Tadao Yamazaki

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.

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.

Small-scale screening method for low-viscosity antibody solutions using small-angle X-ray scattering

Graphical abstract Figure. No caption available. Abstract In this study, we investigated the concentration range in which self‐association starts to form in humanized IgG monoclonal antibody (mAb) solutions. Furthermore, on the basis of the results, we developed a practical method of screening for low‐viscosity antibody solutions by using small‐angle X‐ray scattering (SAXS) measurements utilizing small quantities of samples. With lower‐viscosity mAb3, self‐association was not detected in the range of 1–80 mg/mL. With higher‐viscosity mAb1, on the other hand, self‐association was detected in the range of 10–20 mg/mL and was clearly enhanced by a decrease in temperature. The viscosities of mAb solutions at 160, 180, and 200 mg/mL at 25 °C quantitatively correlated very well with the particle size parameters obtained by SAXS measurements of mAb solutions at 15 mg/mL at 5 °C. The quantity of mAb sample required for the SAXS measurements was only 0.15 mg, which is about one‐hundredth of that required for actual viscosity measurements at a high concentration, and such quantities could be available even at an early stage of development. In conclusion, the SAXS analysis method proposed in this study is a valuable tool for the development of concentrated mAb therapeutics with high manufacturability and high usability for subcutaneous injection. Abbreviations: Dmaxapp: apparent maximum dimension; Rgapp: apparent radius of gyration; EMS: electromagnetically spinning; IFT: indirect Fourier transformation; p(r): pair–distance distribution function; q.s.: quantity sufficient; SAXS: small‐angle X‐ray scattering.