Betacyanins enhance vitexin-2-O-xyloside mediated inhibition of proliferation of T24 bladder cancer cells.

Betacyanins (BC) were purified from beetroot (Beta vulgaris var. rubra L.) and tested, alone or in combination with vitexin-2-O-xyloside (XVX) from Beta vulgaris var. cicla L., for their ability to reduce the proliferation rate in T24 bladder cancer cells. Combination of BC and XVX exhibited a synergistic effect concerning the inhibition of proliferation in T24 cancer cells at 24 and 48 h but not after 72 h of incubation. The induction of apoptosis was evidenced by means of fluorescence activated cell sorting (FACS) analysis, as well as through the increase in caspase 3 and 8 activities. Using RTqPCR experiments, it was shown that the combination of XVX + BC was able to enhance the expression levels of pro-apoptotic BAX and downregulate anti-apoptotic BIRC5 (survivin), as well as pro-survival CTNNB1 (ß-catenin). The most evident effect of BC was the increase of the activity of caspase 8, leading to induction of extrinsic apoptosis. Moreover, XVX, BC and their combination showed no cytotoxic effect on normal human skin NCTC 2544 keratinocytes. These results demonstrated the efficacy and the mechanisms of the action of BC and XVX, extracted from edible plants, and suggested that a diet or a nutrition supplement, enriched with these bioactive molecules, could be used in the prevention of human bladder cancer.

1-(2-Alkanamidoethyl)-6-methoxyindole derivatives: a new class of potent indole melatonin analogues.

A new series of indole melatonin analogues, bearing the amido ethyl side chain attached at the N-1 position of the indole nucleus, were synthesized and tested for their affinity for the melatonin receptor isolated from quail optic tecta in a series of in vitro ligand-binding experiments using 2-[125I]iodomelatonin as the labeled ligand. The biological activity was evaluated using two models: effects on the forskolin-stimulated cAMP accumulation in explants from quail optic tecta and evaluation of the GTP gamma S index derived from competition experiments performed in the absence or presence of GTP gamma S. Compounds 2a and 2k-n, obtained by shifting the methoxy group and the ethylamido side chain from the C-5 and C-3 positions of melatonin to the C-6 and N-1 positions of the indole nucleus, exhibited an affinity similar to that of melatonin itself, as well as full agonist activity. Optimization of the C-2 substituent by introducing Br, phenyl, or COOCH3 (2b-d) resulted in a significantly enhanced affinity (in the picomolar range) and improved agonist biological activity. Compounds lacking the methoxy group and bearing an N-alicyclic group (2h-j) behaved as partial agonists or antagonists.

(E)-3-(2-(N-phenylcarbamoyl)vinyl)pyrrole-2-carboxylic acid derivatives. A novel class of glycine site antagonists.

The synthesis and preliminary biological evaluation of novel (E)-3-(2-(N-phenylcarbamoyl)-vinyl)pyrrole-2-carboxylic acids bearing alkyl, acyl, alkoxy, phenyl, and halo substituents at the 4- and 5-positions of the pyrrole ring are reported. These compounds were studied for their in vitro affinity at the strychnine-insensitive glycine-binding site of the N-methyl-D-aspartate (NMDA) receptor complex. In the [3H]glycine binding assay (E)-4,5-dibromo-3-(2-(N-phenylcarbamoyl)vinyl)pyrrole-2-carboxylic acid 6w (pKi = 7.95 +/- 0.01) and the 4-bromo-5-methyl 6j (pKi = 7.24 +/- 0.01) and 4,5-dimethyl 6g (pKi = 6.70 +/- 0.03) analogues were the most active compounds of the series. Qualitative structure-activity analysis points to a negative correlation between bulk of the C-4 and C-5 substituents and affinity which is enhanced by halo-substituents. QSAR analysis by the Hansch descriptors F, R, pi, and MR, on a subset of compounds with pKi > or = 4, indicates that electron-withdrawing groups at C-4 and C-5 enhance the affinity. Bulk and lipophilicity are also relevant for the substituents at these positions. 6g was found to be a full antagonist (alpha = 0; enhancement of the [3H]TCP binding). The in vivo potency of 6g, 6j, and 6w was evaluated by the inhibition of NMDA-induced convulsions in mice by both the i.v. and po routes; 6w was the most active compound (ED50 = 3 x 10(-3) (0.8-10) g/kg, i.v. and 30 x 10(-3) (4.5-61) g/kg, p.o.). The results of this study indicate that the 3,4-disubstitutedpyrrole-2-carboxylate represents a novel template for the design of new glycine antagonists.

2-N-acylaminoalkylindoles: design and quantitative structure-activity relationship studies leading to MT2-selective melatonin antagonists.

Several indole analogues of melatonin (MLT) were obtained by moving the MLT side chain from C(3) to C(2) of the indole ring. Binding and in vitro functional assays were performed on cloned human MT1 and MT2 receptors, stably transfected in NIH3T3 cells. Quantitative structure-activity relationship studies showed that 4-methoxy-2-(N-acylaminomethyl)indoles, with a benzyl group in position 1, were selective MT2 antagonists and, in particular, N-[(1-p-chlorobenzyl-4-methoxy-1H-indol-2-yl)methyl]propanamide (12) behaved as a pure antagonist at MT1 and MT2 receptors, with a 148-fold selectivity for MT2. We present a topographical model that suggests a lipophilic group, located out of the plane of the indole ring of MLT, as the key feature of the MT2 selective antagonists.

Conformationally restrained melatonin analogues: synthesis, binding affinity for the melatonin receptor, evaluation of the biological activity, and molecular modeling study.

The design, synthesis, and biological profile of several indole melatonin analogues with a conformationally restricted C3 amidoethane side chain are presented. Examination of the accessible conformations of the melatonin side chain led us to explore some of its fully or partially restricted analogues, 2-12, the binding affinity values of which were utilized to gain further insight on the melatonin binding site. Two pharmacophoric models have been devised for melatonin and the active compounds by conformational analysis and superimposition performed using the DISCO program. In these models, the melatonin side chain can adopt a gauche/anti conformation out of the indole plane. Another contribution of this study regards the observation of a possible binding point interaction around the C2 position of the indole, as suggested by the remarkably increased binding affinity observed in the C2-substituted analogues 6 and 9 and especially in the more rigid analogue 5. The biological activity and the efficacy of the new compounds were tested by measuring the inhibition of the forskolin-stimulated cAMP accumulation and the GTP gamma S index. Both analyses demonstrated that all of the compounds were full agonists with the exception of 4 and 9, which showed a slight reduction in efficacy and would seem to be partial agonists.

2-[N-Acylamino(C1-C3)alkyl]indoles as MT1 melatonin receptor partial agonists, antagonists, and putative inverse agonists.

The synthesis of several novel indole melatonin analogues substituted at the 2-position with acylaminomethyl (8-11), acylaminoethyl (5a-k), or acylaminopropyl (13) side chains is reported. On the basis of a novel in vitro functional assay (specific binding of [35S]GTPgammaS), which can discriminate agonist from partial agonist, antagonist, and inverse agonist ligands, 5a,g, h,j and 13 were shown to be partial agonists, 5d,e and 8-11 competitive antagonists, and 5b,c,k putative inverse agonists. Binding and functional assays were performed on cloned human MT1 receptor. Structure-activity relationship considerations indicate that N-[1-aryl-2-(4-methoxy-1H-indol-2-yl)(C1-C2)alkyl]alkanamides represent a lead structure for this type of ligands.

Melatonin receptor ligands: synthesis of new melatonin derivatives and comprehensive comparative molecular field analysis (CoMFA) study.

The CoMFA methodology was applied to melatonin receptor ligands in order to establish quantitative structure-affinity relationships. One hundred thirty-three compounds were considered: they were either collected from literature or newly synthesized in order to gain information about the less explored positions. To this end, various melatonin derivatives were prepared and their affinity for quail optic tecta melatonin receptor was tested. Compounds were aligned on the putative active conformation of melatonin proposed by our previously reported pharmacophore search, and their relative affinities were calculated from the displacement of 2-[125I]-iodomelatonin on different tissues expressing aMT receptors. Compounds were grouped into three sets according to their topology. Subset A: melatonin-like compounds; subset B: N-acyl-2-amino-8-methoxytetralins and related compounds; subset C:N-acyl-phenylalkylamines and related compounds. CoMFA models were derived for each set, using the steric, electrostatic, and lipophilic fields as structural descriptors; the PLS analyses were characterized by good statistical parameters, taking into account the heterogeneity of the binding data, obtained with different experimental protocols. From the CoMFA model for the melatonin-like compounds, besides the well-known positive effect of 2-substitution, a low steric tolerance for substituents in 1, 6, and 7, and a negative effect of electron-rich 4-substituents were observed; the information provided by the newly synthesized compounds was essential for these results. Moreover, a comprehensive model for the 133 compounds, accounting for a common alignment and a common mode of interaction at the melatonin receptor, was derived (Q2 = 0.769, R2 = 0.905). This model validates our previously reported pharmacophore search and offers a clear depiction of the structure-affinity relationships for the melatonin receptor ligands.

The anticoagulant, hepatic lipase-releasing and lipoprotein lipase-releasing activities of several natural and chemically modified heparins differ.

Several 'natural' heparins have been found to have different potencies for releasing hepatic lipase and lipoprotein lipase. These differences can also be obtained by treating heparins with physical and chemical methods, which also affect the anticoagulant activity. These differences in potency in hepatic lipase-releasing activity are discussed in terms of the role of this lipase in lipoprotein and cholesterol metabolism.

Profibrinolytic activities of chemically modified heparins with very low anticoagulant activities.

Many evidences indicate that heparin is an activator of fibrinolysis, but the most important side effect of heparin is bleeding which is a problem particularly in the high risk patient. Here we describe how chemical modifications, associated with separation techniques, can sharply reduce the anticoagulant activities of a heparin while its ability to stimulate fibrinolysis is retained.

Antimetastatic action of a new analog of dacarbazine in mice bearing Lewis lung carcinoma.

We report the selective antimetastatic properties of 3-(3,3-dimethyl-1-triazenyl)-5-methyl-4,5-dihydroisoxazole in the murine transplantable tumor model Lewis lung carcinoma. The compound verifies a previous study on the correlation of antimetastatic, antitumor and cytotoxic properties of aryl- and heteroaryltriazenes with their Electron Ionization Mass Spectrometry (EI-MS) behavior. The new analog of dacarbazine exhibits a selective antimetastatic activity accompanied by limited thymus toxicity. The mechanism of action is unclear nevertheless any antiproliferative or cytotoxic effect is excluded.

Amides of beta-(4-chlorophenoxy)-alpha-phenyl-ethylamines are inhibitors of cholesterol biosynthesis in vitro.

Amides of beta-(4-chlorophenoxy)-alpha-phenyl-ethylamines were prepared and tested for their inhibitory activity on cholesterol biosynthesis in vitro. Among the substances prepared, (II e), (II g), (II k) and (II m) had much greater inhibitory activity than clofibrate.

Design and synthesis of melatonin receptors agonists and antagonists.

We review our work towards the design and synthesis of high-affinity melatonin (N-acetyl-5-methoxytryptamine) agonist and antagonist compounds. High affinity melatonergic agonists were obtained by shifting the melatonin side chain from C3 to N1 of the indole ring system. Conversely, by moving the side chain from C3 to C2 it was possible to obtain melatonin antagonist compounds, albeit of moderate affinity.

7-Substituted theophyllines. Part VI--N-[2-(4-chlorophenoxy)-1-phenyl-alkyl]-alkylamido derivatives.

The new amides are inhibitors of cholesterol biosynthesis in vitro as well as in vivo. These compounds are also inhibitors in vivo of triglyceride biosynthesis and of platelet aggregation. All tests showed activity (values expressed as percentage variation) much greater than clofibrate. Besides, all compounds have no effect on coagulation or diuresis and showed low acute toxicity.

3-(2-Carbamoylvinyl)-4,5-dimethylpyrrole-2-carboxylic acids as ligands at the NMDA glycine-binding site: a study on the 2-carbamoylvinyl chain modification.

Twenty 4,5-dimethylpyrrole-2-carboxylic acids (5a-t) with different 2-carbamoylvinyl chains in position 3 were prepared to further investigate the relationships between structure and in vitro affinity for the strychnine-insensitive glycine-binding site. None of these compounds was superior to (E)-3-(N-phenyl-2-carbamoylvinyl)-4,5-dimethylpyrrole-2-carb oxylic acid III (pKi = 6.70), which was taken as a reference standard, but overall the results obtained indicate that the N-phenyl-2-carbamoylvinyl substituent of III may be replaced with the N-(1-adamantyl)-2-carbamoylvinyl group as in 5h (pKi = 6.20) without considerable loss of affinity. This finding adds to previous knowledge.

[Diagnostic problems and results of laparoscopic cholecystectomy in chronic acalculous cholecystitis]. / Problemi diagnostici e risultati della colecistectomia laparoscopica nella colecistite cronica alitiasica.

We report on our experience with laparoscopic cholecystectomy in 15 patients, 12 females and 3 males (mean age: 44 years), with chronic acalculous cholecystitis. These patients presented with recurrent episodes of biliary colic together with a dysmorphic or dysfunctioning gallbladder as confirmed by ultrasound and/or cholescintiscan with 99m-Tc HIDA performed in fasting conditions and after meals. First of all, we considered the possible presence of concomitant digestive disease (peptic ulcer disease, recurrent pancreatitis, irritable bowel syndrome, chronic hepatitis) potentially responsible for the pain. Ultrasound investigations revealed a pathological gallbladder in 10 patients. Cholecystectomy was curative in 8/10. Cholescintiscan revealed a pathological gallbladder in 8 patients and cholecystectomy was curative in only 5 of these. No postoperative deaths or significant complications occurred. The mean duration of the operation (35 vs 48 min) and hospital stay (2.1 vs 2.8 days) were reduced in comparison to 346 cholecystectomies performed for gallstones. After 6-36 months' follow-up, resolution of symptoms was successful in 10/15 cases (66.6%); in 3 cases, only dyspepsia was reduced, whilst in the other 2 cases, who also presented concomitant irritable bowel syndrome and gastroduodenitis, there was no improvement in pain. In all but the latter two cases (86.6%), histological examination revealed chronic gallbladder inflammation. In conclusion, laparoscopic cholecystectomy was curative (66.6%) or led to an improvement in symptoms (20%) in patients with chronic acalculous cholcystitis. Cholescintiscans were not always diagnostic for the disease, whereas ultrasound findings were more useful as an indication for surgery.

[Multicenter prospective study of informed consent in general surgery]. / Studio prospettico multicentrico sul consenso informato in chirurgia generale.

To understand the level of acceptance, awareness and usefulness of informed consent, a group of 119 patients (59 men and 60 women) from different types of hospitals were given a questionnaire which required only 'YES or NO' answers, both before and after surgery. The questionnaire concerned the patient's knowledge about pathology, operative risks, approval, anxiety caused, understanding of information received and consent given, and also if he would inform a relative in the same condition. From the analysis of the results it was established that: the more information a patient has about his illness and operation risks, the more he will want to have; the less he knows the less he will want to know, and he will also have more faith in the doctors. Some patients would not inform a relative with a similar pathology. To conclude, informed consent, instead of being a right of the patient is progressively becoming more a right of the doctor. It does not have any real effect on the patient's choice but is useful, as it represents a moment of personalised attention from medical personnel, though the patient may not completely understand the information received. There are few advantages in strictly medical terms but informed consent has increased malpractice litigation.

Indole melatonin agonists and antagonists derived by shifting the melatonin side chain from the C-3 to the N-1 or to the C-2 indole position.

This article reviews our efforts in the development of indole melatonin (MLT) agonist and antagonist compounds. Evidence is presented which indicates that high-affinity melatonergic agonists were obtained by shifting the MLT amido side chain from the C-3 to the N-1 indole position. Conversely, by moving the side chain from the C-3 to the C-2 indole position it is possible to produce MLT antagonist compounds.

[7-Substituted theophyllines. V. Azabenzhydrylaminoalkyl derivatives]. / Teofilline 7-sostituite. Parte V--Azabenzidrilamminoalchilderivati.

Some 7-(azabenzhydrylaminoalkyl)theophyllines were prepared and pharmacologically screened. They showed low toxicity, antidepressant properties and, in some cases, antithrombotic activity.

Absorption by the rat intestinal tract of fluorescein-labelled pig duodenal glycosaminoglycans.

Fluorescein-labelled glycosaminoglycans (F-GAG) were absorbed by the rat intestinal tract when administered in an anhydrous suspension consisting of vegetable fats and sodium taurocholate. A statistically significant regression between plasma levels of F-GAG and plasma lipoprotein lipase activities was found.

Retention of antilipemic activity by periodate-oxidized non-anticoagulant heparins.

Heparin preparations with different anticoagulant and antilipemic (fat-clearing) activities were oxidized with periodate under conditions of cleavage of all the C(2)-C(3) bonds of non-sulfated uronic acid residues, while preserving the original molecular weight of the polysaccharide. Periodate-oxidised heparins (oxyheparins, O-HEP) and the corresponding borohydride-reduced products (reduced oxyheparins, RO-HEP) were compared with the original heparins for their content in trisulfated disaccharide sequences (as determined by 13C-nuclear magnetic resonance) and in active sites for antithrombin-III (as determined indirectly by affinity chromatography), and for their anticoagulant and antilipemic (lipoprotein lipase-releasing) activities. The drop of anticoagulant activity induced by periodate oxidation was paralleled by a substantial decrease of affinity for antithrombin, and is thought to arise from glycol splitting at the level of the D-glucuronic acid residue that is part of the active site for antithrombin. The trisulfated disaccharide sequences and the associated antilipemic activities were substantially unaffected by periodate oxidation. The residual anticoagulant activity of periodate-oxidized heparins obtained from preparations - such as those from beef lung - rich in trisulfated disaccharide sequences is discussed in terms of the influence of charge density on heparin-protease interactions not mediated by antithrombin.