Quality of different chondroitin sulfate preparations in relation to their therapeutic activity.

OBJECTIVES: Chondroitin sulfate is currently recommended by the European League Against Rheumatism (EULAR) as a SYSADOA (symptomatic slow acting drug for osteoarthritis) in Europe in the treatment of knee and hand osteoarthritis based on research evidence and meta-analysis of numerous clinical studies. Furthermore, recent clinical trials demonstrated its possible structure-modifying effects. Chondroitin sulfate, alone or in combination with glucosamine or other ingredients, is also utilized as a nutraceutical in dietary supplements in Europe and the USA. However, it is derived from animal sources by extraction and purification processes. As a consequence, source material, manufacturing processes, the presence of contaminants and many other factors contribute to the overall biological and pharmacological actions of these agents. We aim to review the quality control of chondroitin sulfate in pharmaceutical-grade preparations and nutraceuticals. KEY FINDINGS: Pharmaceutical-grade formulations of chondroitin sulfate are of high and standardized quality, purity and properties, due to the stricter regulations to which this drug is subjected by local national health institutes as regards production and characteristics. On the contrary, as several published studies available in literature indicate, the chondroitin sulfate quality of several nutraceuticals is poor. Additionally, there are no definite regulations governing the origin of the ingredients in these nutraceuticals and the origin of the ingredients in natural products is the most important factor ensuring quality, and thus safety and efficacy, in particular for chondroitin sulfate, due to its extraction from different sources. CONCLUSIONS: Due to the poor chondroitin sulfate quality of some nutraceuticals, we conclude that stricter regulations regarding their quality control should be introduced to guarantee the manufacture of high quality products for nutraceutical utilization and to protect customers from low-quality, ineffective and potentially dangerous products. There is a need for specific and accurate analytical procedures, which should be enforced to confirm purity and label claims both for raw materials and finished chondroitin sulfate products, and also to govern the origin of ingredients. Until these stricter regulations are in place, then it is strongly recommended that pharmaceutical-grade chondroitin sulfate is used rather than food supplements.

Chondroitin-4-sulphate inhibits NF-kB translocation and caspase activation in collagen-induced arthritis in mice.

OBJECTIVE: Free radical damage, inflammation, and apoptosis play a critical role in the onset and progression of cartilage erosion in arthritis. Many studies have demonstrated that glycosaminoglycans (GAGs), and chondroitin-4-sulphate (C4S) in particular, possess antioxidant activity that is able to inhibit lipid peroxidation which is the main mechanism of free radical-mediated biological injury. In addition to the effect directly exerted by reactive oxygen species (ROS), the activation of nuclear factor kB (NF-kB) and caspases may contribute substantially to increase inflammation and cell damage. We studied whether the antioxidant action of chronic C4S treatment to reduce ROS injury involves NF-kB and caspases modulation using an experimental model of collagen-induced arthritis in mice. METHODS: Arthritis was induced in mice via an intradermal injection at the base of the tail of 100 microl of emulsion containing bovine type II collagen in complete Freund's adjuvant. RESULTS: Arthritis provoked the following: severe oedema and inflammation in the hind paws; lipid peroxidation in the joints [measured by 8-isoprostane (8-IPE) levels]; reduction of the endogenous antioxidants catalase (CAT) activity and reduced glutathione (GSH) levels; induction of NF-kB translocation; a loss of cytoplasmic NF-kB inhibitor alpha (IkBalpha); an increase in metalloproteinase-13 (MMP-13), caspase-3 and caspase-7 gene expression and their related protein; the induction of cartilage polymorphonuclear (PMN) activation and infiltration [evaluated by elastase (ELA) assay] and cartilage alterations evaluated by histological analysis. Intraperitoneal administration of different doses of C4S (for 25 days), ameliorated all the symptoms of inflammation in the articular knee and paw joints, limited lipid peroxidation, inhibited NF-kB activation and IkBalpha protein loss, decreased mRNA MMP-13 and caspases expression and their related protein, restored endogenous antioxidants, and reduced PMN accumulation in the damaged cartilage. CONCLUSION: The evidence that C4S was able to inhibit NF-kB and apoptosis activation supports the hypothesis that the C4S effect depends on reduction of ROS production, although other direct effects cannot be excluded.

Metabolism and biochemical/physiological roles of chondroitin sulfates: analysis of endogenous and supplemental chondroitin sulfates in blood circulation.

Chondroitin sulfate (CS) is a linear heteropolysaccharide consisting of repeating disaccharide units of glucuronic acid and galactosamine, which is commonly sulfated at C-4 and/or C-6 of galactosamine. The administration of CS as a supplement or a drug for the treatment of osteoarthrosis, the prevention of subsequent coronary events, treatment of psoriasis and ophthalmic diseases has been suggested. Much debate on the metabolism of CS and therefore the effectiveness of these treatments, especially after oral administration, has arisen due to the macromolecular nature of CS. Difficulties in analysing CS in blood due to the low endogenous concentrations and the covalent and anionic complexes with proteins have hampered the resolution of these issues. In this review, the information on the pharmacokinetics of CS obtained from studies in experimental animals and in humans is presented. Emphasis has been given to the analytical methods used for the determination of glycosaminoglycans, intact CS and CS-derived disaccharides in blood serum and plasma.

[Chondromodulation in 2003: dream or reality?]. / Chondromodulation en 2003: rêve ou réalité?

The use of drugs for the treatment of osteoarthritis, with a view to obtain a long-term symptomatic as well as structural benefit, is still in a preliminary state. Nowadays, matricial precursors, such as glucosamine sulfate, are an interesting approach in this indication. They have demonstrated a symptomatic efficacy comparable to that of N.S.A.I.D., with significantly reduced side-effects. Glucosamine sulfate also appears to positively interfere with the structural evolution of osteoarthritis by preventing joint space narrowing which characterizes the evolution of the disease. Encouraging results have also been obtained with chondroitin sulfate and diacerein. As for other molecules, new studies should be undertaken to confirm the results already obtained.

Tenascin glycoproteins and the complementary ligand DSD-1-PG/ phosphacan--structuring the neural extracellular matrix during development and repair.

The differentiation and morphogenesis of neural tissues involves a diversity of interactions between neural cells and their environment. Many potentially important interactions occur with the extracellular matrix (ECM), a complex association of extracellular molecules organised into aggregates and polymers. The large modular glycoprotein, Tenascin-C, and the chondroitin sulphate proteoglycan, DSD-1-PG/Phosphacan, have complex and frequently overlapping expression patterns in the developing CNS. Their presence in zones of cell proliferation, migration, and differentiation, as well as in boundary structures, suggest that they may be involved in the modulation of an extensive range of cellular processes. They are both strongly up-regulated in a range of CNS lesions and pathologies, being components of the glial scar, and expressed by gliomas. Functional roles in many cellular processes are possible through their extensive molecular interaction sites, both with each other, and with many of the same cell surface receptors, adhesion molecules, growth factors and other matrix proteins. These multiple interactions involve sites on both their protein domains and on the heterogeneous carbohydrate groups with which they are post-translationally modified. In vitro assays demonstrate cell-type specific effects on adhesion, migration and the formation and extension of cellular processes, including neurites and axons.

Recombinant human thrombomodulin(csa+): a tool for analyzing Plasmodium falciparum adhesion to chondroitin-4-sulfate.

The proteoglycan thrombomodulin has been shown to be involved, via its chondroitin-sulfate moiety, in the cytoadhesion of chondroitin-4-sulfate-binding-Plasmodium falciparum-infected erythrocytes to endothelial cells and syncytiotrophoblasts. We cloned and expressed in CHO and COS-7 cells a gene encoding soluble human recombinant thrombomodulin, with a chondroitin-4-sulfate moiety. This system is complementary to the in vitro cell models currently used to study the chondroitin-4-sulfate-binding phenotype. It also provides a means of overcoming the lack of specificity observed in interactions of infected erythrocytes with modified chondroitin-4-sulfate. This thrombomodulin displayed normal activity in coagulation, indicating that it was in a functional conformation. The recombinant protein, whether produced in CHO or COS-7 cells, inhibited cytoadhesion to Saimiri brain microvascular endothelial cells 1D infected with Palo-Alto(FUP)1 parasites selected for chondroitin-4-sulfate receptor preference. Thus, the recombinant protein was produced with a chondroitin-sulfate moiety, identified as a chondroitin-4-sulfate, in both cell types. In both cases, the recombinant protein bound to the chondroitin-4-sulfate phenotype, but not to CD36- and ICAM-1-binding parasites. The chondroitin-4-sulfate was 36 kDa in size for CHO and 17.5 kDa for COS-7 cells. There was, however, no difference in the capacities of the recombinant proteins produced by the two cell types to inhibit the cytoadhesion of infected erythrocytes. Thrombomodulin immobilized on plastic or coupled to Dynabeads was used to purify specifically the infected erythrocytes that bind to chondroitin-4-sulfate. These infected erythrocytes were cultured to establish parasite lines of this phenotype. We then showed that the thrombomodulin, labeled with FITC, could be used to detect this phenotype in blood samples. Finally, the direct binding of infected erythrocytes to immobilized thrombomodulin was used to screen for anti-chondroitin-4-sulfate-binding antibodies.

The influence of corneal stromal matrix proteins on the migration of human corneal fibroblasts.

Motivated by the alterations seen in the corneal matrix composition after photorefractive keratectomy and the migration of corneal keratocytes seen following this procedure, the locomotor response of corneal stromal fibroblasts to various extracellular matrix proteins was determined. In addition, the involvement of integrin mediated attachment to the matrix proteins was investigated. Quantitative invasion assays were performed using collagen gels, supplemented with either fibronectin, tenascin, collagen type V, collagen type VI, chondroitin sulfate or keratan sulfate. The ultrastructure of the gels was visualized by scanning electron microscopy and related to the migration results. The extent of alpha(1)beta(1), alpha(2)beta(1), alpha(3)beta(1)and alpha(5)beta(1)integrin mediated attachment to the matrix proteins was evaluated using blocking antibodies. Fibronectin increased corneal fibroblast migration significantly, and served as an excellent substrate for cellular attachment, mediated by the alpha(5)beta(1)integrin. Addition of tenascin to the fibronectin-containing gels disrupted these effects, while attachment to this matrix also involved the integrins alpha(2)beta(1)and alpha(3)beta(1). Chondroitin sulfate and collagen types V and VI primarily altered the structure of the collagen matrix, resulting in an inhibition of migration by the collagens and an increase by chondroitin sulfate. They all served as poor substrates for attachment. Thus, the migratory activity of corneal fibroblasts in vitro is influenced by the composition of the surrounding extracellular matrix, either by integrin mediated cell-matrix interactions or through matrix-matrix interactions. This study provides evidence that the provisional matrix deposited in a corneal stromal wound may facilitate the entry of migrating corneal fibroblasts.

Inhibitors and promoters of thalamic neuron adhesion and outgrowth in embryonic neocortex: functional association with chondroitin sulfate.

When embryonic thalamic neurons are plated onto living slices of mouse forebrain, cell attachment and neurite outgrowth on different layers of the developing cerebral cortex vary dramatically, in ways that correlate with the timing and pattern of thalamocortical innervation. These layer-specific differences can be eliminated from embryonic day 16 slices by enzymatic removal of chondroitin sulfate (CS). The cortical plate (a zone avoided by thalamic axons in vivo) possesses inhibitory activity (anti-adhesive, neurite repelling) and the intermediate zone and subplate (in which thalamic axons normally grow) possess stimulatory activity (adhesive, neurite promoting), both of which are chondroitinase sensitive. These opposing activities appear not to reflect the presence of different CS proteoglycans (CSPGs) in different zones, but rather the presence of differentially localized CS-binding molecules, which can be competed away by soluble CS. This model reconciles conflicting reports on the actions of CSPGs in neural development, and suggests a role for CSPGs in the organization of matrix-bound cues in the brain.

Modification of lymphocyte migration by sulfated polysaccharides.

The role of sulfated polysaccharides in lymphocyte migration has been analyzed in vivo using lymphocytes labeled with an intracellular DNA-binding fluorochrome Hoechst 33342. The influence of a panel of sulfated polysaccharides on entry (by injecting the sulfated polysaccharide prior to the labeled cells) and displacement from lymphoid organs (by injecting the sulfated polysaccharide after the labeled cells have localized) indicated that different sulfated polysaccharides have selective effects on entry and displacement, and furthermore positioning of subpopulations within organs. Additional experiments suggested that receptors for sulfated polysaccharides on high endothelial venules may interact with complementary structures on lymphocytes. The data supporting this conclusion were: (a) the normal localization behavior of lymphocytes preincubated with sulfated polysaccharides; (b) an inverse relationship between the expression of lymphocyte surface receptors for sulfated polysaccharides and the ability of the lymphocytes to enter lymphoid organs and (c) the selective binding of sulfated polysaccharide-coupled fluoresceinated beads to high endothelial venules. In this case only the beads coupled with the sulfated polysaccharides that inhibited entry bound to the high endothelial venules. These findings are discussed in terms of a fundamental cellular recognition system utilizing sulfated polysaccharides.