Synthesis and optimization of hyaluronic acid-methotrexate conjugates to maximize benefit in the treatment of osteoarthritis.

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.

Influence of knee alignment on quadriceps cross-sectional area.

Previous studies of methods for stimulating the individual muscles composing the quadriceps femoris have not considered the structural features of a subject's knee joint. In this study, we compared the ratios of the individual muscles composing the quadriceps between subjects with different knee alignments using magnetic resonance (MR) imaging. A total of 18 healthy males were examined: 6 normal knees (age, 23.0+/-0.6 yr; femorotibial angle (FTA), 176.8+/-0.4 degrees), 6 genu varum (age, 21.8+/-2.9 yr; FTA, 181.7+/-2.6 degrees) and 6 genu valgum (age, 21.0+/-1.6 yr; FTA, 172.3+/-1.5 degrees). The cross-sectional areas (CSAs) of quadriceps muscles were obtained by MR imaging of the entire left thigh. The CSAs of the vastus lateralis (VL), rectus femoris (RF), vastus medialis (VM) and vastus intermedius (VI) muscles were obtained by MR imaging of the entire left thigh in a supine position. The VM/VL ratio was also obtained by dividing the CSA of the VM by that of the VL and compared among the three groups of subjects with different knee alignments. The genu varum group showed a significantly higher %CSA of VM in the CSA of the quadriceps (VM/Quad) (49.0+/-2.6%) than values for the other two groups. The genu valgum group showed significantly higher values of RF/Quad (15.2+/-2.1%) and VL/Quad (40.6+/-4.0%) than the other groups. The VM/VL ratio was significantly higher in the genu varum than in values for the other two groups. This difference in CSA, in respect to knee alignment, may be considered when devising muscle training programs.

Novel hyaluronic acid-methotrexate conjugates for osteoarthritis treatment.

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.