Purpose: In the pathophysiology of osteoarthritis (OA) several mediators are involved, however the attempts to develop a drug acting on specific targets involved in OA progression (e.g. Matrix Metalloproteases: MMPs) failed at various stages due to unfavourable pharmacokinetics or chronic toxicity. On the other hand Hyaluronic Acid (HA) is widely used as an intra-articular therapy for OA thanks to its capacity of restoring viscoelasticity and lubrication in degraded synovial fluid and to its beneficial effects on inflammation and cartilage metabolism.
We describe here the selection of a HA alkylamide derivative (HYADD®4) with inhibitory activity in the micromolar range vs MMP13, for the intra-articular treatment of OA patients.
Methods: Chlostridium histolyticum Collagenase (ChC) was used as model of MMP. A number of selected glycosaminoglycans currently used in viscosupplements were compared for their inhibitory effect on ChC enzymatic activity: Chondroitin Sulfate (CS), unmodified HA, HA cross-linked and HA amidated with hexadecylamine, all formulated at 8 mg/ml in PBS pH 7, were incubated in presence of ChC at 37°C and samples withdrawn at different timepoints were tested in a ChC activity assay. In order to abolish the effect of physical entrapment of the enzyme, the test was repeated after depolymerization of the materials by heating at 105°C for 24 h before incubation with ChC.
For the screening of the modified HAs: six amide derivatives of HA were synthesized by alkylation at the carboxylic group of the D-Glucuronic acid unit with a series of linear alkyl chains C8, C12, C15, C16, C18 and a benzyl group, at the same amidation degree. The inhibition constant (Ki) of the depolymerized HA amide derivatives, versus ChC was measured by steady-state kinetics experiments at several inhibitor concentration.
The compound selected as the best ChC inhibitor (HYADD®-C16 or HYADD®4) was then tested in vitro vs the catalytical subunits of 10 human MMPs using a MMP Inhibitor Profiling Kit. The selective inhibitory activity of HYADD®4 vs MMP13 observed in the screening test was finally validated on the human enzyme in an ex-vivo experiment with human inflamed synovial fluid (SF), collected by arthrocentesis and analyzed by means of a human MMP13 assay kit.
Results: In the first screening test the cross-linked HA and HA hexadecylamide, but not CS and high MW HA, showed a relevant decrease of the ChC activity (probe metalloprotease). In order to rule out the hypothesis that the inhibition was due to simple physical entrapment of the enzyme in the test materials, the experiment was repeated after depolymerization of the polysaccharides: in these conditions ChC activity was almost completely recovered in the case of cross-linked HAs, while depolymerized HA hexadecylamide (HYADD®4) caused a further loss of enzyme activity, as a proof of specific inhibition.
Since the HA alkylamide showed this unique property, in the second part of the work six different amide derivatives of HA, synthesized with C8, C12, C15, C16, C18 linear alkyl amines and a benzyl amine, were tested for the inhibitory activity against ChC. The inhibitor potency showed to be directly correlated to the length of the alkyl chain. The C16 alkyl amide derivative HYADD®4 was identified as lead compound (Ki= 6.2±1.6 μM), on the basis of the best compromise between potency and solubility.
With the aim of assessing the specificity of the effect, HYADD®4 was then screened vs 10 different human MMP catalytic subunits, showing higher selectivity for MMP8 and MMP13. The Ki against human MMP13 was found to be in the micromolar range (61.7 ± 7.0 μM).
Finally, since MMP13, unlike MMP8, is involved in OA progression, an ex vivo study was performed by incubating HYADD®4 in SF from patients with inflammatory arthritis: the Ki value obtained (106.1 ± 9.2 μM) was comparable to that observed in vitro, thus validating the inhibitory activity against MMP13.
Conclusions: These data suggest that the extremely hydrophobic side chain of HYADD®4, whose water solubility is boosted by the HA backbone, is pivotal in the mechanism of MMP competitive inhibition.
The polymer structure suggests that the alkyl side chain could easily and selectively dock the hydrophobic S1' pocket in the MMP catalytic domain.
The hexadecyl derivative shows the best performance against MMP13 and MMP8, as confirmed also in an ex-vivo experiment in SF vs human MMP13. Furthermore, the intra-articular administration of the alkyl-modified HA as a treatment for OA, thanks to its site-selectivity and solubility, can overcome the common issues of the small molecule MMP inhibitors, such as systemic distribution and toxicity.
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