Development and validation of novel enzyme activity methods to assess inhibition of matrix metalloproteinases (MMPs) in human serum by antibodies against enzyme therapeutics.

This paper summarizes the development and validation of five enzyme activity methods to assess the specific inhibition of human endogenous matrix metalloproteinases MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), MMP-8 (collagenase 2) and MMP-13 (collagenase 3) by anti-Collagenase Clostridium histolyticum (CCH) antibodies in human serum. These MMPs are of interest since antibodies against a therapeutic enzyme may cross-react with, and inactivate, the MMPs. The validated methods utilize spiked exogenous individual MMPs added to serum to determine if the serum inhibits MMP enzyme activity. Factors evaluated and optimized during development include pH, reaction time and temperature, inhibitor concentration for the positive control, and substrate and serum concentration. Characteristics established during validation for each MMP activity inhibition method included intra- and inter-assay precision and recovery, recovery in the pooled normal human serum samples, bench-top stability at room temperature and on wet ice, and assay cut-point determination. Precision results ranged from ~1 to 12% CV, recoveries of the activities of the exogenous MMPs ranged from ~84 to 90% and cut-point values ranged from 67 to 91%.

Use of optical biosensor technology to study immunological cross-reactivity between different sulfonamide drugs.

Adverse reactions to medications account for a substantial number of hospitalizations and in some cases fatalities. The nature of the many drug-drug interactions caused by the inhibition of drug-metabolizing enzymes can now be predicted and examined with a greater deal of accuracy due to research developments in the understanding of the drug-metabolizing enzymes. However, the more troubling aspects of drug-drug interactions are the idiosyncratic reactions that are unpredictable and quite often life-threatening. These reactions are often caused by a prior sensitization of a person's immune system to a given drug or class of drugs. The following work offers a technique to examine in a medium-throughput system the cross-reactivity of drugs to antibodies in order to predict if structures share the same antigenic potential toward a sensitized individual. Two commercially important sulfonamide drugs, sulfamethazine and furosemide, were taken and their binding to their respective antibodies were tested in the presence of other structurally related sulfonamide drugs. The BIACORE 3000 biosensor was used for the study and the solution-phase equilibrium assay principle was employed. The data obtained help us determine which drugs can react, and to what extent, with sulfamethazine and furosemide, giving rise to possible allergic or hypersensitivity reactions. Though sulfamethazine and furosemide were used in this study; this principle and methodology can be applied to study any drug molecule-antibody pair.