Chemical Modification of the N-Acetyl Moieties of Hyaluronic Acid from Streptococcus equi for Studies in Cytokine Production.

Partial N-deacetylation and certain N-reacylations of low-molecular-weight hyaluronic acid (hyaluronan) abate its proinflammatory properties in mammalian systems. Here, we describe the treatment of bacterial hyaluronic acid by hydrazine or NaOH to yield smaller partially deacetylated polymers. These N-deacetylated polymers can be reacylated with acyl anhydrides to yield substituted hyaluronic acid derivatives of equivalent size and equimolar N-acyl substitutions.

Rheological study of hyaluronic acid derivatives.

The viscoelastic properties of four novel, low molecular weight hyaluronic acid derivatives were investigated and compared to the parent hyaluronic acid compound. Briefly, all derivatives were synthesized by first deacetylating the parent hyaluronic acid. One sample was left as such, while two others were reacytelated. The final compound, of particular interest for its anti-inflammatory properties, was butyrylated. The compounds were dissolved in phosphate buffer solution (PBS) and studied at a concentration of 5 mg/mL. Shear thinning behaviour was observed for all compounds, however, derivative samples had a lower viscosity than the parent compound at high shear rates. Viscoelastic properties were also observed to decrease as a result of the derivative preparation method. It is believed that these changes are primarily caused by a decrease in hyaluronic acid molecular weight. By increasing the concentration of the anti-inflammatory compound, it may be possible to modulate the viscoelastic properties to more closely resemble those of commercial viscosupplements. As a result, an anti-inflammatory derivative of hyaluronic acid may potentially improve upon existing viscosupplements used to treat patients who are susceptible to flare up.

Chemically modified N-acylated hyaluronan fragments modulate proinflammatory cytokine production by stimulated human macrophages.

Low molecular mass hyaluronans are known to induce inflammation. To determine the role of the acetyl groups of low molecular mass hyaluronan in stimulating the production of proinflammatory cytokines, partial N-deacetylation was carried out by hydrazinolysis. This resulted in 19.7 ± 3.5% free NH2 functional groups, which were then acylated by reacting with an acyl anhydride, including acetic anhydride. Hydrazinolysis resulted in bond cleavage of the hyaluronan chain causing a reduction of the molecular mass to 30-214 kDa. The total NH2 and N-acetyl moieties in the reacetylated hyaluronan were 0% and 98.7 ± 1.5% respectively, whereas for butyrylated hyaluronan, the total NH2, N-acetyl, and N-butyryl moieties were 0, 82.2 ± 4.6, and 22.7 ± 3.8%, respectively, based on (1)H NMR. We studied the effect of these polymers on cytokine production by cultured human macrophages (THP-1 cells). The reacetylated hyaluronan stimulated proinflammatory cytokine production to levels similar to LPS, whereas partially deacetylated hyaluronan had no stimulatory effect, indicating the critical role of the N-acetyl groups in the stimulation of proinflammatory cytokine production. Butyrylated hyaluronan significantly reduced the stimulatory effect on cytokine production by the reacetylated hyaluronan or LPS but had no stimulatory effect of its own. The other partially N-acylated hyaluronan derivatives tested showed smaller stimulatory effects than reacetylated hyaluronan. Antibody and antagonist experiments suggest that the acetylated and partially butyrylated lower molecular mass hyaluronans exert their effects through the TLR-4 receptor system. Selectively N-butyrylated lower molecular mass hyaluronan shows promise as an example of a novel semisynthetic anti-inflammatory molecule.