In vitro study of biofunctional indicators after exposure to asbestos-like fluoro-edenite fibres.

The in vitro biological response to fluoro-edenite (FE) fibres, an asbestos-like amphibole, was evaluated in lung alveolar epithelial A549, mesothelial MeT-5A and monocyte-macrophage J774 cell lines. The mineral has been found in the vicinity of the town of Biancavilla (Catania, Sicily), where an abnormal incidence of mesothelioma has been documented. Cell motility, distribution of polymerized actin, and synthesis of vascular endothelial growth factor (VEGF) and of beta-catenin, critical parameters for tumour development, progression and survival, were investigated in A549 and MeT-5A cells exposed to 50 microg/ml FE fibres for 24 hr and 48 hr. The levels of cyclooxygenase (COX-2) and prostaglandin (PGE2), two molecules involved in cancer pathogenesis by affecting mitogenesis, cell adhesion, immune surveillance and apoptosis, were measured in J774 cells treated with FE fibres under the same experimental conditions. Finally, FE fibres were studied by SEM and EDS analysis to investigate their chemical composition. Exposure of A549 and MeT-5A cells to FE fibres affected differentially phalloidin-stained cytoplasmic F-actin networks, cell motility and VEGF and beta-catenin expression according to the different sensitivity of the two cell lines. In J774 cells it induced a significant increase in COX-2 expression, as assessed by Western blot analysis, and in the concentration of PGE2, measured in culture media by ELISA. SEM-EDS investigations demonstrated two types of FE fibres, edenite and fluoro-edenite, differing in chemical composition and both recognizable as calcic amphiboles. Fibre width ranged from less than 1 microm (prevalently 0.5 microm) to 2-3 microm (edenite) up to several microm (fluoro-edenite); length ranged from about 6 to 80 microm (edenite) up to some hundred microm (fluoro-edenite). Results provide convincing evidence that FE fibres are capable of inducing in vitro functional modifications in a number of parameters with crucial roles in cancer development and progression. Inhaled FE fibres have the potential to induce mesothelioma, even though their ability to penetrate lung alveoli depends on their aerodynamic diameter.

Effect of hyaluronic acid and polysaccharides from Opuntia ficus indica (L.) cladodes on the metabolism of human chondrocyte cultures.

Conventional medications in articular disease are often effective for symptom relief, but they can also cause significant side effects and do not slow the progression of the disease. Several natural substances have been shown to be effective as non-steroidal anti-inflammatory drugs at relieving the symptoms of osteoarthritis (OA), and preliminary evidence suggests that some of these compounds may exert a favourable influence on the course of the disease. In this study, we assay the anti-inflammatory/chondroprotective effect of some lyophilised extracts obtained from Opuntia ficus indica (L.) cladodes and of hyaluronic acid (HA) on the production of key molecules released during chronic inflammatory events such as nitric oxide (NO), glycosaminoglycans (GAGs), prostaglandins (PGE(2)) and reactive oxygen species (ROS) in human chondrocyte culture, stimulated with proinflammatory cytokine interleukin-1 beta (IL-1 beta). Further the antioxidant effect of these extracts was evaluated in vitro employing the bleaching of the stable 1,1-diphenyl-2-picrylhydrazyl radical (DPPH test). All the extracts tested in this study showed an interesting profile in active compounds. Particularly some of these extracts were characterized by polyphenolic and polysaccharidic species. In vitro results pointed out that the extracts of Opuntia ficus indica cladodes were able to contrast the harmful effects of IL-1 beta. Our data showed the protective effect of the extracts of Opuntia ficus indica cladodes in cartilage alteration, which appears greater than that elicited by hyaluronic acid (HA) commonly employed as visco-supplementation in the treatment of joint diseases.

Protective effect of Capparis spinosa on chondrocytes.

The aim of the present study was to evaluate the in vitro chondroprotective effects of the lyophilised methanolic extract from flowering buds of Capparis Spinosa L (LECS). This plant, common to the Mediterranean basin, has been used by the traditional medicine for its diuretic and antihypertensive effects and also in certain pathological conditions related to uncontrolled lipid peroxidation. The extract contains many constituents, in particular some flavonoids (kaempferol and quercetin derivatives) and hydrocinammic acids with several known biological effects such as the anti-inflammatory and the antioxidant ones. In this study, we assayed the effect of LECS on human chondrocytes cultures stimulated by proinflammatory cytokine interleukin-1beta (IL-1beta) and we determined the production of key molecules released during chronic inflammatory events (nitric oxide, glycosaminoglycans, prostaglandins and reactive oxygen species). We observed that LECS was able to counteract the harmful effects induced by IL-1beta. This protection appeared to be greater than that elicited by indomethacin, which is usually employed in joint diseases. Since LECS possess a chondroprotective effect, it might be used in the management of cartilage damage during the inflammatory processes.

"In vitro" differences among (R) and (S) enantiomers of profens in their activities related to articular pathophysiology.

An important group of non steroidal antinflammatory drugs (NSAIDs), which have been used for the symptomatic treatment of various forms of arthritis, are the 2-arylpropionic acid derivatives, 'profens'. By virtue of a chiral carbon atom on the propionic acid side chain, they exist as enantiomeric pairs. Whereas the S (+) enantiomer could be represented as an effective, but unselective COX inhibitor, the R (-) enantiomer could be much less active in this respect. However, recent findings suggest that certain pharmacological effects of profens cannot be attributed exclusively to the S (+) enantiomer. To obtain further insights into the pharmacological effects of profens, this study investigated the influence of pure enantiomers (S), (R), and racemic flurbiprofen and ketoprofen on the production of NO, MMP-3, PGE(2), ROS and GAGs, key molecules involved in cartilage destruction. Our results show that (S) flurbiprofen and ketoprofen decrease, at 1- and 10-microM concentrations, the interleukin-1beta induced cartilage destruction.

Investigations on behavioral effects of an extract of Cannabis sativa L. in the rat.

The behavioral responses of the rat to an extract of Cannabis sativa were examined after IP injection of 5, 15 and 30 mg/kg (expressed as delta 9 tetrahydrocannabinol). The lowest dose of the extract induced stereotyped behavior (rhythmic head movements, intermittent gnawing and sniffing) together with hypersensitivity to stimuli and hyperthermia. The administration of higher doses of the extract resulted, initially, in similar behavioral effects but of greater intensity, followed by a cataleptic state alternating with atonic muscular prostration; rectal temperature was decreased. Pre-treatment with 6-hydoxydopamine (6-OHDA, which produces degeneration of catecholamine-containing nerve terminals)or pimozide (blocker of dopamine receptors) significantly reduced both stereotype and hyperreactivity. Thermic effects were also antagonized by 6-OHDA pre-treatment. Cannabis-induced catalepsy was enhanced by pimozide but reduced by atropine (3 mg/kg SC). These results support the hypothesis that catecholamines play an important role in the complex behavioral effects of cannabis.

Protective effects of benzisothiazolylamidines on IL-1 beta induced alterations in human articular chondrocyte metabolism.

The in vitro effects on human articular chondrocytes were evaluated for a series of N-benzo[d]isothiazol-3-yl-amidines, bearing as pharmacophoric moiety the nonacidic isosteric nitrogen analogue of the carboxylic group. The aim was to verify their effectiveness in articular diseases, such as arthritis. Human chondrocytes were treated with IL-1beta in the presence of a series of N-benzo[d]isothiazol-3-yl-amidines at a concentration of 100 microg/mL. After 120 h, the amount of glycosaminoglycans (GAGs), the production of nitric oxide (NO) and the inhibition of metalloproteinases (MMP-3) and prostaglandin (PGE2) were measured. Nitrite production induced by inflammatory IL-1beta on cultured chondrocytes was inhibited by the N-benzo[d]isothiazol-3-yl-amidines tested, in particular by N-benzo[d]isothiazol-3-yl-benzamidine, which was the most active. Concerning the effects on GAGs, all the tested benzisothiazolylamidines, and in particular N-benzo[d]isothiazol-3-yl-acetamidine, prevented the depletion of proteoglycan induced by IL-1beta. Inhibitory effects of the tested compounds on MMP-3 activity and on PGE2 production were also observed.

Characterization and in-vivo ocular absorption of liposome-encapsulated acyclovir.

The potential of liposomes as an in-vivo ophthalmic drug delivery system for acyclovir was investigated. The drug-membrane interaction was evaluated by means of differential scanning calorimetry analysis. These experiments showed that acyclovir is able to interact with both positively and negatively charged membranes via electrostatic or hydrogen bonds. No interaction was observed with neutral membranes made up of dipalmitoylphosphatidylcholine. Different liposome preparation procedures were carried out to encapsulate acyclovir. The drug encapsulation mainly depends on the amount of water which the liposome system is able to entrap. In the case of multilamellar vesicles, charged systems showed the highest encapsulation efficiency. No particular difference in the encapsulation efficiency was observed for oligolamellar vesicles prepared with the reverse-phase evaporation technique. Oligolamellar liposomes showed the highest acyclovir encapsulation parameters and had release profiles similar to those of multilamellar liposomes. In-vivo experiments using male New Zealand albino rabbits were carried out to evaluate the aqueous humour concentration of acyclovir bioavailability. The most suitable ophthalmic drug delivery system was oligolamellar systems made up of dipalmitoylphosphatidylcholine-cholesterol-dimethyldioctadecyl glycerole bromide (7:4:1 molar ratio), which presented the highest encapsulation capacity and were able to deliver greater amounts of the drug into the aqueous humour than a saline acyclovir solution or a physical liposome/drug blend.

Enhancement of 4-biphenylacetic acid bioavailability in rats by its beta-cyclodextrin complex after oral administration.

4-Biphenylacetic acid, a potent non-steroidal anti-inflammatory agent forms a solid inclusion complex with beta-cyclodextrin in a 1:1 molar ratio, which exhibits better solubility and dissolution characteristics than the uncomplexed drug. Following oral administration of the complex to rats, quicker and higher drug plasma concentrations can be achieved than with the drug alone. Parallel studies, using the carrageenan paw oedema test, demonstrate a greater anti-inflammatory activity of the complex (ED50 of 2.9 mg kg-1 for the complex and of 6.2 mg kg-1 for the free drug). The complex displayed a better gastric tolerability in the rat than drug alone.

In vitro evaluation of thiazolyl and benzothiazolyl Schiff bases on pig cartilage.

A series of anti-inflammatory agents known as Schiff bases, combining thiazolyl and benzothiazolyl ring and vanillin moieties in the same molecule, was synthesized and evaluated for screening anti-degenerative activity on nasal pig cartilage cultures treated with interleukin 1beta, (IL-1beta). The amount of glycosaminoglycans (GAGs), the production of nitric oxide (NO) and prostaglandin E2 (PGE2), released into the culture medium, were detected. The tested Schiff bases decreased, dose-dependently, the NO and PGE2 production and the GAGs release with respect to samples treated with IL-1beta alone, showing a different behavior correlated to their structure. These results suggest that thiazolyl and benzothiazolyl Schiff bases in general, and particularly the Schiff base with bromine and methoxyl group in position three would protect cartilage matrix from degenerative factors induced by IL-1beta.

Different in vitro activity of flurbiprofen and its enantiomers on human articular cartilage.

The 2-arylpropionic acid derivatives or 'profens' are an important group of non-steroidal anti-inflammatory drugs that have been used for the symptomatic treatment of various forms of arthritis. These compounds are chiral and the majority of them are still marketed as racemate although it is known that the (S)- form is the principal effective in the cyclooxygenase inhibition. However, recent findings suggest that certain pharmacological effect of 2-arylpropionic acids cannot be attributed exclusively to the (S)-(+) enantiomer. To obtain further insights into the pharmacological effect of profens, the present study investigated the influence of racemic and pure enantiomers of flurbiprofen on the production of nitric oxide and glycosaminoglycans, key molecules involved in cartilage destruction. The culture of human articular cartilage stimulated by interleukin-1beta (IL-1beta), which plays an important role in the degradation of cartilage, has been established, as a profit experimental model, for reproducing the mechanisms involved in the pathophysiology of arthritic diseases. Our results show that mainly (S)-(+)-flurbiprofen decreases, at therapeutically concentrations, the IL-1beta induced cartilage destruction.

Carboxymethyl- and carboxy-derivatives of 7H- and 5H-1,2,4-triazolo [3,4-b][1,3,4] thiadiazine: synthesis and biological evaluation.

Some carboxymethyl- and carboxy-derivatives of 7H- and 5H-1,2,4-triazolo [3,4-b][1,3,4] thiadiazine were prepared. Their structure are discussed on the basis of IR, NMR and mass spectra. The synthesized compounds were subjected to a preliminary pharmacological screening for antiinflammatory-analgesic activity and to microbiological test on various species of mycetes and bacteria.

Liposomes as a potential drug carrier for citicoline (CDP-choline) and the effect of formulation conditions on encapsulation efficiency.

In this paper we report the investigation of the potential of liposomes as drug carrier for citicoline (1; CDP-choline). The aim of our work is to improve the pharmacokinetic and pharmacodynamic parameters of the drug to facilitate the overcoming of the blood-brain barrier. The thermotropic behaviour of hydrated dispersions of various phospholipids and their mixtures containing 1 have been investigated by differential scanning calorimetry (DSC) to have a clear view of the interaction between the drug and the liposome phospholipids. By the values of transition peak temperature (Tm) and transition enthalpy (delta H) we note a strong interaction between 1 and the polar heads of L-alpha-dipalmitoylphosphatidic acid (DPPA) and L-alpha-dipalmitoylphosphatidylserine (DPPS), whereas there is not any considerable interaction between the drug and L-alpha-dipalmitoylphosphatidylcholine (DPPC) or L-alpha-dimyristoylphosphatidylcholine (DMPC); in any case no interaction occurs between 1 and the hydrophobic part of the phospholipid. So we conclude that all the drug is fitted into the aqueous spaces. The results of the encapsulation efficiency experiments demonstrate how the encapsulation capacity increase with using charged phospholipids, reaching the top with DPPA. Moreover, it was noted that the presence of Cholesterol (Chol) enhances the encapsulation capacity (EC) and drug content (DC) values of DPPC, a neutral phospholipid. The size of the liposomes was determined by light scattering (LS).

Involvement of inducible nitric oxide synthase and cyclooxygenase-2 in the anti-inflammatory effects of a red orange extract in human chondrocytes.

In the present study, a complex of compounds (red orange complex, ROC), obtained from three red orange varieties (Citrus sinensis varieties: Moro, Tarocco and Sanguinello), containing cyanidin glycosides, hydroxycinnamic acids, flavanone glycosides and ascorbic acid, was screened to discover new lead compounds in the suppression of the production of key molecules released during inflammatory events in interleukin-1beta (IL-beta) stimulated human primary chondrocytes. The expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX)-2 and intercellular adhesion molecule-1 (ICAM-1), and the release of nitric oxide, prostaglandin (PG)E(2) and interleukin-8 (IL-8) were determined. Indomethacin was used as an anti-inflammatory drug reference. ROC acts as a potent inhibitor of iNOS and COX-2 gene expression while also suppressing the production of PGE(2) and nitrite in human chondrocytes. In addition, ROC induces a significant decrease in ICAM expression and IL-8 release. These findings suggest that ROC exerts anti-inflammatory effects probably through the suppression of COX-2 and iNOS expression.

The in vitro effect of a lyophilized extract of wine obtained from Jacquez grapes on human chondrocytes.

The present work was aimed at evaluating the in vitro effects of a lyophilized extract of wine (JW-E) obtained from Jacquez grapes (Vitis aestivalis-cinereaxVitis vinifera grapes) on the production of key molecules released in inflammatory disease utilising interleukin-1beta (IL-1beta) activated chondrocytes. The extract contains large amounts of phenolic components, in particular some flavonoids (flavan-3-ols, also known as catechins) and proanthocyanidins, as hydroxycinnamic acids and anthocyanins, that possess several biological features such as antiinflammatory and antioxidant effects and a "radical scavenger" activity too. In this study, we assayed the effect of JW-E on the production of key molecules released during chronic inflammatory events as nitric oxide (NO), prostaglandins E(2) (PGE(2)) and reactive oxygen species (ROS) in human chondrocytes culture, stimulated with proinflammatory cytokine interleukin-1beta. The JW-E proved to possess good ability against the harmfull effects of IL-1beta. Our data showed the protective effects of JW-E in cartilage alteration, that appears greater than that elicited by indomethacin, a not steroidal antiinflammatory drug (NSAID), commonly employed in joint diseases.

[Synthesis of 2-methoxynaphthalene derivatives as potential anti-inflammatory agents]. / Sintesi di derivati della 2-metossinaftalina come potenziali antiinfiammatori.

Compounds having 2-methoxynaphthalene as their parent nucleus were synthesized and evaluated for antiinflammatory effect according to the carrageenin paw edema method in rats. The synthetic routes for the preparation of isomeric 1,2- and 2,6-disubstituted derivatives are described. Replacement of the alpha-methylacetic moiety in naproxen by 4-hydroxybutyric acid side chain did not cause loss of activity.

[2,5-diaryl-substituted 1,3,4-oxadiazoles: synthesis and preliminary pharmacological investigation]. / 1,3,4-ossadiazoli 2,5-diarilsostituiti: sintesi e ricerca farmacologica preliminare.

Seven 2,5-diarylalkyloxysubstituted 1,3,4-oxadiazoles were synthesized. They showed pronounced antiphlogistic action together with central activity (sedative, analgesic) that principally seems to be connected with the presence in the molecule of the 3,4-dioxymethylenephenyl radical.

[Reactivity of 3-aryl-4-amino-5-mercapto-4H-1,2,4-triazoles: synthesis and biological evaluation of 3,6-diaryl derivatives of 7H-1,2,4-thiazole[3,4-b][1,3,4]thiadiazines, of 3-aryl-4-amino-5-carboxymethylthio-4H-1,2,4-triazoles]. / Reattivita' di 3-aril-4-ammino-5-mercapto-4H-1,2,4-triazoli: sintesi e valutazione biologica di 3,6-diarilderivati della 7H-1,2,4-triazolo[3,4-b] [1,3,4]tiadiazina, di 3-aril-4-ammino-5-carbossimetiltio-4H-1,2,4-triazoli e di alcuni 3-aril-4H-1,2,4-triazoli.

Starting from 3-aryl-4-amino-5-mercapto-4H-1,2,4-triazoles (II), two series of 3,6-disubstituted 7H-1,2,4-triazol [3,4-b] [1,3,4] thiadiazines (III) and their 5-carboxymethylthio derivatives (IV) were prepared. From the mercapto-amino-triazoles (II) because of their reactivity in some oxidising media, were obtained the triazole derivatives (V), (VI) and (VII). The synthesis of the alpha-thioketones (VIII) of 3-aryl-5-mercapto-1,3,4-oxadiazoles (I), used in alternative synthesis of triazole-thiadiazines (III), is also reported. All the substances described were subjected to biological screening. In the tests, the carboxymethylthiotriazole (IV) showed weak antiinflammatory activity (carrageenin edema) and more consistent scavanger activity, in vitro, on superoxide anions. The triazole-thiadiazines (III) and triazoles (II), (V) and (VI) showed moderate antimycotic activity.

[Synthesis and preliminary biological investigation of 2,6-diaryl substituted imidazo(1,2-b)-1,3-4-thiadiazoles]. / Sintesi e valutazione biologica preliminare di imidazo [2,1-b]-1,3,4-tiadiazoli-2,6-diarilsostituiti.

The synthesis of imidazo [2,1-b]-1,3,4-thiadiazole derivatives and preliminary pharmacological screening are reported. The compounds studied showed mainly antiphlogistic, antipyretic and analgesic activity. From the results it appears that analgesic activity is peripheral.

[Hyperprolactinemia and catalepsy induced by haloperidol]. / Iperprolattinemia e catalessi indotta da aloperidolo.

The effect of endogenous hyperprolactinemia induced by pituitary transplantation under the kidney capsule on haloperidol induced catalepsy was evaluated in male Wistar rats treated with two doses of the drug (500 gamma/kg; 2 mg/kg i.p.). Rats of 220 +/- 30 g received intraperitoneal injection of haloperidol. Every five minutes following drug administration the rats were assessed for catalepsy by placing the forepaw on a horizontal bar, and observed for two minutes. Data obtained show that hyperprolactinemia potentiates the cataleptic score in rats treated with dose of 500 gamma/kg i.p., while no significant difference was observed between hyperprolactinemic rats and control rats, at the dose of 2 mg/kg i.p. of haloperidol.

Synthesis and biological activity of acetamides, arylureas and 2-alkoxyphenyl-6-phenyl-1,3,4-oxa(thia)diazole-[3,2-a]-S- triazin-5,7-diones derived from 2-amino-5-alkoxyphenyl-1,3,4-oxa(thia)diazole.

The syntheses of some acetamides (II) and phenylureas (III) derived from 2-amino-5-alkoxyphenyl-1,3,4-oxa(thia)diazoles (I) and some 2-alkoxyphenyl-6-phenyl-1,3,4-oxa(thia)diazole-[3,2-a]-s- triazin-5,7-diones (IV) are described. Pharmacological tests showed antiinflammatory and analgesic activities of bicyclic derivatives. A probable influence of alkoxyphenyl substituents, present in three kinds of structures, was observed.