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1.
J Pharm Biomed Anal ; 70: 408-14, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22854133

ABSTRACT

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%.


Subject(s)
Antibodies, Bacterial/blood , Biological Assay , Clostridium histolyticum/enzymology , Matrix Metalloproteinase Inhibitors/pharmacology , Matrix Metalloproteinases/analysis , Microbial Collagenase/immunology , Antibody Specificity , Biological Assay/methods , Biological Assay/standards , Calibration , Cross Reactions , Enzyme Stability , Humans , Hydrogen-Ion Concentration , Matrix Metalloproteinase 1/analysis , Matrix Metalloproteinase 1/immunology , Matrix Metalloproteinase 13/analysis , Matrix Metalloproteinase 13/immunology , Matrix Metalloproteinase 2/analysis , Matrix Metalloproteinase 2/immunology , Matrix Metalloproteinase 3/analysis , Matrix Metalloproteinase 3/immunology , Matrix Metalloproteinase 8/analysis , Matrix Metalloproteinase 8/immunology , Matrix Metalloproteinases/immunology , Microbial Collagenase/therapeutic use , Recombinant Proteins/analysis , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/immunology , Reference Standards , Reproducibility of Results , Temperature , Time Factors
2.
Clin Vaccine Immunol ; 19(4): 562-9, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22357647

ABSTRACT

Collagenase Clostridium histolyticum (CCH) contains a fixed ratio of class I (AUX-I) and class II (AUX-II) collagenases and is used as treatment for Dupuytren's contracture. These two Zn-dependent enzymes, produced by the Gram-positive bacterium Clostridium histolyticum, are related functionally to matrix metalloproteinases (MMPs) which, among other functions, degrade the extracellular matrix. Since AUX-I and AUX-II exhibit sequence similarities to human MMPs, we assessed MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), MMP-8 (collagenase 2), and MMP-13 (collagenase 3) for cross-reactivity with anti-AUX-I and anti-AUX-II antibodies in patient serum. Serum samples from 71 subjects enrolled in a long-term clinical study (58 males and 13 females; 63 ± 10 years old [mean ± standard error]) were evaluated for cross-reactivity with the five MMPs using the two validated enzyme-linked immunosorbent assays (ELISAs). Inhibition cutoff points for anti-AUX-I and anti-AUX-II antibodies were based on assay inhibition obtained with a nonspecific protein, bovine gamma globulin, which was tested for each clinical sample. No MMP cross-reactivity was found for any of the 71 clinical antibody-positive sera evaluated. Sequence identity assessments indicated minimal, nonmeaningful alignments of the MMPs and AUX-I/AUX-II. Furthermore, clinical adverse event assessments indicated no safety signals related to MMP inhibition. The bioanalytical results, sequence identity, and clinical assessments consistently did not demonstrate cross-reactivity between CCH antidrug antibodies and endogenous human matrix metalloproteinases. The results presented here suggest that treatment of Dupuytren's contracture patients with CCH does not lead to any clinical adverse events associated with MMP inhibition.


Subject(s)
Antibodies, Bacterial/blood , Cross Reactions , Dupuytren Contracture/immunology , Matrix Metalloproteinases/immunology , Microbial Collagenase/immunology , Aged , Enzyme-Linked Immunosorbent Assay , Female , Humans , Male , Middle Aged , Sequence Homology, Amino Acid
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