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Original Article Optimized Alkylated Cyclodextrin Polysulphates with Reduced Risks on Thromboembolic Accidents Improve Osteoarthritic Chondrocyte Metabolism

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Original Article Optimized Alkylated Cyclodextrin Polysulphates with Reduced Risks on Thromboembolic Accidents Improve Osteoarthritic Chondrocyte Metabolism Rheumatology 2011;50:12261235 RHEUMATOLOGY doi:10.1093/rheumatology/keq396 Advance Access publication 23 February 2011 Original article Optimized alkylated cyclodextrin polysulphates with reduced risks on thromboembolic accidents improve osteoarthritic chondrocyte metabolism Sara Groeneboer1, Stijn Lambrecht1, Aad Dhollander1, Peggy Jacques1, Bert Vander Cruyssen1, Rik J. Lories2, Katrien Devreese3, Koen Chiers4, Dirk Elewaut1 and Gust Verbruggen1 Abstract Objectives. To compare the ability of different cyclodextrin polysulphate (CDPS) derivatives to affect human articular cartilage cell metabolism in vitro. Methods. OA chondrocytes were cultured in alginate and exposed to 5 mg/ml of 2,3,6-tri-O-methyl-b- cyclodextrin (ME-CD), 2,3-di-O-methyl-6-sulphate-b-cyclodextrin (ME-CD-6-S), 2,6-di-O-methyl-3- sulphate-b-cyclodextrin (ME-CD-3-S), (2-carboxyethyl)-b-CDPS (CE-CDPS), (2-hydroxypropyl)-b-CDPS (HP-CDPS), 6-monoamino-6-monodeoxy-b-CDPS (MA-CDPS) or b-CDPS for 5 days. Effects on IL-1-driven chondrocyte extracellular matrix (ECM) metabolism were assayed by analysis of the accumu- lation of aggrecan in the interterritorial matrix, IL-6 secretion and qPCR. MA-CDPS, HP-CDPS, CE-CDPS and CDPS were analysed for their in vitro effect on coagulation and their ability to activate platelets in an in vitro assay to detect possible cross-reactivity with heparin-induced thrombocytopenia (HIT) antibodies. Results. The monosulphated cyclodextrins ME-CD-6-S and -3-S failed to affect aggrecan synthesis and IL-6 secretion by the OA chondrocytes. Polysulphated cyclodextrins MA-CDPS, HP-CDPS, CE-CDPS and CDPS at 5 mg/ml concentrations, on the other hand, significantly induced aggrecan pro- duction and repressed IL-6 release by the chondrocytes in culture. aPTT and PT for all derivatives were lengthened for polysaccharide concentrations >50 mg/ml. Five micrograms per millilitre of b-CDPS con- centrations that significantly modulated ECM ground substance production in vitro did not affect aPTT or PT. Furthermore, CE-CDPS, in contrast to MA-CDPS, HP-CDPS and CDPS, did not significantly activate BASIC platelets, suggesting a minimal potential to induce HIT thromboembolic accidents in vivo. SCIENCE Conclusions. CE-CDPS is a new, structurally adjusted, sulphated b-cyclodextrin derivative with preserved chondroprotective capacity and a promising safety profile. Key words: Chondroprotection, Osteoarthritis, Cyclodextrin polysulphates. Introduction OA is characterized by the degeneration and eventual loss 1 Department of Rheumatology, Laboratory of Connective Tissue of cartilage with concurrent changes in the subchondral Biology, Ghent University Hospital, Ghent, 2Department of Musculoskeletal Sciences, Division of Rheumatology, Laboratory for bone. While it is generally accepted that mechanical Skeletal Development and Joint Disorders, KU Leuven, 3Department of stress may result in the damage that will set off the OA Clinical Chemistry, Microbiology and Immunology, Coagulation Laboratory, Ghent University Hospital and 4Department of Pathology, process, its progression involves the activities of multiple Bacteriology and Avian Diseases, Ghent University, Ghent, Belgium. auto/paracrine cytokine activities. IL-1 is believed to be a Submitted 5 October 2009; revised version accepted major regulator of matrix degradation by increasing metal- 27 October 2010. loproteinase activity and suppressing the synthesis of Correspondence to: Gust Verbruggen, Department of Rheumatology, matrix components. In addition to significantly higher Laboratory of Connective Tissue Biology, Ghent University Hospital, De Pintelaan, 185 BE-9000, Ghent, Belgium. E-mail: gust.verbruggen@ intracellular levels of IL-1b, chondrocytes from fibrillated ugent.be cartilage show an up-regulation of the signalling IL-1 ! The Author(s) 2011. Published by Oxford University Press on behalf of The British Society for Rheumatology. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Optimized alkylated cyclodextrin polysulphates receptor I (IL-1RI) levels compared with normal cartilage was taken off the market in 1994. Neutraceutical industries [1]. Despite the attempt of chondrocytes to restore the then introduced the naturally occurring chondroitin homeostasis of their matrix by enhancing insulin-like sulphate to replace chondroitin polysulphate for the use growth factor 1 (IGF-1) and TGF-b growth factor activity in degenerative joint disease in humans. In the sole head- [2, 3], the catabolic activity of the OA chondrocyte results to-head confrontation thus far, the naturally occurring in a continuous net loss of matrix components ensuing chondroitin sulphate was inferior to chondroitin polysul- disease progression. phate when the chondroprotective effects of both drugs The search for agents that restore the structural defi- were assessed in a population with hand OA [16]. ciencies underlying OA started in the mid-1970s with the Recently, a novel polysulphated polysaccharide, cyclo- findings that sulphated polysaccharides, e.g. heparin and dextrin polysulphate (CDPS) was reported to induce a chondroitin sulphates, with varying degrees of sulphating, down-regulation of intracellular IL-1a and -b and to enhanced the synthesis of extracellular matrix (ECM) cause a concomitant increase in the synthesis of aggre- substances by connective tissue cells in culture [4, 5]. In can, collagen Type II and fibronectin in the cell-associated particular, the polysulphated polysaccharides provided matrix (CAM) of human chondrocytes cultured in alginate significant effects on the structure and function of articular beads [17]. This CDPS also depressed the IL-6 release cartilage cells in culture [6, 7]. These effects were also of OA chondrocytes by 60% compared with untreated observed in vivo [810]. Consequently, both xylosan poly- OA cartilage cells and equalled the levels secreted by sulphate and chondroitin polysulphate found their place normal cells [17]. in the treatment of osteoarticular pathology in veterinary CDPS, subcutaneously administered in a rabbit model and in human medicine, respectively. of experimental OA, reduced the cartilage lesions and Apart from the chondroprotective capacities, however, osteophyte formation in the affected joints [18]. These these polysulphated polysaccharides were revealed to data suggest that CDPS positively affects the tissue possess important biological activities similar to those of pathology underlying OA and this agent can therefore be heparin. Such biological activities were clearly shown to classified as a structure or disease-modifying OA drug. be related to the molecular structure of the polysaccharide To alleviate possible heparin-related side effects, we and varied following distinct modifications. In vitro and have developed six sulphated b-cyclodextrin derivatives in vivo studies on the effects of these polysacchar- by introducing hydrophobic substituents on the 2, 3 ides on coagulation showed variable effects on thrombin and/or 6 position, assuming they would preserve their clotting times. It was, however, the possibility of such chondroprotective effect. Next, the effect on coagulation polysulphated polysaccharides to induce heparin-induced and the potency to induce thrombocytopenia through thrombocytopenic thrombosis (HITT) through cross- cross-reaction with heparin/PF4 antibodies were assayed. reaction with heparin/platelet factor-4 antibodies that Once optimized, these derivatives were tested for their had raised serious concern [11]. These antibodies arise capacity to restore cartilage damage in vitro. occasionally when activated thrombocytes release platelet factor-4 (PF4) during heparin treatment. Heparin then Materials and methods forms a complex with PF4 that acts as an antigen which triggers the production of auto-antibodies. These antibo- b-Cyclodextrin derivatives 0 dies bind to the complex via their F(ab) region and to the The 2,3,6-tri-O-methyl-b-cyclodextrin (ME-CD) was pur- low affinity immunoglobulin gamma Fc region receptor II-b chased from Cyclolab Ltd (Budapest, Hungary) and (FcgRII) [immunoglobulin G (IgG) CD32] of other platelets served as a non-sulphated control molecule. via their Fc portion, thereby initiating platelet activation, b-Cyclodextrins carrying one sulphate on each glucopyr- aggregation and thromboembolic accidents [12]. It has anose unit included 2,3-di-O-methyl-6-- been shown that some low-molecular weight heparins as sulphate-b-cyclodextrin (ME-CD-6-S: Regis well as other sulphated polysaccharides, e.g. chondroitin Technologies Inc.; Morton Grove, IL, USA) and 2,6-di-O- polysulphates [13], can also bind to heparin-induced methyl-3-sulphate-b-cyclodextrin (ME-CD-3-S: Cyclolab thrombocytopenia (HIT) antibodies in the presence of Ltd). Commercially available (2-carboxyethyl)- PF4 and that their reactivity is dependent on their molecu- b-cyclodextrin (CE-CD containing, on average, three car- lar weight and the sulphating grade [12, 14, 15]. boxyethyl groups: Sigma Chemical Company, St Louis, When, in the 1980s, controversial reports on the occur- MO, USA), (2-hydroxypropyl)-b-cyclodextrin (HP-CD con- rence of HITT following treatment with chondroitin polysul- taining seven hydroxypropyl groups: Sigma Chemical phate came out [13], national and international agencies Company)
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