ABSTRACT
AIM: Hyaluronic acid (HA) is a glycosaminoglycan and is essential for protecting the cartilage surface by its physical property. It is known that serum HA concentration in rheumatoid arthritis (RA) patients is higher than in healthy volunteer. However, molecular weight (MW) of serum HA in RA patients is not clear, since it needs a large sample volume to assay serum HA MW. The aim of this study is to establish the method for measuring serum HA MW in small sample sizes and to assess the association between serum HA MW and hyaluronidase (HAase) activity. METHODS: MW of serum HA in RA patients was measured using high-performance liquid chromatography and HA-binding protein. Additionally, the correlation between serum HA and HAase activity was examined using zymographic measurements. RESULTS: Serum HA MW peaked at 1-2 × 10(5) Da in all cases. However, in certain cases two peaks were observed, one each at low (1-2 × 10(5) Da) and high (8-14 × 10(5) Da) MW. HAase activity was lower in cases exhibiting this two-peaked serum HA MW pattern than in those cases with only a single peak. CONCLUSION: The novel method developed for this study permits accurate measurement of serum HA MW. The correlation observed between serum HA MW and HAase activity suggests that serum HA MW may reflect the condition of subjects' joints.
Subject(s)
Arthritis, Rheumatoid/blood , Hyaluronic Acid/blood , Hyaluronoglucosaminidase/metabolism , Adult , Aged , Arthritis, Rheumatoid/diagnosis , Arthritis, Rheumatoid/enzymology , Chromatography, High Pressure Liquid , Female , Humans , Hyaluronan Receptors/chemistry , Hyaluronic Acid/chemistry , Image Processing, Computer-Assisted , Joints/pathology , Joints/physiopathology , Male , Middle Aged , Molecular Weight , Reproducibility of ResultsABSTRACT
We previously reported that a conjugate of hyaluronic acid (HA) and methotrexate (MTX) could be a prototype for future osteoarthritis drugs having the efficacy of the two clinically validated agents but with a reduced risk of the systemic side effects of MTX by using HA as the drug delivery carrier. To identify a clinical candidate, we attempted optimization of a lead, conjugate 1. Initially, in fragmentation experiments with cathepsins, we optimized the peptide part of HA-MTX conjugates to be simpler and more susceptible to enzymatic cleavage. Then we optimized the peptide, the linker, the molecular weight, and the binding ratio of the MTX of the conjugates to inhibit proliferation of human fibroblast-like synoviocytes in vitro and knee swelling in rat antigen-induced monoarthritis in vivo. Consequently, we found conjugate 30 (DK226) to be a candidate drug for the treatment of osteoarthritis.
Subject(s)
Hyaluronic Acid/analogs & derivatives , Hyaluronic Acid/chemistry , Hyaluronic Acid/therapeutic use , Methotrexate/analogs & derivatives , Methotrexate/chemistry , Methotrexate/therapeutic use , Osteoarthritis/drug therapy , Animals , Cathepsins/metabolism , Cell Line , Fibroblasts/drug effects , Humans , Hyaluronic Acid/pharmacology , Knee Joint/drug effects , Knee Joint/pathology , Male , Methotrexate/pharmacology , Rats , Rats, Inbred Lew , Synovial Fluid/cytologyABSTRACT
Hyaluronic acid (HA) provides synovial fluid viscoelasticity and has a lubricating effect. Injections of HA preparations into the knee joint are widely used as osteoarthritis therapy. The current HA products reduce pain but do not fully control inflammation. Oral methotrexate (MTX) has anti-inflammatory efficacy but is associated with severe adverse events. Based on the rationale that a conjugation of HA and MTX would combine the efficacy of the two clinically evaluated agents and avoid the risks of MTX alone, we designed HA-MTX conjugates in which the MTX connects with the HA through peptides susceptible to cleavage by lysosomal enzymes. Intra-articular injection of our HA-MTX conjugate (conjugate 4) produced a significant reduction of the knee swelling in antigen-induced arthritis rat, whereas free MTX, HA or a mixture of HA and MTX showed no or marginal effects on the model. The efficacy of conjugate 4 was almost the same as that of MTX oral treatment. Conjugate 4 has potential as a compound for the treatment of osteoarthritis.
