The loss of anabolic responses of chondrocytes to IGF-1 is one of the reasons why diseased cartilage cannot counteract the action of catabolic cytokines during OA. The results of the present study show that NBI-31772, a small, nonpeptide compound that binds to endogenous IGFBPs and releases biologically active IGF-1 [32
], can restore proteoglycan synthesis by human OA chondrocytes, therefore opening the possibility of a new field of pharmacological intervention for the treatment of OA and other joint-related diseases.
As previously demonstrated in ovine and bovine chondrocytes [30
], a mutant of IGF-1 with strongly decreased affinity for IGFBPs was able to stimulate the synthesis of proteoglycans by OA chondrocytes to a higher extent than did IGF-1. This simple but convincing experiment was the basis for suspecting the involvement of endogenous IGFBPs in the loss of IGF-1 effects in OA chondrocytes, and it was a prerequisite to justify their targeting. Three forms of IGFBPs were found to be secreted by human OA chondrocytes, the major one being IGFBP-3. OA cartilage explants secreted IGFBP-3 exclusively. In a functional radiobinding assay, NBI-31772 displaced the binding of 125
I-labeled IGF-1 at a molar concentration 20 times that of unlabeled IGF-1. Although the ability of NBI-31772 to displace 125
I-labeled IGF-1 from the other IGFBPs has not been checked in this study, it has been previously shown that it can interact with all six IGFBPs with similar efficacy [32
]. Therefore, it is probable that in the experiments with OA chondrocytes, NBI-31772 also acted by displacing IGF-1 from IGFBP-2 and -4 in addition to IGFBP-3.
In the first set of experiments with rabbit chondrocytes, IGFBP-3 at a concentration four times that of IGF-1 completely abolished the stimulation of proteoglycan synthesis by IGF-1, and NBI-31772 partially restored IGF-1-dependent synthetic activities at 1 μM and almost totally at 10 μM. These concentrations were much higher than those found to displace 125
I-labeled IGF-1 from IGFBP-3 in the radiobinding assay. This discrepancy was also found in a previous study reporting the activity of NBI-31772 on the reversal of IGFBP-3 inhibition of Balb/c3T3 IGF-1-dependent proliferation [38
]. The higher amounts of IGFBP-3 used in the biological assays compared with the radiobinding assay may in part account for this discrepancy. On the other hand, differences may rest on a decreased stability of the chemical compound in a biological environment and/or to its capture or neutralization by chondrocyte-secreted proteins or macromolecules, thus limiting its activity. Similar results were found for human OA chondrocytes, without addition of IGFBP-3. In this cellular model, we asked whether NBI-31772 could displace IGF-1 from endogenous IGFBPs. As also found by others [27
], IGF-1 was either not detectable or poorly detectable in the extracellular medium, reflecting poor ability of chondrocytes to secrete IGF-1. Thus, it was given exogenously. The addition of NBI-31772 was able to activate or increase proteoglycan synthesis, showing that complexes of IGF-1 with IGFBP could be disrupted to release bioactive IGF-1. NBI-31772 also significantly increased the ratio of cell-associated proteoglycans in the presence of IGF-1/IGFBP complexes. When, in these experiments, IGF-1 was replaced by R3
IGF-1, the stimulating effect of NBI-31772 on proteoglycan synthesis was completely abolished, thus ruling out the possibility that NBI-31772 may act independently of the inhibition of IGF-1-binding to IGFBPs. However, an increase of cell-associated proteoglycans was still observed, showing that this effect of NBI-31772 occurred apart from IGFBPs. Although unexplained, this mechanism may be important in a pathological context, since communication between chondrocytes and the extracellular matrix is disrupted in OA, and an increase of pericellular proteoglycans may help to maintain chondrocytes in a more favorable environment.
Under physiological conditions, IGF-1/IGFBP complexes can be disrupted by various proteases [15
], thereby releasing active IGF-1. In a recent report by Fowlkes and co-workers, matrix metalloproteinase 3 was able to disrupt IGF/IGFBP-3 complexes and to liberate free, intact IGFs able to phosphorylate the IGF-1 receptor and to trigger cell proliferation [40
]. A few different approaches have been used to prevent excessive sequestration of IGF-1 and restore its bioavailabity in pathological conditions. Mutant peptides of IGF-1 still able to bind to IGFBPs but with very weak affinity for the IGF-1 receptor were designed to displace native IGF-1 from endogenous IGFBPs [41
]. Phage-displayed peptide libraries yielded small peptide compounds able to selectively displace IGF-1 from IGFBP-1 or IGFBP-3 [45
]. From the last generation, low-molecular-weight compounds (fluorenylmethoxycarbonyl derivatives) were shown to bind to the IGF-1 binding site on IGFBP-5 [47
], and small isoquinoline compounds were shown to displace IGFs from IGFBPs without selectivity for a particular IGFBP [32
]. These molecules have been proposed as potential drug candidates for the treatment of diabetes [32
], renal disease [50
], or neurodegenerative diseases [43
Most of the current pharmacological approaches for the treatment of OA aim to prevent catabolism by blocking actors of the deleterious cascade, while restoration of anabolism or cartilage regeneration still rests on invasive procedures [51
]. In this context, in situ
cartilage regeneration by growth-factor therapy, including targeting of cartilage defects with fibrin–(chondrocyte)–IGF-1 composites [52
], or gene therapy with IGF-1 delivered intra-articularly [54
], have been proposed. To be effective, the amount of IGF-1 delivered within the joint in this way should exceed the binding capacity of local IGFBPs. In addition to noninvasive intervention, one of the main advantages of our proposed strategy would be to target directly the actors of cartilage unresponsiveness to IGF-1, and to bypass the capture of exogenous IGF-1 by IGFBPs. Keeping this objective in mind, it is clear that the small compound used in this study, NBI-31772, should be considered only a tool to demonstrate the concept in vitro
. Indeed, concentrations of NBI-31772 active in the biological assays were relatively high (1–10 μM) and would be incompatible with in vivo
trials. Secondly, NBI-31772 did not penetrate into cartilage. This should not be an exclusion criterion per se, since one may hypothesize that if the compound were capable of reaching the synovial fluid or cartilage surrounding tissues, it might release IGF-1 captured by IGFBPs in these tissues and sustain its action in cartilage. However, the in vivo
duration of action of NBI-31772 was short [48
], which implies that in vivo
trials to assess the therapeutic relevance of this class of molecule in joint diseases need to be performed with more stable molecules. In view of a therapeutic application, the optimization of small chemical inhibitors of IGF/IGFBP interaction would be preferred over the use of IGF-1 peptide mutants (either binding receptors and triggering biological effects without binding IGFBPs, or, conversely, displacing endogenous IGF-1 from IGFBPs without binding receptors), since the former should be more easily optimizable while the latter should be more susceptible to the attack by proteases. In a clinical context, compounds that selectively target IGFBP-3 would be mandatory if oral availability is preferred, in order to avoid or to limit side effects caused by displacing IGF-1 from its binding proteins in undesirable tissues. This question of selectivity may not be so important in the case of topical delivery, and local treatment should be envisaged instead.