Barbara Gentile

Effect of propolis on human cartilage and chondrocytes.

Propolis, a natural product derived from plant resins collected by the honeybees, has been used for thousands of years in folk medicine for several purposes. The extract that contains amino acids, phenolic acids, phenolic acid esters, flavonoids, cinnamic acid, terpenes and caffeic acid, possesses several biological activities such as anti-inflammatory, immunostimulatory, anti-viral and anti-bacterial. In this study, we assay the effects of propolis extract on the production of key molecules released during chronic inflammatory events as nitric oxide (NO) and glycosaminoglycans (GAGs) in cultures of human cartilaginous tissues and chondrocytes, stimulated with interleukin-1beta (IL-1beta). We observed that this natural compound and its active principle, caffeic acid phenethyl ester (CAPE), were able to contrast the harmful effects of IL-1beta. Our data clearly demonstrated the protective action of propolis in cartilage alteration, that appears greater than that elicited by indomethacin, commonly employed in joint diseases.

Amidinobenzisothiazole derivatives with antidegenerative activity on cartilage

N-(Benzo[d]isothiazol-3-yl)amidines were synthesised and evaluated for their antiinflammatory activity. Encouraging results led us to evaluate these derivatives on the prevention of cartilage destruction in articular disease. Antidegenerative activity was assayed on culture of porcine nasal cartilage and diarthroidal joint human cartilage in the presence of interleukin-1beta (IL-1beta). The amount of glycosaminoglycans (GAGs) and the production of nitric oxide (NO) in the culture medium were determined. The obtained results showed that all the compounds, in the presence of IL-beta, blocked the cartilage breakdown, with different behaviour. The antidegenerative activity is more evident in human cartilage.

Thienopyrimidine derivatives prevent cartilage destruction in articular disease.

The effects of a series of thienopyrimidine derivatives on the prevention of cartilage destruction in articular disease were investigated. Anti-degenerative activity was assayed on culture of nasal pig cartilage in the presence or in the absence of interleukin 1beta (IL-1beta). The amount of glycosaminoglycans (GAGs) and the production of nitric oxide (NO) in the culture medium were determined. Some thienopyrimidine derivatives, in the presence of IL-beta, blocked the cartilage breakdown by inhibiting both the NO production and GAGs release in a dose-dependent manner.

Aminothiazole derivatives with antidegenerative activity on cartilage

A series of 2-dialkylamino-N-(4-substituted thiazolyl-2)acetamides and 3-dialkylamino-N-(4-substituted thiazolyl-2)propionamides were synthesized and evaluated for their anti-inflammatory activity. Encouraging results led us to investigate the effect of these compounds on NO production and GAGs release. Their effects were evaluated in vitro on the metabolism of pig cartilage, treated with IL-1beta. The amount of glycosaminoglycans (GAGs) and the production of nitric oxide (NO) in the culture medium were determined. The results, obtained, showed that all compounds, in the presence of IL-1beta, blocked the cartilage breakdown, with different behavior. A quantitative structure-activity relationship (QSAR) study was performed.

Protective effects of benzisothiazolylamidines on IL-1β 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-1β in the presence of a series of N-benzo[d]isothiazol-3-yl-amidines at a concentration of 100 μg/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-1β 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-1β. Inhibitory effects of the tested compounds on MMP-3 activity and on PGE2 production were also observed.

“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, PGE2, ROS and GAGs, key molecules involved in cartilage destruction. Our results show that (S) flurbiprofen and ketoprofen decrease, at 1- and 10-μM concentrations, the interleukin-1β induced cartilage destruction.

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.

Effects of leflunomide on human cartilage.

Modern therapeutic approach in rheumatoid arthritis (RA) includes early use of disease-modifying anti-rheumatic drugs (DMARDs). DMARDs may influence the course of disease progression, and their introduction in early RA is recommended to limit irreversible joint damage. Among DMARDs, leflunomide and methotrexate are more utilised in pharmacological therapy. In the present work, we considered the effects of leflunomide, in comparison with those of methotrexate and to those of leflunomide-methotrexate combination on human cartilage to verify its effectiveness in arthritic disease, simulated by our experimental model. We measured in vitro the amount of glycosaminoglycans (GAGs) and the production of nitric oxide (NO) released into the culture medium of human articular cartilage treated with interleukin-1beta (IL-1beta), which promotes the cartilage destruction during articular disease. Leflunomide, in the presence of IL-1beta decreased NO production and GAGs release respect IL-1beta alone treated samples, in dose-related manner. Our results suggest that leflunomide is able to protect cartilage matrix from degradative factors induced by IL-1beta with respect to methotrexate and leflunomide-methotrexate combination.

Activity of Liposome-entrapped Immunomodulator Oligopeptides on Human Epithelial Thymic Cells

We have studied the in-vitro effects of liposomal formulations loaded with three oligopeptides (Tp3, Tp4, Tp5), synthetic analogues corresponding to the active site of thymopoietin, on human thymic epithelial cells. n n n nThe activity of the peptides entrapped in multilamellar vesicles and in stable plurilamellar liposomal vesicles, with or without cholesterol and containing stearylamine or dipalmitoylphosphatidyl serine, as positive or negative charge-inducer, was tested. The tetrazolium salt assay was performed as a marker of cell growth. To study the response of thymic epithelial cell cultures to liposome addition-dependent stress, we investigated the changes in the level of heat shock proteins (HSPs70). n n n nThe results indicated that in-vitro among the peptides tested only Tp4 increased thymic epithelial cell growth. These stimulating effects were improved further when the peptide was entrapped in neutral 1,2-dipalmitoyl-L-α-phosphatidylcholine/cholesterol liposomes. Tp4 was also effective in modulating HSP70 protective effects.