazole excellent sensitivity (limit of detection (LOD)=60 ng/mL), linearity (0.2-20 µg/mL concentration range; y=23848(±361)X, n=15; r(2) =0.987) and precision (RSD<9%) by using an internal standard method. The PK results were discussed in terms of Area Under the Curve, Cmax and Tmax . Data reliability was demonstrated by comparison with a liquid chromatography-tandem mass spectrometry method (p>0.05). The data achieved demonstrated that the DESI-MS method is suitable for sensitive and fast profiling of a drug and its metabolites at the therapeutic concentration levels.

Novel Potent N-Methyl-d-aspartate (NMDA) Receptor Antagonists or σ1 Receptor Ligands Based on Properly Substituted 1,4-Dioxane Ring.

Two series of 1,4-dioxanes (4-11 and 12-19) were rationally designed and prepared to interact either with the phencyclidine (PCP) binding site of the N-methyl-d-aspartate (NMDA) receptor or with σ1 receptors, respectively. The biological profiles of the novel compounds were assessed using radioligand binding assays, and the compounds with the highest affinities were investigated for their functional activity. The results were in line with the available pharmacophore models and highlighted that the 1,4-dioxane scaffold is compatible with potent antagonist activity at NMDA receptor or high affinity for σ1 receptors. The primary amines 6b and 7 bearing a cyclohexyl and a phenyl ring or two phenyl rings in position 6, respectively, were the most potent noncompetitive antagonists at the NMDA receptor with IC50 values similar to those of the dissociative anesthetic (S)-(+)-ketamine. The 5,5-diphenyl substitution associated with a benzylaminomethyl moiety in position 2, as in 18, favored the interaction with σ1 receptors.