Structure-activity relationships of a lead hydroxamic acid inhibitor of recombinant human stromelysin were systematically defined by taking advantage of a concise synthesis that allowed diverse functionality to be explored at each position in a template. An ex vivo rat model and an in vivo rabbit model of stromelysin-induced cartilage degradation were used to further optimize these analogs for oral activity and duration of action. The culmination of these modifications resulted in CGS 27023A, a potent, orally active stromelysin inhibitor that blocks the erosion of cartilage matrix.
Recent work from our laboratory led to the development of a radioimmunoassay (RIA) for porcine relaxin utilizing [ 12.51]polytyrosyl relaxin (1 ) . Addition of tyrosine to the relaxin molecule proved to be necessary as the purified native hormone does not contain tyrosine or histidine (2) and fails to iodinate when subjected to the Hunter and Greenwood method (1). An impure relaxin extract was used by other workers who claimed successful iodination of the hormone (3).We have previously demonstrated crossreactions of antisera to porcine relaxin with relaxin-con taining tissues of several species including rats, mice, chickens, whales, and human beings (4). Because of this broad range of immunological cross-reactivity of relaxin from different sources, it was of interest to develop a RIA for determination of blood levels of relaxin in species other than the pig. We have now assayed relaxin levels throughout pregnancy in rats, mice, and guinea pigs, and near term in dogs and primates.Materials and methods. Radioimmunoassay. The relaxin RIA has been modified since the publication of the original report Reagents. Two fractions of porcine relaxin prepared according to the procedure of Sherwood and O'Byrne (2) were used in the development of the method. A moderately pure fraction (G-50:1000 U/mg) was used for immunization and a highly purified fraction (CM-A + B: 2500 to 3000 U/mg) was used for standards and preparation of polytyrosyl-relaxin . The assay was equally sensitive and linear when we used a purified relaxin prepared by a different method and kindly supplied by Dr . Christian Schwabe, Medical College of South Carolina. Thus, the antigenic determinants of porcine relaxin do not appear to be altered by differing routes of biochemical purification. How-(1). ever, reduced-alkylated relaxin as well as isolated a and p subunits of relaxin did not compete with [1251]polytyrosyl-relaxin for binding sites on the antibody. Although relaxin precursors and/or metabolic products may be immunoreactive, these observations suggest a requirement for the overall structure of two peptide chains connected by disulfide bonds for antibody recognition. Polytyrosyl-relaxin ,[ 1251]polytyrosyl-relaxin, and relaxin antisera were prepared as previously described (1). Goat anti-rabbit gamma globulin was purchased from Antibodies Incorporated, Davis, California.RIA procedure. 1. One-hundred microliters of relaxin standard solutions [ l o to 2500 pg relaxin in phosphate buffered saline (PBS = 0.01 M sodium phosphate, pH 7.0, 0.14 M sodium chloride)-1% egg albumin] plus a volume of control serum or plasma (male or castrated) equivalent to that of the unknown samples were added to standard curve tubes.2. Unknown serum or plasma samples were added to other tubes.3 . Sufficient PBS-1% egg albumin was added to each tube to bring the volume to 500 pl. 4. One-hundred microliters of [12"I]polytyrosyl-relaxin in PBS-1% egg albumin (30,000 to 60,000 cpm) were added to each tube.5 . One-hundred microliters of relaxin antiserum diluted in 0.05 M EDTA-...
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