Lithocholic Acid Is an Eph-ephrin Ligand Interfering with Eph-kinase Activation

Eph-ephrin system plays a central role in a large variety of human cancers. In fact, alterated expression and/or de-regulated function of Eph-ephrin system promotes tumorigenesis and development of a more aggressive and metastatic tumour phenotype. In particular EphA2 upregulation is correlated with tumour stage and progression and the expression of EphA2 in non-trasformed cells induces malignant transformation and confers tumorigenic potential. Based on these evidences our aim was to identify small molecules able to modulate EphA2-ephrinA1 activity through an ELISA-based binding screening. We identified lithocholic acid (LCA) as a competitive and reversible ligand inhibiting EphA2-ephrinA1 interaction (Ki = 49 µM). Since each ephrin binds many Eph receptors, also LCA does not discriminate between different Eph-ephrin binding suggesting an interaction with a highly conserved region of Eph receptor family. Structurally related bile acids neither inhibited Eph-ephrin binding nor affected Eph phosphorylation. Conversely, LCA inhibited EphA2 phosphorylation induced by ephrinA1-Fc in PC3 and HT29 human prostate and colon adenocarcinoma cell lines (IC50 = 48 and 66 µM, respectively) without affecting cell viability or other receptor tyrosine-kinase (EGFR, VEGFR, IGFR1β, IRKβ) activity. LCA did not inhibit the enzymatic kinase activity of EphA2 at 100 µM (LANCE method) confirming to target the Eph-ephrin protein-protein interaction. Finally, LCA inhibited cell rounding and retraction induced by EphA2 activation in PC3 cells. In conclusion, our findings identified a hit compound useful for the development of molecules targeting ephrin system. Moreover, as ephrin signalling is a key player in the intestinal cell renewal, our work could provide an interesting starting point for further investigations about the role of LCA in the intestinal homeostasis

Distinct Roles of α4β2 and α7 Nicotinic Receptors in the Modulation of Inflammatory Responses in TNBS-Induced Colitis

Background and aim: Accumulating evidence indicates that vagus nerve signaling through nicotinic receptors (nAChRs) negatively modulates the inflammatory and immune responses in various clinical and experimental conditions, such as Inflammatory Bowel Disease (IBD), purportedly through a spleen-dependent pathway. Our aims were: 1)to pharmacologically investigate the role played by α4β2 and α7 nAChRs in the modulation of local and systemic inflammatory responses in 2,4,6-TriNitroBenzene Sulfonic acid (TNBS)-colitis in mice; 2)to assess the involvement of the spleen in nicotinic responses. Methods: Colitis was induced in Swiss mice by enema (i.r.) administration of 5mg/mouse TNBS 6 days after skin sensitiza- tion. Mice were randomly assigned to: CTR: saline (0.9% NaCl) 10 ml/kg TC: α4β2 agonist TC2403 5mg/kg DBE: α4β2 antagonist Dihydro-βErythroidine 1.5mg/kg AR-R: α7 agonist AR-R17779 1.5mg/kg MLA: α7 antagonist MethylLycAconitine 1mg/kg Pharmacological treatments started 8h after TNBS enema and were administered subcutaneously (s.c.) twice daily for 3 days. Normal mice (N) received saline 50 μL i.r. and 10 ml/kg s.c.. In a second series of experiments, mice subjected to splenectomy (SPX), performed 14 days before colitis induction, were administered with saline (SPX-CTR) or with AR-R17779 1.5mg/kg (SPX- AR). We determined clinical outcome as Disease Activity Index (DAI), colonic damage as Macroscopic Score (MS) and thickness and colonic and lung myeloperoxidase (MPO) activity. + Splenic and mesenteric lymph nodes (MLN) CD3 T cells were counted by flow cytometry. All animal experiments were performed according to the guidelines for the use and care of laboratory animals (DL 26/2014). Results: Compared to N, CTR mice showed markedly higher DAI (P<0.001), MS (p<0.001), colonic thickness (p<0.01), colonic (P<0.001) and + lungMPO(p<0.01);CD3 Tcellswereincreasedinthespleen(P<0.01)andslightlydecreased in MLN. Compared to CTR, TC improved only DAI (p<0.01), while DBE significantly reduced colonic MPO and DAI (p<0.05). AR-R significantly ameliorated MS (p<0.05), colonic MPO and thickness (p<0.05), while MLA slightly augmented MS and lung MPO but did not affect the other markers. In SPX-AR mice, α7 agonist lost efficacy in decreasing MS and MPO and worsened DAI but reduced colonic thickness (p<0.01); no effects were produced on T cells, either with or without SPX.Conclusions: The weak and seemingly contradictory effects shown by α4β2 agents indicated a controversial role of this nAChR subtype in TNBS colitis, thus requiring further investigations. On the contrary,α7 nAChRs, either tonically activated by the cholinergic anti-inflammatory reflex or exogenously stimu- lated, play a protective role against the inflammatory responses triggered by TNBS: this effect, not involving changes in T cells trafficking, does not rely exclusively on the splee

Accommodation and peristalsis are functional responses to obstruction in rat hypertrophic ileum

AIM: To investigate the effects of chronic obstruction on enteric reflexes evoked by electrical stimulation (EFS) or intraluminal distension of the rat hypertrophic ileum. METHODS: Motor responses to EFS and to intraluminal distension were studied in the absence and in the presence of various inhibitors of enteric mediators. Ileum segments from operated (chronic ileal obstruction), sham-operated (control) and normal rats were horizontally mounted, connected to a pressure transducer and intraluminally perfused. The effects of selective serotonin receptor (5-HTR) blockers were investigated on distension-induced responses. The cellular localization of 5-HT3Rs was also examined in control and hypertrophic tissues through confocal microscopy. RESULTS: In non-obstructed segments, EFS elicited tetrodotoxin (TTX)-sensitive responses with high amplitude contraction followed by weak relaxation. In hypertrophic tissues, EFS lowered the baseline pressure and evoked TTX-sensitive contractions significantly larger than normal (P < 0.01) or control (P < 0.05), and devoid of any relaxation phase (P < 0.01 vs normal). Incubation with atropine and guanethidine [non-adrenergic non-cholinergic (NANC) conditions] did not modify intestinal tone in normal and control preparations, but reversed the accommodation produced by EFS in hypertrophic tissues, and depressed the amplitude of contractions in all types of tissues. L-NAME and α-chymotrypsin blocked residual NANC motility in all tissues and augmented intraluminal pressure in hypertrophic segments (P < 0.05 vs NANC conditions). Intraluminal distension of the intestinal wall evoked non-propulsive cycles of contractions and relaxations in non-obstructed tissues. In all hypertrophic segments, strong propulsive strokes, markedly wider (P < 0.001), and larger than normal (P < 0.001) or control (P < 0.05) were elicited. Both motor patterns were blocked under NANC conditions and with simultaneous incubation with L-NAME and α-chymotrypsin. In all types of tissues, incubation with ketanserin or GR125487 did not modify distension-induced motility. In contrast, blockade of 5-HT3Rs by ondansetron concentration-dependently inhibited motor responses in normal and control tissues, but only slightly impaired enteric reflexes in the hypertrophic preparations. Finally, confocal microscopy did not reveal a different cellular distribution of 5-HT3Rs in control and hypertrophic ileum. CONCLUSION: Accommodation and distension-induced peristalsis of rat hypertrophic ileum are controlled by cholinergic and peptidergic transmission and are negligibly affected by 5-HT3Rs, which modulate distension-induced motility in non-obstructed tissues

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

Intestinal Hypoxia and reperfusion: role for Eph-ephrin system

Background and aim: Mesenteric ischemia-reperfusion (I/R) is a life-threatening clinical problem consisting in the temporary deprivation of blood flow to the gut. The ensuing local organ damage is followed by the development of a systemic inflammatory response, eventually leading to multiple organ failure. Several neuronal, paracrine and plasmatic messengers are involved in mesenteric I/R injury. However, up to now, no studies have been performed on Eph receptors, the largest family of tyrosine kinase receptors, and their cell-bound ephrin ligands, whose role in cell adhesion-based processes during inflammation and immunity is growingly emerging. Eph-ephrin interaction generates a bidirectional signaling affecting both the receptor bearing (forward signaling) and the ligand bearing cells (reverse signaling). Our aim was therefore to investigate the effects produced by unidirectional activation of forward signaling (administration of chimeric protein ephrinA1-Fc; 50, 100, 200microg/kg), of reverse signaling (EphA2-Fc; 180microg/kg), or inhibition of both signals (monomeric EphA2 at equimolar doses of ephrinA1-Fc) on the inflammatory responses caused by mesenteric I/R in mice. Methods: Eph-ephrin proteins were intravenously administered to Swiss mice 5 min prior to 45 min occlusion of the superior mesenteric artery (SMA) followed by 5h reperfusion: intestinal and lung myeloperoxidase (MPO) activity, index of neutrophil recruit- ment, gut malondialdehyde (MDA) levels, marker of lipoperoxidation, and edema, index of increased vascular permeability, were compared to those of control I/R mice, receiving only vehicle, and of sham operated (SO) mice, subjected to the same manipulation without SMA occlusion and receiving vehicle. All experiments were performed according to the guidelines for the Care and Use of Animals (DL26/2014). Results: I/R mice displayed increased gut (45.3±4.7 vs 0.8±0.5 U/g, P<0.001) and lung (289.9±21.0vs 144.6±45.1 U/g, P<0.05) MPO activity and higher MDA levels (595.0±87.3 vs 203.4±62.6 nmol/g dry tissue, P<0.05) and edema (4.4±0.1 vs 3.4±0.3 wet to dry weight ratio, P<0.01) compared to SO. EphrinA1- Fc markedly reduced leukocyte infiltration in the gut (24.9±2.9 U/g) at 100microg/kg and in the lung (128.0±19.1 U/g) at 200microg/kg, while monomeric EphA2 significantly lowered intestinal edema formation (3.9±0.1g/g, P<0.05 vs I/R) at the highest dose. Neither EphA2- Fc nor equimolar doses of IgG-Fc alone were effective in modifying I/R-induced inflammatory responses. Conclusions: These preliminary findings indicate that pharmacological modula- tion of Eph-ephrin system may be advantageous in limiting the inflammatory responses elicited by mesenteric I/R: forward signaling activation attenuates local and systemic leuko- cytes enrolment and reverse signaling silencing contrasts local changes in vascular perme- ability

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

A promising imidazole-free histamine H3 receptor antagonist with good brain penetration and anticholinesterase activity.

Histamine H3 antagonists, lacking the imidazole ring that is associated with the drawbacks of CYP450 inhibition and low CNS access, have been recently proposed as a new therapeutic approach to several cognitive disorders characterized by deficit of neurotransmitter release such as Alzheimer disease, a pathology currently treated with cholinesterase (ChE) inhibitors. In the search for an improved treatment, a new biphenylic derivative five has been developed (Morini et al., 2006) as agent combining anticholinesterase activity with H3 histamine receptor inhibition. The selectivity, affinity and potency at H3 receptors and the inhibition of ChE were studied in vitro. The ability to enter the CNS (ex vivo binding) and to exert anti-amnesic activity (passive avoidance test) were investigated in 200–250 g female Wistar rats, applying experimental procedures supervised and approved by the Ministero della Salute (DL116/92). The in vitro results are summarized in Table 1. In ex vivo study compound 5 was 3 fold more potent than the conventional H3-blocker thioperamide in inhibiting [3H](R)-a-methylhistamine cortical binding (ED50 = 0.63 vs. 2.04 mg/kg i.p.). At 1.25 mg/kg i.p. it was as effective as the anti- Alzheimer drug donepezil and 7 fold more potent than thioperamide in reverting scopolamine-induced amnesia. These results suggest that the good CNS penetration and the dual inhibition of cholinesterase and histamine H3-receptors could account for the antiamnesic effect of compound 5, a potential benchmark for the development of non-imidazole H3-antagonists with therapeutic potential in cognitive disorders. Reference: Morini et al. Bioorg Med Chem Lett. 2006; 16: 406