Two sides of the same coin: Wnt signaling in neurodegeneration and neuro-oncology.

Wnts function through the activation of at least three intracellular signal transduction pathways, of which the canonical beta-catenin mediated pathway is the best understood. Aberrant canonical Wnt signaling has been involved in both neurodegeneration and cancer. An impairment of Wnt signals appears to be associated with aspects of neurodegenerative pathologies while overactivation of Wnt signaling is a common theme in several types of human tumors. Therefore, although therapeutic approaches aimed at modulating Wnt signaling in neurodegenerative and hyperproliferative diseases might impinge on the same molecular mechanisms, different pharmacological outcomes are required. Here we review recent developments on the understanding of the role of Wnt signaling in Alzheimer's disease and CNS tumors, and identify possible avenues for therapeutic intervention within a complex and multi-faceted signaling pathway.

Can voltammetry measure nitrogen monoxide (NO) and/or nitrites?

Recently, voltammetry with carbon fibre electrodes (CFE) has been implemented for real time measurement of nitrogen monoxide (NO) indicating that it is oxidised at the potential value of nitrites, approximately +700 mV. In contrast, here we show that modified CFE can monitor NO at oxidation potentials different than that of nitrites, i.e. +550 mV. Indeed, at +550 mV a significant increase of amperometric current levels was obtained when NO but not nitrites, were added to a phosphate buffer saline solution (PBS). Differential pulse voltammetry (DPV) supports these findings as two oxidation peaks were obtained when examining air preserved NO; peak 1 at +550 mV and peak 2 at +700 mV, respectively. In contrast, only peak 2 was monitored when nitrites or a solution of NO oxidised in air was added to PBS. Biological support to these in vitro data comes from the observation that the relaxation of an adrenaline-contracted aortic ring produced via addition of NO is concomitant with peak 1 at +550 mV. The relaxation is almost completed before the appearance of peak 2 at +700 mV. Furthermore, in vivo experiments performed in the striatum of rats show that the amperometric signal monitored at +550 mV is responsive to glutamatergic stimulation or inhibition of NO synthase.

Successful xenografting of cryopreserved primary pancreatic cancers.

In order to assess the suitability of cryopreserved neoplastic tissues for xenografting into nude (nu/nu) mice, we compared the take rate in 28 samples of pancreatic ductal carcinoma. Eleven fresh samples were implanted in nu/nu mice, and 17 were frozen in cryopreserving solution and implanted at a later time. All samples were examined for the presence of neoplastic tissue in cryostat sections. A total of 15 tumors grew in the animals; five from the freshly implanted samples and ten from those cryopreserved. Ten xenografted tumors were characterized for alterations in p53, K-ras, and p16 genes, which were found in six, eight, and nine cases, respectively. Our results demonstrate that the take rate for xenografting is comparable between cryopreserved and fresh tissue samples. The procedure allows for the exchange of tumor material between institutions and permits the establishment of centralized facilities for the storage of an array of different primary tumor samples suitable for the production of in vivo models of cancers.

Electrochemical evidence that lacidipine stimulates release of nitrogen monoxide in rat aorta.

Dihidropyridines (DHPs) such as amlodipine, lercanidipine and lacidipine, are compounds capable of vascular protection via their calcium antagonist activity. In addition, they present vascular dilatation function, which has been related to an anti endothelin efficacy, particularly for lacidipine. Recent works have suggested that DHPs modulate vascular relaxation via increase in the release of nitrogen monoxide (NO). Using voltammetry with selective biosensors the present experiments performed in rat aortic rings demonstrate the capability of DHPs to implement endothelial NO at 'useful' and not toxic nanomolar levels, with a maximum efficacy for lacidipine. This activity joins the already described positive effects of these compounds upon vascular functions.

Lacidipine [correction of Lalsoacidipine] modulates the secretion of matrix metalloproteinase-9 by human macrophages.

Activated macrophages within the arterial wall secrete matrix-degrading metalloproteinases (MMPs) that weaken the atherosclerotic plaque and contribute to its fissuration. Preclinical studies have shown that calcium antagonists may reduce atherogenesis in the arterial wall. In the present study we evaluated the effect of lacidipine on 92-kDa gelatinase B (MMP-9) expression in human macrophages in cultures. Cells were treated for 24 h with lacidipine and the conditioned media were analyzed. Lacidipine (1-20 microM) significantly reduced, in a dose-dependent manner, MMP-9 potential gelatinolytic capacity up to 50%. When MMP-9 expression was stimulated by treatment with phorbol esters or tumor necrosis factor-alpha, lacidipine was able to inhibit this enhanced gelatinolytic capacity up to 50 and 60%, respectively. Western blot analysis and enzyme-linked immunosorbent assay showed a reduction of MMP-9 protein actually released by cells. The addition of lacidipine in the incubation media determined no significant variation in Ca(2+) concentration. The drug did not affect MMP-9 mRNA levels, but it effectively reduced the amount of both active and total free MMP-9 secreted by human macrophages. Lacidipine reduced also the secretion of the tissue inhibitor of metalloproteinase-1 (TIMP-1); however we observed an overall reduction of the gelatinolytic activity of the cells. Finally, peritoneal macrophages, obtained from mice treated with lacidipine, showed a reduced secretion of MMP-9. Together, our data indicate that lacidipine may potentially exert an antiatherosclerotic activity by modulating the secretion of MMP-9 by macrophages. This, in addition to the previously demonstrated inhibition of cholesterol esterification, may contribute to increase plaque stability.

The calcium-channel blocker lacidipine reduces the development of atherosclerotic lesions in the apoE-deficient mouse.

BACKGROUND: Lacidipine is a widely used calcium-channel blocker, which has both long-lasting antihypertensive activity and also antioxidant properties. Previous studies have demonstrated the ability of lacidipine to reduce the development of atherosclerotic lesions in several animal models. OBJECTIVE: The present study investigated the antiatherosclerotic potential of lacidipine in the apoE-deficient mouse, an experimental model of atherosclerosis showing progressively complex and widespread lesions which closely resemble the inflammatory-fibrous plaques seen in humans. METHODS: Lacidipine was administered daily by gavage for 10 weeks at dose levels of 0 (control), 0.3, 1.0 and 3.0 mg/kg. RESULTS: Lacidipine administration reduces the extension of atherosclerotic lesions in the aorta of the apoE-deficient mouse without affecting plasma lipid levels. We also show that apoE-deficient mice have four-fold higher values of the proatherogenic peptide, endothelin, compared with the wild-type C57BL/6 mouse and that lacidipine administration reduced, in a dose-dependent manner, the concentrations of plasma endothelin. CONCLUSION: Lacidipine has anti-atherogenic effects in the apoE-deficient mouse, and reduces plasma endothelin concentrations.

Involvement of cholecystokinin within craving for cocaine: role of cholecystokinin receptor ligands.

In the brain, cholecystokinin (CCK) has been described to act as a central neurotransmitter or neuromodulator involved in functions such as food consumption, stress and anxiety. Recently, the CCK system has been involved in drug dependence phenomena and proposed to be correlated to a putative state of 'drug preferring' phenotype within free choice tests. CCK exerts its action in the CNS through at least two different G-protein coupled high affinity receptors, CCK1 and CCK2. Various selective CCK receptor agonists and antagonists have been synthesised. In particular, L-364,718 has been demonstrated to be a potent and selective CCK1 receptor antagonist, whereas L-365,260 is a potent and selective CCK2 receptor antagonist. More recently, GV150013 has been reported to be a highly selective CCK2 receptor antagonist. This paper reviews the putative role of the CCK system within drug dependence phenomena. In particular, it analyses the relationship between central CCK activity and the exhibition of spontaneous preference for drugs of abuse, such as cocaine or alcohol. The potential therapeutic role for CCK receptor antagonists is also discussed.

Synthesis and SAR of new 5-phenyl-3-ureido-1,5-benzodiazepines as cholecystokinin-B receptor antagonists.

A series of 5-phenyl-3-ureidobenzodiazepine-2,4-diones was synthesized and evaluated as cholecystokinin-B (CCK-B) receptor antagonists. Structure-activity relationship (SAR) studies revealed the importance of the N-1 substituent for potent and selective CCK-B affinity. Addition of substituents at the urea side chain provided in some cases more potent compounds. Moreover the introduction of bulky substituents such as adamantylmethyl at N-1 and resolution of the racemic ureas resulted in our lead compound GV150013.

Anti-atherosclerotic activity of the calcium antagonist lacidipine in cholesterol-fed hamsters.

We have investigated the activity of the calcium antagonist lacidipine in male hamsters fed an atherogenic diet containing 2% cholesterol and 5% butter. Animals were examined at 14, 20 and 24 weeks of treatment. At 14 weeks, in hamsters fed the atherogenic diet and without lacidipine treatment, there were significant increases in serum levels of total cholesterol, triglycerides and lipoproteins; these values were approximately similar at week 24. Lacidipine treatment at 0.3, 1.0 and 3.0 mg/kg/d did not affect levels of serum cholesterol, triglycerides and lipoproteins. At 24 weeks, in hyperlipidemic hamsters fed the atherogenic diet, the area of the fatty streak in the aortic arch covered a mean area of 375 +/- 145 micron2 x 100, which accounted for 2.7% of the total surface area of the aortic arch. In hamsters fed the atherogenic diet and treated with lacidipine at 0.3, 1.0 and 3.0 mg/kg, at 24 weeks, the surface area of the aortic arch lesion was significantly reduced by 41 to 71%. In the thoracic aorta at 24 weeks, in lacidipine-treated animals, both the incidence and degree of severity of the lesions was reduced, the area of the fatty streak being lowered by 78 to 97% in comparison with non-lacidipine-treated control animals. Ultrastructural examination demonstrated that the early changes in the aorta in hamsters fed the atherogenic diet involved the intima and smooth muscle cells; lacidipine treatment reduced the severity of the intimal lesions significantly. With SEM, lacidipine administration was seen to reduce endothelial irregularity and the presence of crater-like lesions. At TEM, treatment with lacidipine reduced the number of foam cells and the presence of liposomes in the subendothelium. This investigation demonstrates that in the hyperlipidemic hamster, lacidipine treatment decreases atheromatous lesions without lowering serum lipids. It is suggested that lacidipine influences the atherogenic process by an unusual mechanism which may be related to a combination of both the long-lasting calcium antagonism of the drug and significant antioxidant activity.

Extracellular matrix gene expression in the left ventricular tissue of spontaneously hypertensive rats.

The aim of this study was to investigate the extracellular matrix gene expression in the hypertrophied left ventricular tissue of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats, at early and mature ages. Interestingly, with age, a marked increase (+85% and +187% at 25 and 30 weeks of age, respectively, p < 0.01, vs 5 weeks) in matrix metalloproteinase-1 (MMP-1) mRNA levels in SHR and a progressive decrease (-50%, -70%, -78%, -70% at 10, 15, 25 and 30 weeks, respectively, p < 0.01, vs 5 weeks) in WKY were seen. Moreover, mRNA levels were significantly lower in SHR at 5 weeks. The analysis of mRNA expression for the tissue inhibitor of metalloproteinase-1 (TIMP-1) showed a significant increase in WKY (+44% and +44%, vs 15 and 25 weeks, respectively, p < 0.05), whereas there were no significant changes in SHR with development. At 30 weeks TIMP-1 mRNA levels were significantly reduced in SHR. Temporal trends of procollagen alpha1(I) and procollagen alpha1(III) mRNA levels were similar in both strains, but lower levels for procollagen alpha1(III) were found in SHR at 5 and 30 weeks. Although no significant differences were measured between the strains, mRNA levels for fibronectin were found decreased in WKY and increased in SHR with age. The results of the present study suggest an altered balance between collagen deposition and collagen degradation with development in this model of left ventricular hypertrophy and hypertension.

Cycloalkyl indole-2-carboxylates as useful tools for mapping the "north-eastern" region of the glycine binding site associated with the NMDA receptor.

A novel series of indole-2-carboxylate analogues of GV 150526 (1) in which the terminal phenyl ring belonging to the side chain present in the position C-3 has been replaced with a bridged cycloalkyl group was synthesized and evaluated for its pharmacological profile. Modelling studies on this class of novel glycine antagonist allowed us to identify an asymmetric lipophilic pocket present in the "North-Eastern" region of the pharmacophoric model of the glycine binding site associated to the NMDA receptor. Among the derivatives prepared, 3-[2-(1-adamantylaminocarbonyl)ethenyl]-4,6-dichloroindole-2 -carboxylic acid 6b and 3-[2-(norbornylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-c arboxylic acid 6l were found to be antagonists acting at the strychnine-insensitive glycine binding site, showing nanomolar affinity for the glycine binding site (Ki = 63 and 19 nM, respectively), coupled with high glutamate receptor selectivity (IC50 > 10(-5) M at the NMDA, AMPA, KA binding sites) and high in vivo potency after systemic administration by inhibition of convulsion induced by NMDA in mice.

Comparative effects of different dihydropyridines on the expression of adhesion molecules induced by TNF-alpha on endothelial cells.

OBJECTIVE: Lacidipine has already been demonstrated to reduce the expression of some adhesion molecules induced by pro-oxidant signals on endothelial cells. In order to verify if this effect is a peculiarity of this molecule, or belongs to other dihydropyridinic compounds (DHPs), the activity of lacidipine was compared with that of lercanidipine, amlodipine, nimodipine and nifedipine. DESIGN AND METHODS: The compounds were incorporated in human umbilical vein endothelial cells (HUVECs) using native low-density lipoprotein as a carrier. The drug concentrations in HUVECs were measured by mass spectrometry. Human recombinant tumour necrosis factor-alpha was then incubated with HUVECs for 7 h at 37 degrees C for adhesion molecule expression. RESULTS: The cellular amount of lacidipine, lercanidipine and amlodipine was similar, while nimodipine and nifedipine were almost undetectable or undetectable, respectively. Lacidipine, at any concentration, determined a dose-dependent significant decrease of the expression of intercellular adhesion molecule-1 (ICAM-1) ICAM-1, vascular cell adhesion molecule-1 (VCAM-1) VCAM-1 and E-selectin (P < 0.01). Lercanidipine and amlodipine determined variable decreases of adhesion molecules at the intermediate and highest concentrations. Nimodipine and nifedipine determined no effect on ICAM-1, VCAM-1 and E-selectin. The lowest IC50, i.e. the concentration determining the 50% reduction of ICAM-1, VCAM-1 and E-selectin expression was obtained with lacidipine for all the adhesion molecules considered (P < 0.01). CONCLUSIONS: It is concluded that the effect of the DHPs used in this study on adhesion molecule expression is determined first by their lipophilicity and then by their intrinsic antioxidant activity.

Oxidized low-density lipoprotein increases the production of intracellular reactive oxygen species in endothelial cells: inhibitory effect of lacidipine.

OBJECTIVE: The mechanisms by which oxidized low-density lipoprotein (ox-LDL) induces the expression of adhesion molecules on endothelial cells (HUVECs) are still not clear. The signal transduction pathways for these binding molecules include the translocation of the transcription factor NF-kB and the intracellular reactive oxygen species (ROS) are said to play a key role in this process. Aim of this study was (1) to evaluate the effect of ox-LDL on intracellular production of ROS in culture of HUVECs; (2) to evaluate if the intracellular increase of ROS induced by ox-LDL is mediated by the binding to a specific endothelial receptor; (3) to ascertain if lacidipine can decrease ox-LDL-induced ROS production in HUVECs. METHODS: Five microM Cu2+ ox-LDL were incubated with HUVECs for 5 min. 2',7'-Dichlorofluorescein (DCF) as an expression of intracellular ROS production, was measured by flow cytometry. RESULTS: ox-LDL induced a significant dose-dependent increase in DCF production (P < 0.001) through the binding to a specific receptor. The preincubation of HUVECs with radical scavengers compounds and lacidipine significantly reduced (P < 0.001) the ox-LDL-induced DCF production. CONCLUSIONS: ox-LDL increased the intracellular formation of ROS through the ligation to a specific endothelial receptor. Preincubation of HUVECs with lacidipine, a calcium antagonist with antioxidant properties, significantly reduced the intracellular ROS formation induced by ox-LDL. We propose that the effect of lacidipine on adhesion molecule expression and on NF-kB activation can be explained by its effect on intracellular ROS formation.

Spontaneous preference for ethanol in naive rats is influenced by cholecystokinin A receptor antagonism.

Naive adult male Wistar rats free to choose between water or 10% ethanol (v/v) spontaneously became water-preferring (WP) rats, as they drank mainly water (approximately 35 ml per day), or alcohol-drinking (ED) rats, as they also drank a significant amount of ethanol (approximately 14 ml per day). The selective CCKA receptor antagonist L-364,718 at doses selective for the CCKA receptor (5 micrograms/kg, IP) halved the consumption of alcohol of the ED rats without modifying their total liquid in-take. In contrast, the CCKB antagonists L-365,260 or GV150013 were without effect when used at doses selective for the CCKB receptor. These data indicate that the CCK system could be involved in the modulation of alcohol intake. In particular, they suggest that CCKA receptors could play a role in the ethanol preference.

Structural changes of small resistance arteries in spontaneously hypertensive rats after treatment with various doses of lacidipine.

OBJECTIVE: To evaluate the modifications of the morphology of mesenteric small resistance vessels in spontaneously hypertensive rats (SHR) induced by lacidipine treatment. METHODS: Lacidipine was administered at three different dosages, 20, 10, and 0.3 mg/kg per day. Fifty rats were studied. Nine SHR and 11 Wistar-Kyoto (WKY) rats were not treated. Each lacidipine dose was administered to 10 SHR. The drug and the placebo were administered by gavage from age 4 to age 12 weeks. The blood pressure was measured noninvasively every week. The animals were killed when they were aged 13 weeks, and the relative left ventricular mass (left ventricular weight plus septum weight/body weight) was calculated. Small mesenteric resistance vessels were dissected and mounted on a micromyograph (Mulvany's technique), and morphological parameters of the vessels were studied (media thickness and media: lumen ratio). RESULTS: The systolic blood pressure of SHR administered 20 and 10 mg/kg lacidipine per day was reduced significantly during the treatment period, whereas that of rats treated with 0.3 mg/kg lacidipine per day did not change. A significant reduction in media: lumen ratio was observed for all three groups of treated rats, including those to which 0.3 mg/kg lacidipine per day had been administered, and no reduction in systolic blood pressure could be detected. The relative left ventricular mass was reduced significantly only in rats to which 20 and 10 mg/kg lacidipine per day had been administered. CONCLUSION: A significant reduction in magnitude of vascular structural alternations was observed even in SHR treated with a low, nonhypotensive dose of lacidipine.

Substituted indole-2-carboxylates as in vivo potent antagonists acting as the strychnine-insensitive glycine binding site.

A series of indole-2-carboxylates bearing suitable chains at the C-3 position of the indole nucleus was synthesized and evaluated in terms of in vitro affinity using [3H]glycine binding assay and in vivo potency by inhibition of convulsions induced by N-methyl-D-aspartate (NMDA) in mice. 3-[2-[(Phenylamino)carbonyl]ethenyl]-4,6-dichloroindole-2-carboxyl ic acid (8) was an antagonist at the strychnine-insensitive glycine binding site (noncompetitive inhibition of the binding of [3H]TCP, pA2 = 8.1) displaying nanomolar affinity for the glycine binding site (pKi = 8.5), coupled with high glutamate receptor selectivity (> 1000-fold relative to the affinity at the NMDA, AMPA, and kainate binding sites). This indole derivative inhibited convulsions induced by NMDA in mice, when administered by both iv and po routes (ED50 = 0.06 and 6 mg/kg, respectively). The effect of the substituents on the terminal phenyl ring of the C-3 side chain was investigated. QSAR analysis suggested that the pKi value decreases with lipophilicity and steric bulk of substituents and increases with the electron donor resonance effect of the groups present in the para position of the terminal phenyl ring. According to these results the terminal phenyl ring of the C-3 side chain should lie in a nonhydrophobic pocket of limited size, refining the proposed pharmacophore model of the glycine binding site associated with the NMDA receptor.

Lacidipine inhibits the activation of the transcription factor NF-kappaB and the expression of adhesion molecules induced by pro-oxidant signals on endothelial cells.

OBJECTIVE: The adhesion of monocytes to endothelium, an early event in atherosclerosis is mediated by cell adhesion molecules. Signal-transduction pathways for these binding molecules include the translocation of the transcription factor NF-kappaB; moreover, intracellularly generated oxygen-derived free radicals play a major role in this process. In this study we evaluated the extent to which lacidipine, a calcium antagonist with antioxidant properties, affects the expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin on human umbilical vein endothelial cells, induced by different pro-oxidant signals such as oxidized low density lipoprotein (LDL) and tumor necrosis factor-alpha (TNF-alpha). METHODS: We incubated 5 micromol/l Cu2+-oxidized LDL and TNF-alpha (2 ng/ml) with human umbilical vein endothelial cells for 48 and 6 h, respectively. ICAM-1, VCAM-1 and E-selectin were measured by flow cytometry. NF-kappaB was evaluated by electrophoretic mobility shift assay. RESULTS: The incubation of 5 micromol/l Cu2+-oxidized LDL not only caused a dose-dependent increase in ICAM-1, VCAM-1 and E-selectin (P < 0.001), but also synergically increased their TNF-alpha-induced expression (P < 0.001). The addition of lacidipine to human umbilical vein endothelial cells significantly reduced the expression of ICAM-1, VCAM-1 and E-selectin induced by TNF-alpha alone or with oxidized LDL (P < 0.001). The reduction in adhesion molecule expression caused by lacidipine was paralleled by a significant fall in NF-kappaB translocation. CONCLUSIONS: The results suggest that lacidipine may have prevented NF-kappaB-mediated adhesion molecule expression by exerting its effects on oxygen-derived free radicals. The results support previous observations that lacidipine may have therapeutic effects in atherosclerosis.

Synthesis and antimicrobial evaluation of 2 beta-chloromethyl-6 beta-carbamoylmethyl-penam-1,1-dioxide-3-carboxylic acid.

A new 2 beta-Chloromethyl-6 beta-carbamoylmethyl-penam-1,1-dioxide-3-carboxylic acid was synthesised and evaluated for its antimicrobial and beta-lactamase inhibitory activity. The compound was found to be inactive.

Synthesis of novel 6-alpha and 6-beta-alkylcarbonylmethyl substituted penems.

6-alpha and 6-beta Alkylcarbonylmethyl penems were synthesized from 6-alpha-bromo and 6-oxo penicillanates respectively and their in vitro antibacterial activity was studied. The compounds were generally active against Gram-positive but not against Gram-negative strains, the compounds of the 6-beta series being more active. Relatively to imipenem, taken as reference compound, the penems resulted more stable towards chemical hydrolysis in Tris-HCl buffered medium (pH 7.4) but more sensitive towards dehydropeptidase-I (DHP-I).