Comparison of Hyaluronidase-Mediated Degradation Kinetics of Commercially Available Hyaluronic Acid Fillers In Vitro.

BACKGROUND: The ability to degrade hyaluronic acid (HA)-based fillers with hyaluronidase allows for better management of adverse effects and reversal of suboptimal treatment outcomes. OBJECTIVES: The aim of this study was to compare the enzymatic degradation kinetics of 16 commercially available HA-based fillers, representing 6 manufacturing technologies. METHODS: In this nonclinical study, a recently developed in vitro multidose hyaluronidase administration protocol was used to induce degradation of HA-based fillers, enabling real-time evaluation of viscoelastic properties under near-static conditions. Each filler was exposed to repeated doses of hyaluronidase at intervals of 5 minutes to reach the degradation threshold of G' ≤ 30 Pa. RESULTS: Noticeable differences in degradation characteristics were observed based on the design and technology of different filler classes. Vycross fillers were the most difficult to degrade and the Cohesive Polydensified Matrix filler was the least difficult to degrade. Preserved Network Technology products demonstrated proportional increases in gel degradation time and enzyme volume required for degradation across the individual resilient hyaluronic acid (RHA) products and indication categories. No obvious relationship was observed between gel degradation characteristics and the individual parameters of HA concentration, HA chain length, or the degree of modification of each filler when analyzed separately; however, a general correlation was identified with certain physicochemical properties. CONCLUSIONS: Manufacturing technology was the most important factor influencing the reversibility of an HA product. An understanding of the differential degradation profiles of commercially available fillers will allow clinicians to select products that offer a higher margin of safety due to their preferential reversibility.

Insights into the source, mechanism and biotechnological applications of hyaluronidases.

It has long been found that hyaluronidases exist in a variety of organisms, playing their roles in various biological processes including infection, envenomation and metabolic regulation through degrading hyaluronan. However, exploiting them as a bioresource for specific applications had not been extensively studied until the latest decades. In recent years, new application scenarios have been developed, which extended the field of application, and emphasized the research value of hyaluronidase. This critical review comprehensively summarizes existing studies on hyaluronidase from different source, particularly in their structures, action patterns, and biological functions in human and mammals. Furthermore, we give in-depth insight into the resource mining and protein engineering process of hyaluronidase, as well as strategies for their high-level production, indicating that mixed strategies should be adopted to obtain well-performing hyaluronidase with efficiency. In addition, advances in application of hyaluronidase were summarized and discussed. Finally, prospects for future researches are proposed, highlighting the importance of further investigation into the characteristics of hyaluronidases, and the necessity of investigating their products for the development of their application value.

2-Acetamido-2-deoxy-d-glucono-1,5-lactone Sulfonylhydrazones: Synthesis and Evaluation as Inhibitors of Human OGA and HexB Enzymes.

Inhibition of the human O-linked ß-N-acetylglucosaminidase (hOGA, GH84) enzyme is pharmacologically relevant in several diseases such as neurodegenerative and cardiovascular disorders, type 2 diabetes, and cancer. Human lysosomal hexosaminidases (hHexA and hHexB, GH20) are mechanistically related enzymes; therefore, selective inhibition of these enzymes is crucial in terms of potential applications. In order to extend the structure-activity relationships of OGA inhibitors, a series of 2-acetamido-2-deoxy-d-glucono-1,5-lactone sulfonylhydrazones was prepared from d-glucosamine. The synthetic sequence involved condensation of N-acetyl-3,4,6-tri-O-acetyl-d-glucosamine with arenesulfonylhydrazines, followed by MnO2 oxidation to the corresponding glucono-1,5-lactone sulfonylhydrazones. Removal of the O-acetyl protecting groups by NH3/MeOH furnished the test compounds. Evaluation of these compounds by enzyme kinetic methods against hOGA and hHexB revealed potent nanomolar competitive inhibition of both enzymes, with no significant selectivity towards either. The most efficient inhibitor of hOGA was 2-acetamido-2-deoxy-d-glucono-1,5-lactone 1-naphthalenesulfonylhydrazone (5f, Ki = 27 nM). This compound had a Ki of 6.8 nM towards hHexB. To assess the binding mode of these inhibitors to hOGA, computational studies (Prime protein-ligand refinement and QM/MM optimizations) were performed, which suggested the binding preference of the glucono-1,5-lactone sulfonylhydrazones in an s-cis conformation for all test compounds.

Structure and cleavage pattern of a hyaluronate 3-glycanohydrolase in the glycoside hydrolase 79 family.

Hyaluronidases have attracted a great deal of interest in the field of medicine due to their fundamental roles in the breakdown of hyaluronan. However, little is known about the catalytic mechanism of the hyaluronate 3-glycanohydrolases. Here, we report the crystal structure and cleavage pattern of a leech hyaluronidase (LHyal), which hydrolyzes the ß-1,3-glycosidic bonds of hyaluronan. LHyal exhibits the typical structural features of glycoside hydrolase 79 family but contains a variable 'exo-pocket' loop where basic residues R102 and K103 are the structural determinants of hyaluronan binding. Through analysis of the hydrolysis of even- and odd-numbered hyaluronan oligosaccharides, we demonstrate that hexasaccharide is the shortest natural substrate, which can be cleaved from both the reducing and non-reducing ends to release disaccharides, and pentasaccharides are the smallest fragments for recognition and hydrolysis. These observations provide new insights into the degradation of hyaluronan and the evolutionary relationships of the GH79 family enzymes.

ß-elimination of hyaluronate by red king crab hyaluronidase.

Crustacean hyaluronidases are poorly understood both in terms of their enzymatic properties and in terms of their structural features. In this work, we show that the hepatopancreas homogenate of the red king crab has a hyaluronidase activity that is an order of magnitude higher than its commercial counterpart. Zymography revealed that the molecular weight of a protein with hyalorunidase activity is 40-50 kDa. Analysis of the hepatopancreas transcriptome and results of cloning and sequencing of cDNA revealed a hyaluronidase sequence with an expected molecular weight of 42.5 kDa. Further analysis showed that hyaluronat enzymatic cleavage follows the [Formula: see text]-elimination mechanism, which is well known for bacterial hyaluronidases. The results of ion-exchange chromatography showed that the final product of hyaluronate degradation is unsaturated tetrasaccharide. Thus, we identified a new hyaluronidase of higher eukaryotes, which is not integrated into the modern classification of hyaluronidases.

Hyaluronan Hydrogels for Injection in Superficial Dermal Layers: An In Vitro Characterization to Compare Performance and Unravel the Scientific Basis of Their Indication.

BACKGROUND: Skinboosters represent the latest category of hyaluronan (HA) hydrogels released for aesthetic purposes. Different from originally developed gels, they are intended for more superficial injections, claiming a skin rejuvenation effect through hydration and possibly prompting biochemical effects in place of the conventional volumetric action. Here, three commercial skinboosters were characterized to unravel the scientific basis for such indication and to compare their performances. METHODS: Gels were evaluated for water-soluble/insoluble-HA composition, rheology, hydration, cohesivity, stability and effect, in vitro, on human dermal fibroblasts towards the production of extracellular matrix components. RESULTS: Marked differences in the insoluble-hydrogel amount and in the hydrodynamic parameters for water-soluble-HA chains were evidenced among the gels. Hydration, rigidity and cohesivity also varied over a wide range. Sensitivity to hyaluronidases and Reactive Oxygen Species was demonstrated allowing a stability ranking. Slight differences were found in gels' ability to prompt elastin expression and in ColIV/ColI ratio. CONCLUSIONS: A wide panel of biophysical and biochemical parameters for skinboosters was provided, supporting clinicians in the conscious tuning of their use. Data revealed great variability in gels' behavior notwithstanding the same clinical indication and unexpected similarities to the volumetric formulations. Data may be useful to improve customization of gel design toward specific uses.

3D structure prediction, dynamic investigation and rational construction of an epitope-masked thermostable bovine hyaluronidase.

Hyaluronidase (HAase) from bovine testes (BTH) has long been used in broad pharmaceutical areas, while it is associated with drawbacks in aspects of solubility, immunogenicity and pharmacokinetics. These issues can be addressed by gaining structural insights and designing rational modifications to the enzyme structure, as proposed in this study. A 3D structural model was built for HAase and underwent 40 ns of molecular dynamic simulation to examine its thermostability under normal, melting, and extreme conditions. The enzyme activity of BTH was measured against temperature and pH by kinetic assays. The interaction of bovine HAase with HA and chondroitin was defined by molecular docking. Furthermore, immunogenic properties of the enzyme were explored by immunoinformatics. Thermal effects on bovine HAase structural model and the HAase interactions with its substrates were described. We identified some B- and T-cell epitopes and showed that the protein could be recognized by human immune receptor molecules. Epitope masking by adding polyethylene glycol (PEG) to amine groups of residues presenting on the surface of the protein structure was adopted as a surface modification to enhance pharmacological properties of BTH. Assays showed that PEGylated BTH had higher thermostability and similar activity compared to the native enzyme.

Quantifying the Digestion of Cross-Linked Hyaluronic Acid Fillers With Hyaluronidase.

BACKGROUND: Adverse events due to hyaluronic acid fillers (HAFs) may be treated with hyaluronidase, an enzyme that cleaves bonds within hyaluronic acid. This study reviews the efficacy of currently available hyaluronidase preparations in breaking down commercial, cross-linked HAFs. METHODS: Three HAFs were used in this study (Restylane, Juvederm Voluma, and Belotero [BEL] Balance). A laser-based particle size analyzer (Malvern Mastersizer 3000) was used to calculate particle sizes in untreated HAFs (controls) and those treated with 450 units of hyaluronidase (Hylenex) for 5 and 30 minutes. RESULTS: Particle size analysis revealed that when Restylane was treated with hyaluronidase for 5 minutes, the average particle size reduced modestly, from 472 to 440 µm. At 30 minutes, the average particle size was 419 µm. For Juvederm, the average size of particles reduced from 703 µm in controls to 676 µm after treatment with hyaluronidase for 5 minutes and 635 µm after treatment for 30 minutes. For Belotero, the average size of control particles was 410 µm, reducing to 376 µm after treatment with hyaluronidase for 5 minutes and 345 µm after treatment for 30 minutes. CONCLUSION: After treatment with hyaluronidase for up to 30 minutes, there was only a modest breakdown of all 3 HAFs used. The results of this study raise questions regarding the efficacy of hyaluronidase in degrading cross-linked HAFs.

ATP/Hyals dually responsive core-shell hyaluronan/chitosan-based drug nanocarrier for potential application in breast cancer therapy.

The stability of self-assembled drug nanocarriers during blood circulation and the controlled intracellular drug delivery are two challenges in cancer therapy. In this paper, we constructed an adenosine triphosphate (ATP)/hyaluronidase(Hyals) dually responsive core-shell hyaluronan/chitosan-based drug nanocarrier for breast cancer therapy, using SNX-loaded 3-fluoro-4-carboxyphenylboronic acid-conjugated quaternary ammonium chitosan nanoparticles (SNX@HTCC-FPBA NPs) as the core and crosslinked polyethylene glycol-/methacrylate-modified hyaluronic acid (mHA-PEG) as the shell. The formed SNX@HTCC-FPBA/mHA-PEG NPs were stable against salt ion strength, pH values and human plasma mimicking the bloodstream, but ATP/Hyals dually sensitive with a drug delivery of 85% within 48 h in the mimicking intracellular environment of breast cancer cells. These nanoparticles showed a low hemolysis of less than 3%, a high resistance to bovine serum albumin adsorption of 0.06 mg/mg, and an efficient internalization by two breast cancer cell lines (MCF-7 and MDA-MB-453). The cell culture indicated that they were friendly to human skin fibroblasts, but presented a close IC50 value to SNX for MCF-7 (0.14 µg mL-1) and MDA-MB-453 (0.05 µg mL-1) at 48 h, respectively. Thus, SNX@HTCC-FPBA/mHA-PEG NPs were potential drug nanocarriers for breast tumor therapy.

Antihyaluronidase and Alkaline Phosphatase (ALP) Activities of Medicinal Plants to Combat Echis carinatus Venom-Induced Toxicities.

Snakebite is one of the most neglected diseases of developing countries. Deaths due to snakebite envenoming are quite high in Pakistan, and many deaths are caused by Echis carinatus envenomation. Traditional use of medicinal plants against snakebites is a common practice in Pakistan due to countless benefits. The current study was performed with the objective to evaluate eighteen Pakistani medicinal plants inhibitory potential against hyaluronidase and alkaline phosphatase enzymes of Pakistani Echis carinatus venom. Hyaluronidase activity (0.2-1.6 mg/0.1 mL) and alkaline phosphatase activity (0.1-0.8 mg/0.1 mL) were measured in dose-dependent manner. Crude methanolic extracts of medicinal plants were used for in vitro investigation of their inhibitory activity against toxic enzymes. All active plants were fractioned using different solvents and were again analyzed for inhibitory activity of same enzymes. Results indicated all plants were able to neutralize hyaluronidase that Swertia chirayita (Roxb. ex Flem.) Karst., Terminalia arjuna Wight and Arn, Rubia cordifolia Thumb., and Matthiola incana (L.) R.Br. inhibited maximum hyaluronidase activity equivalent to standard reference (p > 0.5). Pakistani medicinal plants are dense with natural neutralizing metabolites and other active phytochemicals which could inhibit hyaluronidase activity of Pakistani Echis carinatus venom. Further advanced studies at molecular level could lead us to an alternative for envenoming of Pakistani Echis carinatus venom.

Supramolecular Structuring of Hyaluronan-Lactose-Modified Chitosan Matrix: Towards High-Performance Biopolymers with Excellent Biodegradation.

Non-covalent interactions in supramolecular chemistry provide useful systems to understand biological processes, and self-assembly systems are suitable assets to build-up innovative products for biomedical applications. In this field, polyelectrolyte complexes are interesting, especially when polysaccharides are involved, due to their non-toxicity and bio-absorbability. In this work, we investigated a polyelectrolyte formed by hyaluronic acid (HA), a negatively charged linear polysaccharide, with Chitlac (Ch), a positively charged lactose-modified chitosan. The aim of the study was the investigation of a novel Ch-HA polyelectrolyte complex, to understand the interaction between the two polysaccharides and the stability towards enzymatic activity. By means of gel permeation chromatography-triple detector array (GPC-TDA), nuclear magnetic resonance (NMR), dynamic viscosity, Zeta Potential and scanning electron microscopy (SEM), the polyelectrolyte complex properties were identified and compared to individual polysaccharides. The complex showed monodisperse molecular weight distribution, high viscosity, negative charge, and could be degraded by specific enzymes, such as hyaluronidase and lysozyme. The results suggest a close interaction between the two polysaccharides in the complex, which could be considered a self-assembly system.

Hyaluronidases in Human Diseases.

With the burgeoning interest in hyaluronic acid (HA) in recent years, hyaluronidases (HYALs) have come to light for their role in regulating catabolism of HA and its molecular weight (MW) distribution in various tissues. Of the six hyaluronidase-like gene sequences in the human genome, HYALs 1 and 2 are of particular significance because they are the primary hyaluronidases active in human somatic tissue. Perhaps more importantly, for the sake of this review, they cleave anti-inflammatory and anti-fibrotic high-molecular-weight HA into pro-inflammatory and pro-fibrotic oligosaccharides. With this, HYALs regulate HA degradation and thus the development and progression of various diseases. Given the dearth of literature focusing specifically on HYALs in the past decade, this review seeks to expound their role in human diseases of the skin, heart, kidneys, and more. The review will delve into the molecular mechanisms and pathways of HYALs and discuss current and potential future therapeutic benefits of HYALs as a clinical treatment.

Target protein deglycosylation in living cells by a nanobody-fused split O-GlcNAcase.

O-linked N-acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification that is presented on thousands of nucleocytoplasmic proteins. Interrogating the role of O-GlcNAc on a single target protein is crucial, yet challenging to perform in cells. Herein, we developed a nanobody-fused split O-GlcNAcase (OGA) as an O-GlcNAc eraser for selective deglycosylation of a target protein in cells. After systematic cellular optimization, we identified a split OGA with reduced inherent deglycosidase activity that selectively removed O-GlcNAc from the desired target protein when directed by a nanobody. We demonstrate the generality of the nanobody-fused split OGA using four nanobodies against five target proteins and use the system to study the impact of O-GlcNAc on the transcription factors c-Jun and c-Fos. The nanobody-directed O-GlcNAc eraser provides a new strategy for the functional evaluation and engineering of O-GlcNAc via the selective removal of O-GlcNAc from individual proteins directly in cells.

Mammal hyaluronidase activity on chondroitin sulfate and dermatan sulfate: Mass spectrometry analysis of oligosaccharide products.

Mammalian hyaluronidases are endo-N-acetyl-D-hexosaminidases involved in the catabolism of hyaluronic acid (HA) but their role in the catabolism of chondroitin sulfate (CS) is also examined. HA and CS are glycosaminoglycans implicated in several physiological and pathological processes, and understanding their metabolism is of significant importance. Data have been previously reported on the degradation of CS under the action of hyaluronidase, yet a detailed structural investigation of CS depolymerization products remains necessary to improve our knowledge of the CS depolymerizing activity of hyaluronidase. For that purpose, the fine structural characterization of CS oligosaccharides formed upon the enzymatic depolymerization of various CS subtypes by hyaluronidase has been carried out by high-resolution Orbitrap mass spectrometry (MS) and extreme UV (XUV) photodissociation tandem MS. The exact mass measurements show the formation of wide size range of even oligosaccharides upon digestion of CS-A and CS-C comprising hexa- and octa-saccharides among the main digestion products, as well as formation of small quantities of odd-numbered oligosaccharides, while no hyaluronidase activity was detected on CS-B. In addition, slight differences have been observed in the distribution of oligosaccharides in the digestion mixture of CS-A and CS-C, the contribution of longer oligosaccharides being significantly higher for CS-C. The sequence of CS oligosaccharide products determined XUV photodissociation experiments verifies the selective ß(1 â†’ 4) glycosidic bond cleavage catalyzed by mammal hyaluronidase. The ability of the mammal hyaluronidase to produce hexa- and higher oligosaccharides supports its role in the catabolism of CS anchored to membrane proteoglycans and in extra-cellular matrix.

A halotolerant hyaluronidase from newly isolated Brevibacterium halotolerans DC1: Purification and characterization.

An enzyme hyaluronidase (hyase) producing halotolerant bacterium was isolated from dental caries and identified as Brevibacterium halotolerans DC1. Higher growth and hyase production were observed in nutrient broth fortified with hyaluronic acid at pH 7.0, temperature 37 °C, 120 rpm upon 48 h of incubation. Hyase was purified using salt precipitation, DEAE cellulose ion exchange, and Sephadex G-100 gel filtration chromatography. The enzyme was purified to 13-fold with 67.19% recovery of activity and 26.37 U/mg of specific activity. SDS-PAGE and zymography revealed it to be near to homogeneity showing a relative molecular weight of about 43 kDa that was confirmed by MALDI-TOF MS. This hyase was very active and stable at pH 7.0 and temperature 40 °C. The presence of metal ions Ca2+ and Mg2+ increased its activity while Zn2+ and Cu2+ severely inhibited it. Being stable at 2 M NaCl, hyase exhibited its halotolerant nature. This enzyme showed wide substrate specificity where hyaluronic acid (HA) was the best substrate. The kinetic studies revealed that Km and Vmax were 91.3 µg/mL and 306.2 µg/mL/min respectively. This is the first report of hyaluronidase from a halotolerant Brevibacterium spp. which can find applications under high salinity.

Kinetic theory of hyaluronan cleavage by bovine testicular hyaluronidase in standard and crowded environments.

In this paper, we introduce a comprehensive kinetic model describing the enzymatic cleavage of hyaluronan (HA) by bovine testicular hyaluronidase (BTH). Our theory focuses specifically on the late stage of the hydrolysis, where the concentrations of a limited number of oligomers may be determined experimentally with accuracy as functions of time. The present model was applied to fit different experimental sets of kinetic data collected by capillary electrophoresis at two HA concentrations and three concentrations of PEG crowder (0, 10, 17% w/w). Our theory seems to apply universally, irrespective of HA concentration and crowding conditions, reproducing to an excellent extent the time evolution of the individual molar fractions of oligomers. Remarkably, we found that the reaction mechanism in the late degradation stage essentially reduces to the cleavage or transfer of active dimers. While the recombination of dimers is the fastest reaction, the rate-limiting step turns out to be invariably the hydrolysis of hexamers. Crowding, HA itself or other inert, volume-excluding agents, clearly boosts recombination events and concomitantly slows down all fragmentation pathways. Overall, our results bring a novel and comprehensive quantitative insight into the complex reaction mechanism underlying enzymatic HA degradation. Importantly, rationalizing the effect of crowding not only brings the intricate conditions of in-vivo settings a little closer, but also emerges as a powerful tool to help pinpointing relevant kinetic pathways in complex systems.

Response of twelve different hyaluronic acid gels to varying doses of recombinant human hyaluronidase.

PURPOSE: Hyaluronic acid (HA) fillers can be reversed with hyaluronidase, but their dose responses are not well-characterized. We evaluated 12 fillers' in vitro responses to varying doses of recombinant human hyaluronidase (RHH). METHODS: For the 12 HA gels, 0.2 mL aliquots were placed on six slides. Samples received no injection, saline injection, or RHH (2.5, 5, 10, or 20 units). The most resistant gels received 40 units of RHH on a seventh slide. Photos of gels were taken from bird's-eye and lateral views with a ruler at multiple time points. RESULTS: Restylane-L and Restylane Lyft were the most easily dissolvable HA fillers. Both demonstrated a significant response to 2.5 units RHH/0.2 mL. Juvéderm Ultra, Belotero, Restylane Silk, and Restylane Defyne had moderate resistance to RHH. Restylane Refyne, Juvéderm Ultra Plus, Vollure, Versa, and Voluma were most resistant, requiring more than 20 units RHH/0.2 mL for complete dissolution. Volbella was moderately resistant up to 20 units RHH but demonstrated pronounced dissolution with 40 units RHH. CONCLUSIONS: This study visually and objectively demonstrates the in vitro response of HA to dissolution by hyaluronidase. Fillers were categorized into least, moderate, and most resistant to RHH. Interesting findings include markedly different degrees of liquefaction for products used for similar indications (such as Restylane Lyft being more liquefied than Voluma), and Belotero easily flattening but retaining texture. Combined with in vivo data, this may help calculate hyaluronidase doses needed for controlled, partial reversal of commercially available HA gels.

One-pot synthesis of hyaluronic acid-coated gold nanoparticles as SERS substrate for the determination of hyaluronidase activity.

A novel ultrasensitive surface-enhanced Raman spectroscopy (SERS)-based method was developed for the determination of hyaluronidase (HAase), which was based on hyaluronic acid-coated gold nanoparticles (HA-AuNPs) as a substrate, via a facile one-pot method. The detection mechanism is based on HAase which can hydrolyze HA on HA-AuNPs into hyaluronic acid oligomers, causing the originally uniformly dispersed HA-AuNPs to be disintegrated into many smaller HA-AuNPs. These oligomers in turn increase the surface shielding of AuNPs, resulting in high aggregation tendencies. As a result, the original SERS substrate was disassembled, leading to a weakening of the SERS signal at 1173 cm-1. Malachite green was also used as a Raman probe to detect the change of SERS peak intensity and to quantify HAase. Compared with other methods for the determination of HAase, this method is more convenient and efficient; its determination limit was 0.4 mU mL-1. The recoveries of HAase spiked into human urine samples ranged from 97.2 to 103.9%.

Diversity of compounds in Vespa spp. venom and the epidemiology of its sting: a global appraisal.

Poisonous animals imply a risk to human life, because their venom is a complex mixture of low molecular weight components, peptides and proteins. Hornets use the venom for self-defence, to repel intruders and to capture prey, but they can cause poisoning and allergic reactions to people. In particular, they seem to be a health problem in the countries where they are native due to their sting, which in the most severe cases can lead to severe or fatal systemic anaphylaxis. But this situation is being an emerging problem for new countries and continents because hornet incursions are increasing in the global change scenario, such as in Europe and America. Furthermore, 55 detailed cases of hornet sting were found in 27 papers during the current review where 36.4% died due to, mainly, a multi-organ failure, where renal failure and liver dysfunction were the most common complications. Moreover, the great taxonomic, ecological diversity, geographical distribution and the wide spectrum of pathophysiological symptoms of hornets have been the focus of new research. Considering this, the present systematic review summarizes the current knowledge about the components of Vespa venom and the epidemiology of its sting to serve as reference for the new research focused on the development of techniques for diagnosis, new drugs and treatments of its sting.