Amphiphilic peptide-based MMP3 inhibitors for intra-articular treatment of knee OA.

Since 2007, Metalloproteases (MMPs) have been considered potential targets for treating osteoarthritis (OA), for which the primary pathogenic event is the extensive degeneration of articular cartilage. MMP3 is an enzyme critical for these degenerative changes. However, problems of selectivity, low bioavailability and poor metabolic profile during clinical trials of MMPs inhibitors (MMPIs) led to limited beneficial effect and thus did not justify further pursuit of the clinical studies. In a previous work, a new alkyl derivative of hyaluronic acid (HA), HYADD4®, previously approved as intra-articular treatment for knee OA, was studied in vitro and in vivo as MMP3I. Molecular simulation studies confirmed the interaction between the alkyl side chain of this HA derivative and the additional S1' pocket of MMP3. However, the high MW and the polar HA backbone of HYADD4® imply a high desolvation energy cost, which can potentially decrease its inhibitory potency. In this study, a new class of MMP3Is based on a small peptide backbone (CGV) chemically derivatized with an alkyl chain was developed through interactive cycles of design, synthesis and screening, accompanied by computational evaluation and optimization. Two MMP3Is, e(I) and l(II), were selected because of their effective inhibitory activity (3.2 and 10.2 µM, respectively) and water solubility. Both MMPIs showed a broad range of inhibitory effects against almost all the MMPs tested. In an in vitro model of inflammatory OA, e(I) was the most effective MMPI: at the concentration of 93 µM, it reversed inflammatory outcomes. Moreover, because of its amphiphilic structure, the e(I) MMPI promoted stable micellar formulation at concentrations higher than 0.2 mg/mL in water. The findings were confirmed by TEM and Nile red staining analysis. Based on these results, the e(I) MMPI can be considered a good candidate for the intra-articular treatment of OA, and the micellar formulation of this peptide in an aqueous buffer can potentially increase the bioavailability and, thus, the efficacy of the MMPIs.

Dopamine-functionalized sulphated hyaluronic acid as a titanium implant coating enhances biofilm prevention and promotes osseointegration.

A series of new hyaluronan derivatives was synthesized and tested as an antibiotic release system by antibacterial and osseointegration assays. Specifically, partially sulphated hyaluronic acid (sHA) was functionalized with dopamine (DA). The DA moiety guarantees good performance as a binding agent for coating a titanium alloy surface; furthermore, the negatively charged sHA has bone regenerative effects and a high binding affinity for positively charged antibiotics. A sHA scaffold with a defined degree of sulphation (DS =2) was selected as a good compromise between a high negative charge density and poor heparin-like anticoagulant activity, while the degree of DA derivatization (17.1%mol) was chosen based on the absence of cytotoxic activity and the promotion of osteoblast proliferation. The titanium alloy coating was investigated indirectly using a fluorescent probe and directly by environmental scanning electron microscope (ESEM) analysis. Long-duration antibiotic release was demonstrated in vitro, and antibacterial efficacy against a Staphylococcus aureus culture was shown.

A new potential spreading factor: Streptomyces koganeiensis hyaluronidase. A comparative study with bovine testes hyaluronidase and recombinant human hyaluronidase of the HA degradation in ECM.

BACKGROUND: Recombinant human hyaluronidase has been used in the interstitial matrix to promote the dispersion of therapeutics. The production and isolation of an extracellular hyaluronidase from Streptomyces koganeiensis (rHyal_Sk) has recently been described. METHODS: The specificity of rHyal_Sk has been assessed against heparan sulfate, chondroitin sulfates and sulfated HAs. The oligomers generated by HA degradation have been investigated by MALDI-TOF MS analysis. rHyal_Sk has been compared with BTH and PH20 in vitro, against cross-linked HA (ACP) and HA-aggrecan complex, and in vivo, by means of a diffusion assay in nude mice. RESULTS: Depolymerization of HA by rHyal_Sk gave tetra-, hexa- and octasaccharides in high yields. The reaction mechanism and the high HA specificity were demonstrated. The in vivo diffusion assay, supported by the in vitro tests, evidenced an initially enhanced enzymatic activity of rHyal_Sk compared to BTH and PH20. CONCLUSIONS: rHyal_Sk, compared to BTH and PH20, showed higher substrate specificity and no inhibition from GAGs sulfate, together with a superior performance for HA depolymerization in ECM. As better predictive tests for the in vivo activity of hyaluronidase we developed two assays based on the degradation of ACP or of the HA-aggrecan complex. GENERAL SIGNIFICANCE: rHyal_Sk is a new potential spreading factor for intradermal drug administration. Hyaluronidases of distinct classes, that show equivalent activities in a common turbidimetric assay, could have different potencies and dose-efficacies in vivo which influences the therapeutic effect. The new proposed in vitro tests are designed to obtain a predictive characterization of the enzyme activity in vivo.

The role of high molecular weight hyaluronic acid in mucoadhesion on an ocular surface model.

Hyaluronic acid (HA) is frequently formulated in eye drops to improve the stability of the tear film by hydration and lubrication. Mucoadhesion is related to the ocular residence time and therefore to the effectiveness of the eye drops. The ocular residence time of the HA formulation is correlated with the ability of HA to create specific strong interactions in the ocular surface with the mucus layer, mainly composed of a mixture of secreted mucins (MUC; gel forming MUC5AC and MUC2) and shed membrane-bound soluble mucins (MUC1, MUC4, and MUC16). Dry eye disease (DED) is a multifactorial pathology of the preocular tear film with possible damage to the ocular surface classified in two types: (1) aqueous-deficient dry eye and (2) evaporative dry eye, caused by a decrease in goblet cell density that reduces MUC expression and/or by meibomian gland dysfunction, that results in a drop in the lipidic fraction of the tear film. In this work, the binding affinity between HA and MUC2 has been evaluated with three complementary approaches because the secreted MUCs play a pivotal role in the viscoelastic properties of the tear film: 1. Rheological analysis, measuring the mucoadhesive index and the complex viscosity in relation to MM (Molecular Mass) and concentration; 2. Fluorescence analysis, using a fluorescent hydrophobic probe, to investigate the conformational change of MUC2 during the interaction with the HA polymer; 3. Surface plasmon resonance analysis, used to measure the affinity between MUC2 (immobilized on the surface of a sensor chip) and the HA polymers that flowed on it at the molecular level. For all these tests, the mucoadhesive performance of the natural HA linearly increases with the MM, whereas cross-linked HA and other emollient and gelling agents (formulated in artificial tears) do not show the same mucoadhesive properties (with the exception of xanthan gum). The mucoadhesive performance of high MM HA has also been confirmed in conditions that simulate the pathological condition of the tear film during DED by decreasing the MUC2 or oleic acid concentration. Physico-chemical analysis of a series of marketed artificial tears confirms the linear correlation between the MM of the HA used in the products and the mucoadhesive index measured on the ocular surface model.

Effect of intra-articular injection of a hyaluronic acid-alendronate conjugate on post-traumatic osteoarthritis induced by destabilization of the medial meniscus in rats.

Osteoarthritis (OA) is a chronic degenerative joint disease characterized by pain and cartilage damage. Intra-articular (i.a) viscosupplementation with hyaluronic acid (HA) is frequently used for the management of OA. Preclinical studies have reported that bisphosphonates (BPs) may have a therapeutic potential to slow down or reverse the progression of OA. Among these, alendronate (ALN) has demonstrated chondroprotective effects in both in vitro and vivo experiments. This study evaluated the effects of a novel alendronate-hyaluronic acid (ALN-HA) conjugate on an OA in vivo model induced by medial meniscus destabilization (DMM). DMM surgery was performed on the knees of Sprague Dawley rats that received, after four weeks, one intra-articular (i.a.) injection of: (1) ALN-HA; (2) HA; (3) sodium chloride (NaCl). Sham-operated rats were used as control. Allodynia was assessed by Von Frey test. Joint degeneration was evaluated eight weeks after treatment by micro-computed tomography (micro-CT), histology, and immunohistochemistry. Collagen cross-linked C-telopeptides (CTX-I and CTX-II) serum levels were determined by ELISA. Paw withdrawal threshold increased in ALN-HA group when compared to rats treated with NaCl or HA. Micro-CT did not show differences between ALN-HA, HA and NaCl groups. ALN-HA injection produced significant improvements in articular cartilage degeneration showing an OARSI score lower than those of HA and NaCl, and reduced matrix metalloproteinase (MMP)-13, MMP-3, interleukin-6, vascular endothelial growth factor and Caspase-3 expression. CTX-I was reduced after ALN-HA treatment when compared to NaCl. Our results indicate that i.a. use of ALN after conjugation with HA limits OA development and progression in the rat DMM model, and may lead to the development of novel therapeutic strategies in OA management.

A Photopolymerizable Biocompatible Hyaluronic Acid Hydrogel Promotes Early Articular Cartilage Repair in a Minipig Model In Vivo.

Articular cartilage defects represent an unsolved clinical challenge. Photopolymerizable hydrogels are attractive candidates supporting repair. This study investigates the short-term safety and efficacy of two novel hyaluronic acid (HA)-triethylene glycol (TEG)-coumarin hydrogels photocrosslinked in situ in a clinically relevant large animal model. It is hypothesized that HA-hydrogel-augmented microfracture (MFX) is superior to MFX in enhancing early cartilage repair, and that the molar degree of substitution and concentration of HA affects repair. Chondral full-thickness defects in the knees of adult minipigs are treated with either 1) debridement (No MFX), 2) debridement and MFX, 3) debridement, MFX, and HA hydrogel (30% molar derivatization, 30 mg mL-1 HA; F3) (MFX+F3), and 4) debridement, MFX, and HA hydrogel (40% molar derivatization, 20 mg mL-1 HA; F4) (MFX+F4). After 8 weeks postoperatively, MFX+F3 significantly improves total macroscopic and histological scores compared with all other groups without negative effects, besides significantly enhancing the individual repair parameters "defect architecture," "repair tissue surface" (compared with No MFX, MFX), and "subchondral bone" (compared with MFX). These data indicate that photopolymerizable HA hydrogels enable a favorable metastable microenvironment promoting early chondrogenesis in vivo. This work also uncovers a mechanism for effective HA-augmented cartilage repair by combining lower molar derivatization with higher concentrations.

Aerosolized sulfated hyaluronan derivatives prolong the survival of K18 ACE2 mice infected with a lethal dose of SARS-CoV-2.

Despite several vaccines that are currently approved for human use to control the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is an urgent medical need for therapeutic and prophylactic options. SARS-CoV-2 binding and entry in human cells involves interactions of its spike (S) protein with several host cell surface factors, including heparan sulfate proteoglycans (HSPGs), transmembrane protease serine 2 (TMPRSS2), and angiotensin-converting enzyme 2 (ACE2). In this paper we investigated the potential of sulphated Hyaluronic Acid (sHA), a HSPG mimicking polymer, to inhibit the binding of SARS-CoV-2 S protein to human ACE2 receptor. After the assessment of different sulfation degree of sHA backbone, a series of sHA functionalized with different hydrophobic side chains were synthesized and screened. The compound showing the highest binding affinity to the viral S protein was further characterized by surface plasmon resonance (SPR) towards ACE2 and viral S protein binding domain. Selected compounds were formulated as solutions for nebulization and, after being characterized in terms of aerosolization performance and droplet size distribution, their efficacy was assessed in vivo using the K18 human (h)ACE2 transgenic mouse model of SARS-CoV-2 infection.

Hydrophobic Derivatives of Sulfated Hyaluronic Acid as Drug Delivery Systems for Multi-Target Intra-Articular Treatment of Post-Traumatic Osteoarthritis.

During osteoarthritis (OA) development, chondrocytes progressively decompensate, upregulating proteolytic enzymes and reducing the key growth factors involved in promoting chondrocyte anabolism. A combined therapeutic approach is needed to address this multifactorial pathology, which affects the whole joint. Based on the literature, three promising targets for OA treatment have been selected: MMP3 (matrix metallopeptidase 3), TRPV4 (transient receptor potential cation channel subfamily V member 4) and mTOR (mammalian target of rapamycin). In this study, a novel water-soluble and biocompatible amphiphilic polymer named "sHA-oleylamide" was synthesized and screened from a series of hyaluronic acid derivatives for its anticatabolic activity. This MMP inhibitor showed no cytotoxicity, and in an in vitro model of inflammatory OA, it reversed the inflammatory outcome at a concentration of 0.011 mg/mL. The ability of sHA-oleylamide to form 20-50 nm micelles in water with a critical micelle concentration of 0.27±0.1 mg/mL, was confirmed by TEM images and measured by Nile red staining. RN-1747 and rapamycin molecules were successfully loaded in sHA-oleylamide, previously prepared at 12 mg/mL in PBS; both formulations were stable, sterile and confirmed in vitro to have mTOR inhibition by rapamycin and TRPV4 activation activity by RN-1747. The controlled release of RN-1747 from the micellar formulation with sHA-oleylamide showed that only approximately 60% of the total loaded RN-1747 was released within 7 days. These micellar formulations can potentially increase the bioavailability and pharmaceutical efficacy of the selected active molecules, combining their anti-catabolic and pro-anabolic activities and making them suitable for i.a. administration as OA treatments.

A versatile and robust analytical method for hyaluronan quantification in crosslinked products and complex matrices.

Hyaluronic acid (HA), a naturally occurring biopolymer composed of repeating units of d-glucuronic acid and N-acetyl-glucosamine, is widely used as principal component of drugs, medical devices, nutraceuticals and cosmetics. Chemical modifications of HA or the presence of unmodified HA in complex matrices often brings common analytical techniques to fail its identification or quantification. In this work, a specific method for the quantification of HA and HA derivatives was developed and tested. After strong acid hydrolysis, polysaccharide depolymerization and N-acetylglucosamine deacetylation, quantitatively yielded glucosamine residues were derivatized using Fluorenylmethyloxycarbonyl chloride (FMOC), separated and quantitated by means of HPLC equipped with UV detection. The method was partially validated according to ICH Q2(R1) and successfully applied on different viscosupplements composed by modified HA or medical devices containing unmodified HA in complex matrices.

Titanium implant coating based on dopamine-functionalized sulphated hyaluronic acid: in vivo assessment of biocompatibility and antibacterial efficacy.

The number of total knee and/or hip replacements are expected to exceed 5 million a year by 2030; the incidence of biofilm-associated complications can vary from 1% in primary implants to 5.6% in case of revision. The purpose of this study was to test the ability of sHA-DA, a partially sulphated hyaluronic acid (sHA) functionalized with a dopamine (DA) moiety, to prevent acute bacterial growth in an in vivo model of an intra-operatively highly contaminated implant. Previously, in vitro studies showed that the DA moiety guarantees good performance as binding agent for titanium surface adhesion, while the negatively charged sHA has both a high efficiency in electrostatic binding of positively charged antibiotics, and bone regenerative effects. The in vitro testing also highlighted the effectiveness of the sHA-DA system in inhibiting bacterial spreading through a sustained release of the antibiotic payload from the implant coating. In this study the chemical stability of the sHA-DA to ß-ray sterilization was demonstrated, based on evaluation by NMR, SEC-TDA Omnisec and HPLC-MS analysis, thus supporting the approach of terminal sterilization of the coated implant with no loss of efficacy. Furthermore, an in vivo study in rabbits was performed according to UNI EN ISO 10993-6 to assess the histocompatibility of titanium nails pre-coated with sHA-DA. The implants, placed in the femoral medullary cavity and harvested after 12 weeks, proved to be histocompatible and to allow bone growth in adhesion to the metal surface. Finally, an in vivo model of bacterial contamination was set up by injecting 1 mL of bacterial suspension containing 104 or 106 CFU of methicillin-resistant Staphylococcus aureus (MRSA) into the femoral medullary cavity of 30 rabbits. Titanium nails either uncoated or pre-coated with sHA-DA and loaded directly by the surgeon with 5% vancomycin were implanted in the surgical site. After 1 week, only the animals treated with pre-coated nails did not show the presence of systemic or local bacterial infection, as confirmed by microbiology and histology (Smeltzer score). Further insights into the animal model setup are crucial, however the results obtained suggest that the system can be effective in preventing the onset of the bacterial infective process.

Hyaluronic acid-alendronate conjugate: A macromolecular drug delivery system for intra-articular treatment of osteoarthritis.

Objective: Osteoarthritis (OA) is a painful degenerative disease of the whole joint structure, including articular cartilage, synovial fluid, and subchondral bone. Hyaluronic acid (HA), an anionic non-sulfated glycosaminoglycan, is commonly used for intra-articular (IA) treatment in OA, while bisphosphonates (BPs) are anti-resorptive drugs that act on the bone. Here, a novel conjugate with a covalent and hydrolysable linker between HA and alendronate (ALD) was designed as an attractive therapeutic strategy for IA drug delivery. Design: The HA-ALD derivative was synthesized and tested in comparison with a simple mixture of HA and ALD for in vitro ALD release, rheological properties, cytotoxicity towards osteoblasts and chondrocytes and in an in vitro efficacy assay of OA inflammatory model on bovine cartilage explants. Results: The structure of HA-ALD was elucidated exhibiting no depolymerization and efficient drug incorporation. The controlled ALD release in vitro was slower compared to the simple mixture of HA and ALD; moreover, the derivative showed calcium-tuned rheological properties. The absence of cytotoxicity towards osteoblasts and chondrocytes was shown for up to 7 days, and the viability of chondrocytes was confirmed by fluorescence microscopy. Finally, a reduction in collagen release and MMP-13 expression was measured in the OA inflammatory model. Conclusion: This new HA-ALD derivative opens the door to a new approach for OA treatment, as it combines viscosupplementation and biological effects of HA with the pharmacological activity of BPs. Prolonged ALD release increased rheological properties and beneficial effect against cartilage degradation make it a promising IA therapy for OA.

Synergy of Hydeal-D® and Hyaluronic Acid for Protecting and Restoring Urothelium: In Vitro Characterization.

Interstitial cystitis (IC) or painful bladder syndrome is a chronic dysfunction due to an inflammatory condition, characterized by bladder pain and urinary frequency. Currently, no gold standard therapy is available since IC does not respond to conventional ones. Given these premises, the aim of this work was the in vitro characterization of biological properties (mucoadhesion and anti-inflammatory activity) of a commercial product (HydealCyst-HydC) based on hyaluronic acid (HA) and the benzyl ester of HA (Hydeal-D®) intended for bladder instillation to restore and/or protect the urothelial layer of glycosamino glycans (GAGs). The in vitro characterization demonstrated that an interaction product is formed between HA and Hydeal-D® that has a role in the rheological behavior and mucoadhesive properties. HA was identified as a key component to form the mucoadhesive joint, while the interaction of HA with Hydeal-D® improved polysaccharide stability and prolonged the activity ex vivo. Moreover, HydC is cytocompatible with urothelial cells (HTB-4) and possesses an anti-inflammatory effect towards these cells by decreasing the secretion of IL-6 and IL-8, which were both increased in patients with IC, and by increasing the secretion of sulfated GAGs. These two findings, along with the resilience properties of the formulation due to mucoadhesion, suggest the active role of HydC in protecting and restoring urothelium homeostasis.

HA-based dermal filler: downstream process comparison, impurity quantitation by validated HPLC-MS analysis, and in vivo residence time study.

The success of hyaluronic acid (HA)-based dermal fillers, with more than 2 million minimally invasive procedures conducted in 2016 in the US alone, is due to their hygroscopic properties of biocompatibility and reversibility. The type and density of HA cross-linkage, as well as the manufacturing technology, may influence not only the in vivo persistence but also the safety profile of dermal fillers. 1,4-Butanediol diglycidyl ether (BDDE) is the cross-linker used in most market-leading HA fillers; 1,4-butanediol di-(propan-2,3-diolyl) ether (BDPE) is the major impurity obtained from the HA-BDDE cross-linking (HBC) process. In this work, a new process to obtain high purity HBC fillers was developed. A new HPLC-MS method was validated for the quantification of BDPE content in HBC dermal fillers. In vitro cytotoxicity of BDPE was evaluated in fibroblasts (IC50 = 0.48 mg/mL). The viscoelasticity was monitored during the shelf-life of the HBC-10% hydrogel and was correlated with in vitro hyaluronidase resistance and in vivo residence time in a rabbit model. This analysis showed that elasticity is the best parameter to predict the in vivo residence time. Finally, a series of parameters were investigated in certain marketed dermal fillers and were compared with the results of the HBC-10% hydrogel.

Photocrosslinked hydrogels from coumarin derivatives of hyaluronic acid for tissue engineering applications.

Hydrogels are an increasingly attractive choice in the fields of regenerative medicine, wound care and tissue engineering as important forms of bio-scaffolds. For many clinical needs, injectable in situ crosslinkable hydrogels are strongly preferred, due to treatment effectiveness and ease of use. In this study, hyaluronic acid (HA), containing side-arms linked to photo-active coumarin moieties, was used for the preparation of wall-to-wall hydrogels. This photocrosslinkable HA, hereafter called HA-TEG-coumarin, produces colourless aqueous solutions that solidify upon near-UV irradiation (at a specific wavelength of 365 nm) via a clean [2 + 2] photocycloaddition reaction, without by-products formation. The crosslinking event, a robust and non-cytotoxic process, does not require catalysts or radical initiators: in the field of hyaluronan photocrosslinking, this innovative feature is significant to ensure the whole biocompatibility and to avoid collateral reactions. Mechanical and rheological tests showed that hyaluronan derivatives became hydrogels after 3-5 min of irradiation, with average values for bulk and surface elastic moduli of about 32 kPa and 193 kPa, respectively. Fluorescence recovery after photobleaching (FRAP) assay showed that the hydrogels are porous and allow a good permeation for nutrients and growth factors. Cell metabolism and proliferation assays revealed that hydrogel-encapsulated fibroblasts maintained their viability and that HA-TEG-coumarin sustained the proliferation of non-adherent myoblasts. For all of these reasons and thanks to a safe free-radical approach, this novel hyaluronan coumarin derivative could be a good candidate for tissue engineering and regenerative medicine applications.

Biochemical bases for a widespread tolerance of cyanobacteria to the phosphonate herbicide glyphosate.

Possible non-target effects of the widely used, non-selective herbicide glyphosate were examined in six cyanobacterial strains, and the basis of their resistance was investigated. All cyanobacteria showed a remarkable tolerance to the herbicide up to millimolar levels. Two of them were found to possess an insensitive form of glyphosate target, the shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate synthase. Four strains were able to use the phosphonate as the only phosphorus source. Low uptake rates were measured only under phosphorus deprivation. Experimental evidence for glyphosate metabolism was also obtained in strains apparently unable to use the phosphonate. Results suggest that various mechanisms may concur in providing cyanobacterial strains with herbicide tolerance. The data also account for their widespread ability to metabolize the phosphonate. However, such a capability seems limited by low cell permeability to glyphosate, and is rapidly repressed when inorganic phosphate is available.

Hyaluronic acid auto-crosslinked polymer (ACP): Reaction monitoring, process investigation and hyaluronidase stability.

Hyaluronic Acid (HA) is a non-sulphated glycosaminoglycan that, despite its high molecular weight, is soluble in water and is not resistant to enzymatic degradation, the latter of which hinders its wider application as a biomedical material. Auto-crosslinked polymer (ACP) gels of HA are fully biocompatible hydrogels that exhibit improved viscoelastic properties and prolonged in vivo residence times compared to the native polymer. Crosslinking is achieved through a base-catalysed reaction consisting of the activation of HA carboxyl groups by 2-chloro-1-methylpyridinium iodide (CMPI) and subsequent nucleophilic acyl substitution by the hydroxyl groups of HA in organic solvent. In this study, a number of ACP hydrogels have been obtained via reactions using varying ratios of CMPI to HA. The crosslinking reaction was monitored by rheological measurements in organic solvents during CMPI addition to the reaction mixture. The ACP intermediates, powders and hydrogels were characterized, helping to elucidate the crosslinking process. A two-step mechanism was proposed to explain the observed trends in viscosity and particle size. Syntheses were carried out by varying the reaction temperature, respectively at 0 °C, 25 °C and 45 °C in N-Methyl-2-Pyrrolidone (NMP), as well as the solvent respectively in NMP, DMSO and DMF at 25 °C. Interestingly, varying these parameters did not substantially affect the degree of crosslinking but likely did influence the intra/inter-molecular crosslinking ratio and, therefore, the viscoelastic properties. A wide range of crosslinking densities was confirmed through ESEM analysis. Finally, a comparative hyaluronidase degradation assay revealed that the ACPs exhibited a higher resistance toward enzymatic cleavage at low elastic modulus compared to other more chemically resistant, crosslinked HAs. These observations demonstrated the importance of crosslinking density of matrix structures on substrate availability.

Hyaluronic acid alkyl derivative: A novel inhibitor of metalloproteases and hyaluronidases.

Extracellular matrix (ECM) degradation, one of the main features of osteoarthritis, is driven by at least two major classes of enzymes: matrix metalloproteases (MMPs) and hyaluronidases. Among certain glycosaminoglycans, including natural and chemically cross-linked HAs, which are currently used as viscosupplements, the hyaluronic acid (HA) alkyl-amides (Hyadd) were here selected as the strongest MMP and hyaluronidase inhibitors. We used C. histolyticum collagenase (ChC) and bovine testicular hyaluronidase (BTH) as representative models of human MMPs and hyaluronidases, respectively. The role of the alkyl moiety was investigated using HA derivatives with varying alkyl lengths and degrees of derivatization. The selected compound was then screened against 10 different human MMPs in vitro, and the results were validated ex vivo in human synovial fluid. Hyadd-C16, identified as a lead compound, showed the highest inhibition potency against MMP13 and MMP8. The in vitro results were confirmed by the inhibition of human MMP13 (Ki=106.1 µM) and hyaluronidase-2 in the synovial fluid of patients with osteoarthritis. This study demonstrates the unique properties of Hyadd-C16, including its remarkable enzymatic inhibitory activity, which is conferred by the hydrophobic chain, and its high biocompatibility and water solubility of the HA backbone.

Identification and characterization of a bacterial hyaluronidase and its production in recombinant form.

Hyaluronidases (Hyals) are broadly used in medical applications to facilitate the dispersion and/or absorption of fluids or medications. This study reports the isolation, cloning, and industrial-scale recombinant production, purification and full characterization, including X-ray structure determination at 1.45 Å, of an extracellular Hyal from the nonpathogenic bacterium Streptomyces koganeiensis. The recombinant S. koganeiensis Hyal (rHyal_Sk) has a novel bacterial catalytic domain with high enzymatic activity, compared with commercially available Hyals, and is more thermostable and presents higher proteolytic resistance, with activity over a broad pH range. Moreover, rHyal_Sk exhibits remarkable substrate specificity for hyaluronic acid (HA) and poses no risk of animal cross-infection.

Hyaluronan derivatives: Alkyl chain length boosts viscoelastic behavior to depolymerization.

Five amide derivatives of Hyaluronic Acid (HA) were synthesized with C8, C12, C15, C16 and C18 linear alkyl-amines. These polymers (Hyadd) were tested against thermal, oxidative and hyaluronidase degradation by means of rheological experiments and SEC analysis and compared to non-modified HA. First of all, no free hexadecylamine was detected in the treated samples, meaning that under these stressing conditions only cleavage of glycosidic bonds occurs. Then, viscoelastic properties were assessed during thermal degradation and their variation as a function of time was expressed by means of a decay constant k(G'): while no significant difference in the decrease rate was observed between Hyadd-C8 and Hyadd-C12, a marked stabilization of viscoelastic properties during thermal treatment was detected for Hyadd-C15, Hyadd-C16 and Hyadd-C18. On the other hand, no difference was observed between the MW decrease rate (kMW decay constant) of HA and Hyadd-C12 to-C18; the depolymerization takes place on the backbone of the polymers independently whether they are derivatized or not, but longer alkyl chains lead to higher viscoelasticity in the depolymerized products. Finally, both oxidative and enzymatic degradation were carried out analyzing the changes in elastic modulus and in dynamic viscosity: once again, the amide side chain came out with similar behavior to chemical cross-linked HA (HBC) and with improved performances respect to linear HA in terms of preservation of viscoelasticity after chain depolymerization.