title: Proteinase activity in human and murine saliva as a biomarker for proteinase inhibitor efficacy.
abstract: As molecularly targeted agents reach the clinic, there is a need for assays to detect their presence and effectiveness against target molecules in vivo. Proteinase inhibitors are one example of a class of therapeutic agent for which satisfactory methods of identifying successful target modulation in vivo are lacking. This is of particular importance while these drugs are in clinical trials because standard maximum-tolerated dose-finding studies often are not suitable due to lack of toxicity. Saliva represents a readily accessible bodily fluid that can be repeatedly sampled and used for assaying in vivo effects of systemic drugs. Here we show the development of a simple assay that can be used to measure proteinase activity in saliva and proteinase inhibition after systemic treatment with three different proteinase inhibitors. A variety of gelatinolytic activities present in human and murine saliva have been assayed with a fluorescent dye-labeled substrate and assigned to different proteinase categories by inclusion of specific classes of inhibitors. Treatment of mice with either matrix metalloproteinase inhibitors or a urokinase inhibitor for a period as short as 48 hours results in levels of the drugs that can be detected in saliva by mass spectrometry and concomitant decreases in salivary proteinase activity, thus demonstrating that these inhibitors successfully modulate their targets in vivo.
authors.affiliation: Department of Cancer Biology, Vanderbilt University Medical Center, 771 PRB 2220 Pierce Avenue, Nashville, TN 37232-6840, USA.
authors.forename: Barbara
authors.initials: B
authors.lastname: Fingleton
authors.forename: Ramkumar
authors.initials: R
authors.lastname: Menon
authors.forename: Kathy J
authors.initials: KJ
authors.lastname: Carter
authors.forename: P Dawn
authors.initials: PD
authors.lastname: Overstreet
authors.forename: David L
authors.initials: DL
authors.lastname: Hachey
authors.forename: Lynn M
authors.initials: LM
authors.lastname: Matrisian
authors.forename: J Oliver
authors.initials: JO
authors.lastname: McIntyre
mesh_terms: D000578:Amidines / Q000494:pharmacology; D000681:Amylases / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism*; D001798:Blood Proteins / Q000494:pharmacology; D002853:Chromatography, Liquid; D010450:Endopeptidases / Q000378:metabolism*; D005260:Female; D006801:Humans; D007093:Imidazoles; D008297:Male; D013058:Mass Spectrometry; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000494:pharmacology; D051379:Mice; D009930:Organic Chemicals / Q000494:pharmacology; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology*; D012463:Saliva / Q000187:drug effects* / Q000201:enzymology*; D013876:Thiophenes / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors
chemical_list: C097398:4-iodine-benzo(b)thiophene-2-carboxamidine; D000578:Amidines; D015415:Biomarkers; D001798:Blood Proteins; D007093:Imidazoles; D061965:Matrix Metalloproteinase Inhibitors; D009930:Organic Chemicals; D011480:Protease Inhibitors; D013876:Thiophenes; C031220:urokinase inhibitor; C422302:N-((2S)-2-mercapto-1-oxo-4-(3,4,4- trimethyl-2,5-dioxo-1-imidazolidinyl)butyl)-L-leucyl-N,3- dimethyl-L-Valinamide; D010649:Phenylalanine; C080985:batimastat; D000681:Amylases; D010450:Endopeptidases; D014568:Urokinase-Type Plasminogen Activator; D008666:Metalloendopeptidases; D020780:Matrix Metalloproteinase 9