Depolymerization Of Hyaluronate Research Articles

Formulation and Analgesic Effect of Sodium Hyaluronate and Magnesium Sulfate Combination in Rats Following Intra‐articular Injection

The objective of this study was to formulate hyaluronate (HA) combined with magnesium sulfate (MS) for intra‐articular (IA) injection, to provide rapid and profound analgesia in the treatment of osteoarthritis (OA). The injectable HA/MS combination was optimized with respect to the kinds and amounts of buffering agent (10 mM histidine, pH 7.0) and stabilizer (10 mM sodium citrate), by evaluating changes in the appearance, pH, and intrinsic viscosity at 60°C. Increasing concentrations of sodium citrate significantly inhibited the decline in intrinsic viscosity, denoting the depolymerization of HA, preserving over 91% of initial value. In an efficacy study in iodoacetate‐induced OA rats, treatment with the HA/MS combination (1/2% w/v) significantly reduced joint swelling and pain, compared to the vehicle‐ and HA‐treated groups (p < 0.05) at 7 days postdosing. Thus, IA administration of the combination of HA, a slow‐acting symptom‐modifying agent, with the inorganic analgesic is a potentially effective therapeutic approach for rapid pain relief in the OA knee.

Hyaluronate depolymerization following thermal decomposition of oxytetracycline.

Depolymerization of sodium hyaluronate (HA) by tetracyclines was investigated. Reduction in HA molecular weight was followed by size exclusion chromatography with a low angle laser light scattering detector. On mixing with oxytetracycline hydrochloride (OTC) solution and incubating at 37 degrees C, HA was gradually depolymerized. OTC, a representative antibiotic, is known as a photosensitizer, and phototoxic side effects relevant to radicals have been reported. However, HA depolymerization required no irradiation. As time passed, OTC solution incubated at 37 degrees C got colored reddish brown, even in the dark. With reversed-phase HPLC separation, several peaks derived from decomposed OTC appeared. One of the peaks had an absorbance in the visible range. A quantitative correlation between the discoloration and the HA depolymerization rate was obtained. On the other hand, when samples were incubated below 25 degrees C, change of color was slight, and practically no HA depolymerization was observed after up to 4 h. Oxygen depletion by nitrogen saturation or addition of mannitol also prevented the depolymerization. Under anaerobic conditions, the color of the solution did not change, whereas it turned red under aerobic conditions in the presence of mannitol. The mannitol did not inhibit the OTC decomposition, but it preserved HA from damage. On the basis of the known decomposition of OTC and the results of HPLC separation, anhydrooxytetracycline can be proposed as the derivative causing HA depolymerization.

Depolymerization of hyaluronate by the phototoxic drugs phenothiazines and sulfacetamide

Phenothiazines (promazine, promethazine, chlorpromazine) and sulfacetamide, known as phototoxic drugs, depolymerize aqueous sodium hyaluronate (HA) on exposure to light. The reduction in the HA molecular weight was followed by size-exclusion chromatography with low-angle laser light scattering. In the low-concentration region of the drugs below 0.05 mM, the rate constants of depolymerization increased. The molecular weight of HA was practically unchanged without UV irradiation in the presence of drugs or with UV irradiation in the absence of drugs, indicating the phenothiazines and sulfacetamide require photoenergy to yield any kind of damaging chemical species for HA depolymerization. An involvement of active oxygen radicals in the effects of promazine and promethazine was evidenced by inhibition under anaerobic conditions. Further, addition of mannitol controlled the reaction in the presence of oxygen, pointing to hydroxyl radicals as the damaging agent. Chlorpromazine and sulfacetamide preferably depolymerized HA under anaerobic conditions, suggesting the participation of hydrated electrons.

Hyaluronate depolymerization activity induced by progesterone in cultured fibroblasts derived from human uterine cervix

High-molecular-weight [ 14C]hyaluronate was incubated with cultured fibroblasts from human uterine cervix and skin, and then the depolymerization of the hyaluronate was investigated. [ 14C]Hyaluronate in the medium of skin fibroblasts was depolymerized into a constant molecular weight ( M r about 40,000), whereas that of cervix fibroblasts was not depolymerized, irrespective of incubation period. However, when progesterone was added to the medium of cervix fibroblasts, hyaluronate was depolymerized to the same extent as that in skin fibroblasts. The reducing terminal sugar of the depolymerized hyaluronate was N-acetylglucosamine. These results suggest that a hyaluronate-depolymerizing enzyme, endo-β- N-acetylglucosaminidase, was induced by progesterone in cultured fibroblasts derived from human uterine cervix.

Degradation of Hyaluronate by the Concerted Action of Ozone and Sunlight

The influence of ozone and sunlight in a concerted reaction on hyaluronate solutions was investigated. The kinematic viscosity of hyaluronate solutions is decreased by ozone-air mixtures and simultaneous radiation with sun rays within a few minutes, indicating a depolymerization of the hyaluronate molecule. The reaction is dependent on the concentration of ozone and on the time of exposure to ozone and sunlight. The concerted degradation of hyaluronate is more effective than the reaction with each component, ozone and sun rays, alone. We conclude that hyaluronate depolymerization by ozone and sunlight may be one factor for irritations of the eye by photochemical smog and increased exposure to sun rays.

Differential effects of reactive oxygen species on native synovial fluid and purified human umbilical cord hyaluronate.

The ability of reactive oxygen species produced by triggered neutrophilic leukocytes, hypoxanthine/xanthine oxidase (HX/XAO), hydrogen peroxide, and hypochlorous acid/myeloperoxidase (HOCl/MPO) systems to degrade hyaluronate (HA) in human synovial fluid (SF) and purified umbilical cord HA was compared by measuring the molecular weight distribution of HA using high-performance liquid chromatography with a size-exclusion column. The exposure of noninflammatory SF to phorbol myristic acetate (PMA)-activated neutrophils or to hydrogen peroxide (H2O2) caused depolymerization of SF HA to the degree corresponding to that found in rheumatoid SFs. When HX/XAO was used as radical generator, the molecular weight of SF HA decreased from 3.42 x 10(6) to 1.40 x 10(4) daltons with concomitant decrease of SF viscosity to 36% from the original value. The HOCl/MPO system caused no depolymerization of SF HA, even at very high unphysiological HOCl concentrations that induced the precipitation of SF HA together with SF proteins. This effect was found to be comparable to conventional mucin clot formation in SF. However, purified human umbilical cord HA was easily depolymerized with HOCl/MPO or with H2O2, but these effects were sensitive to the hydroxyl radical scavenger mannitol and iron chelator desferrioxamine, indicating that the formation of reactive hydroxyl radical (OH.) is likely to participate in these reactions. Thus we conclude that in inflammatory SF HA is mainly depolymerized by OH. produced by decomposition of H2O2 catalyzed by iron, free or locally bound to HA itself. In contrast to what has been reported earlier, HOCl/MPO only depolymerizes purified umbilical cord HA (in a hydroxyl radical-dependent manner) but does not depolymerize HA in SF. As a matter of fact, HOCl/MPO has a scavenging action on SF HA by consuming H2O2 and thus preventing the formation of reactive hydroxyl radicals.

Oxidative damage to synovial fluid from the inflamed rheumatoid joint detected by 1H NMR spectroscopy.

The glycosaminoglycan hyaluronate (hyaluronic acid, hyaluronan) [l] is a linear repeating disaccharide, @-D-glucuronyl-p-D-Nacetylglucosamine, of very high molecular mass (>4 X 106) which is a major component of the proteoglycan aggregates required for the functional integrity of extra-cellular matrices such as articular cartilage [2]. Hyaluronan is continually secreted in its unaggregated form by the type II synoviocytes and is largely responsible for the very high viscosity of kneejoint synovial fluid [3]. It has previously been demonstrated that in patients with rheumatoid arthritis or other inflammatory joint diseases, synovial fluid hyaluronate is depolymerized with a corresponding modification in the viscoelastic properties of synovial fluid and an increase in the synovial level of low-molecular-mass hyaluronate-derived saccharides [4]. This fragmentation is presumed to occur by the action of reactive oxygen radical species (RORS) [5] since (1) hyaluronidase activity is absent from both normal and inflammatory synovial fluid samples [6], and (2) there is currently a large quantity of experimental evidence implicating the involvement of these chemically-reactive oxygen metabolites in the pathogenesis of inflammatory joint diseases [7-91. In addition to the presence of potential oxygen radical generating systems within the joint, this hypothesis is also supported by the demonstration of hyaluronate depolymerization by such systems in vivo as evidenced by decreases in apparent hyaluronate molecular mass and viscometric parameters. We have previously suggested that hypoxicreperfusion injury during perambulatory motion of the inflamed rheumatoid joint is a mechanism which perpetuates the deleterious production of oxygen-derived radical species within the joint, giving rise to the chronicity of the inflammation [9]. Much of the toxicity of RORS to biological systems is attributable to the highly reactive hydroxyl radical (*OH) [lo], the formation of which appears to be mediated by low-molecular-mass iron chelates. In this communication we present evidence for RORS-mediated damage to knee-joint synovial fluid in patients with inflammatory synovitis. Nuclear magnetic resonance (NMR) spectroscopy has been extensively used for investigating the metabolic profile of body fluids [ll], particularly for monitoring the status and levels of low-molecular-mass endogenous components. We have employed high field proton Hahn spin-echo NMR spectroscopy to detect and characterize a series of lowmolecular-mass species derived from the attack of radiolytically-generated RORS on hyaluronate. We have also utilized this technique to assess RORS-mediated oxidative damage to synovial fluid hyaluronate in vivo during hypoxic-reperfusion injury. In addition, the

Chemical change involved in the oxidative reductive depolymerization of hyaluronic acid.

The oxidative reductive depolymerization (ORD) of hyaluronate has been investigated. A solution of hyaluronate (Mr 4.07 x 10(5] in phosphate buffer (pH 7.2) was incubated in the presence of Fe2+ for 24 h at 37 degrees C under an oxygen atmosphere to yield depolymerized hyaluronate (ORD fragments; an average Mr of 2,600). The ORD fragments contain 21 and 24% less hexosamine and uronic acid, respectively, but no olefinic linkage. They were exhaustively digested with chondroitinase AC-II. The resulting oligosaccharides and monosaccharides were separated by gel filtration and ion-exchange chromatography, and their structures were determined by proton and carbon-13 NMR, fast atom bombardment mass spectrometry, and chromatographic techniques combined with chemical modifications. The following structures derived from the reducing ends of the ORD fragments were identified: 4,5-unsaturated GlcA(beta 1----3)-N-acetyl-D-glucosaminic acid (where GlcA- represents glucuronosyl-) (21%), 4,5-unsaturated GlcA(beta 1----3)GlcNAc(beta 1----3)-D-arabo-pentauronic acid (24%), and N-acetyl-D-glucosamine (51%). The following structures derived from the nonreducing ends were identified: L-threo-tetro-dialdosyl-(1----3)GlcNAc (a tentative structure, 8%), N-acetylhyalobiuronic acid (20%), and N-acetyl-D-glucosamine (45%). The results indicate that the ORD reaction of hyaluronate proceeds essentially by random destruction of unit monosaccharides due to oxygen-derived free radicals, followed by secondary hydrolytic cleavage of the resulting unstable glycosidic substituents.

Random coil scission rates determined by time‐dependent total intensity light scattering: Hyaluronate depolymerization by hyaluronidase

A recently developed theory of the light scattering by random coils undergoing random scission is applied to the digestion of hyaluronate by hyaluronidase. The time dependence of the scattered light from solutions undergoing digestion was monitored. Working at a high angle with high molecular weight hyaluronate allowed the use of a powerful approximation for determining initial velocities and the Henri-Michaelis-menten coefficients, without explicit knowledge of the hyaluronate molecular weight, radius of gyration, second virial coefficient, or polydispersity. Effects due to a molecular weight dependent second virial coefficient and to non-Gaussian behavior are briefly considered. Assays were performed over nearly two orders of magnitude in substrate concentration. The initial velocities are compared with those obtained by a standard reducing sugar assay, which was performed on identical samples. The main advantages of the light scattering assay procedure over the more traditional assays are that many relatively high-precision data points can be quickly and automatically collected with simple apparatus, and that the technique is most sensitive for the initial period of digestion, where the other assays are least sensitive. The shapes of the scattering curves also provide evidence that hyaluronate in these solutions is not a stable double strand and that the hyaluronidase cleaves bond randomly. The curves also indicate that enzyme deactivation occurs, which accounts for the lower velocities yielded by the slower reductimetric assay, which is measured over longer initial periods.

Effect of oxygen-derived free radicals on hyaluronic acid.

To investigate possible mechanisms of hyaluronic acid depolymerization, superoxide anion and other secondary oxygen-derived free radicals were generated in vitro and allowed to act upon a hyaluronate substrate. Superoxide, generated either enzymatically with xanthine oxidase or by stimulation of polymorphonuclear leukocytes, reduced the viscosity of hyaluronate solutions dramatically while the chromatographic profiles of the glycosaminoglycan shifted toward lower molecular weights. Superoxide-treated hyaluronate also became susceptible to further degradation by beta-N-acetylglucosaminidase A. Experiments with scavengers of various toxic oxygen-derived free radicals clearly implicated these reactants as mediators of hyaluronate depolymerization. Generation of superoxide by leukocytes in vivo may account for the loss of synovial fluid viscosity that accompanies inflammatory joint disease.

Correlation between the autoxidation of ferrous ions or of L-ascorbic acid and the depolymerization of hyaluronate

The depolymerization of hyaluronic acid by reductants in the presence of a variety of buffer systems has been investigated. Many organic buffers inhibit the depolymerization. A parallelism was observed between absorption of oxygen, oxidation of L-ascorbic acid, and depolymerization of hyaluronic acid.