ABSTRACT
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.
Subject(s)
Anti-Inflammatory Agents/therapeutic use , Drug Delivery Systems/methods , Hyaluronic Acid/therapeutic use , Methotrexate/therapeutic use , Osteoarthritis/drug therapy , Animals , Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents/chemistry , Cell Proliferation/drug effects , Cells, Cultured , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/immunology , Humans , Hyaluronic Acid/administration & dosage , Hyaluronic Acid/chemistry , Injections, Intra-Articular , Knee/pathology , Male , Methotrexate/administration & dosage , Methotrexate/chemistry , Osteoarthritis/chemically induced , Osteoarthritis, Knee/chemically induced , Osteoarthritis, Knee/drug therapy , Rats , Rats, Inbred Lew , Synovial Fluid/cytology , Synovial Fluid/drug effects , Tumor Necrosis Factor-alpha/immunologyABSTRACT
A series of novel tetra-peptide motilin agonists, having the general structure H-Phe-Val-X-Ile-NH(2), were designed, on the basis of structure-activity relationship studies of motilin. Peptides, in which X is a side chain substituted tryptophan residue, have agonistic activity. H-Phe-Val-Trp(2'-CH(2)CH(2)OH)-Ile-NH(2)(7), H-Phe-Val-Trp(2'-SCH(3))-Ile-NH(2)(8), and H-Phe-Val-Trp(2'-SCH(2)CH(2)CH(3))-Ile-NH(2)(9), showed an EC(50) for contractile activity in the rabbit smooth muscle of 14.1+/-3.2, 12.9+/-4.1, and 4.6+/-1.6 microM, respectively. Interaction of the tryptophan aliphatic side chain with motilin receptor appears to influence the signal transduction via motilin receptor.
Subject(s)
Drug Design , Motilin/agonists , Oligopeptides/chemical synthesis , Oligopeptides/pharmacology , Amino Acid Sequence , Animals , Gastrointestinal Agents/chemical synthesis , Gastrointestinal Agents/chemistry , Gastrointestinal Agents/metabolism , Gastrointestinal Agents/pharmacology , Gastrointestinal Motility/drug effects , Intestines/drug effects , Intestines/physiology , Male , Motilin/metabolism , Motilin/pharmacology , Muscle Contraction/drug effects , Muscle, Smooth/drug effects , Muscle, Smooth/physiology , Oligopeptides/chemistry , Oligopeptides/metabolism , Rabbits , Receptors, Gastrointestinal Hormone/metabolism , Receptors, Neuropeptide/metabolism , Signal Transduction , Structure-Activity RelationshipABSTRACT
A series of cyclic peptides having the general structure H-Phe-c[-N(epsilon)-Lys-X-NH-(CH(2))(n)-CO-] were designed on the basis of structure-activity relationship studies of motilin. All were motilin antagonists. The cyclic peptides, in which X is a 3-tert-butyl-substituted tyrosine residue (H-Phe-c[-N(epsilon)-Lys-Tyr(3-tBu)-beta Ala-] (3), H-Phe-c[-N(epsilon)-Lys-Tyr(3-tBu)-Gly-] (6), H-Phe-c[-N(epsilon)-Lys-Tyr(3-tBu)-Abu-] (7), and H-Phe-c[-N(epsilon)-Lys-Tyr(3-tBu)-Ahx-] (8)) showed potent motilin receptor antagonistic activity in the rabbit smooth muscle (pA(2) > 7). The 3-tert-butyl Tyr was found to be the moiety responsible for enhanced binding to the motilin receptor, while the size of the ring had little importance.
