Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2000-08-18
2002-07-02
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S024000, C435S004000, C435S968000, C424S009200
Reexamination Certificate
active
06413734
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for judging effectiveness of a drug having protease inhibitory activity. More specifically, the present invention relates to a method for judging effectiveness of a drug having protease inhibitory activity prior to administration of the drug for therapeutic treatment of a disease in which protease participation is suspected.
2. Related Art
Various proteases, for example, matrix metalloproteinases (MMP), plasminogen activators (PA), serine proteases such as plasmin and the like, are known to participate in infiltration and metastasis of cancer cells, progress of periodontal diseases such as periodontitis, progress of tissue destruction such as those in rheumatoid arthritis and the like. As methods for quantitative measurement of such proteases, immunoassay methods and immunoblotting methods utilizing antibodies, electrophoretic zymography methods and so forth are known. Further, as a method for measuring protease activity in tissues, there is known the so-called in situ zymography method described in FASEB Journal, Vol. 9, July, pp.974-980, 1995 or WO 97/32035. For a purpose of inhibiting excessive protease activity and thereby suppressing progress of pathological conditions, various drugs having protease inhibitory activity have been developed or are in a process of development.
The protease activities of MMP, plasmin and the like are controlled by expression amounts and activation degrees of those enzymes, as well as by amounts of endogenous inhibitors. It is known that, among diseases in which participation of a protease is suspected, e.g., rheumatoid arthritis, there are significant differences in degrees of protease activity in lesions, even though the diseases exhibit similar clinical symptoms. In some cases, a drug having protease inhibitory activity exhibits marked efficacy, and in other cases clinical symptoms are scarcely improved by administration of a drug having protease inhibitory activity. Therefore, it is clinically very important to accurately measure protease activity in lesions and judge propriety of administration of a drug having protease inhibitory activity. Although the conventionally known immunoassay methods, immunoblotting methods and electrophoretic zymography methods are effective for detection and quantification of proteases, they cannot accurately measure protease activity, per se. Therefore, they cannot be used for the purpose of judging propriety of administration of a drug having protease inhibitory activity.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a method for judging whether or not a drug having protease inhibitory activity is effective for therapeutic treatment of a disease in which participation of protease is suspected before administration of the drug. More specifically, the object of the present invention is to provide a method for judging whether or not a drug having protease inhibitory activity is effective for therapeutic treatment of a disease in which participation of protease is suspected by utilizing the method for measuring protease activity disclosed in WO97/32035.
The inventors of the present invention conducted various studies to achieve the foregoing object. As a result, they found that comprehensive protease activity, including factors such as expression amount and activation of proteases in inflammatory tissues and so forth, was accurately measurable by using the thin membrane comprising gelatin or the like disclosed in WO97/32035, and accurate judgment was possible as to whether or not a drug having protease inhibitory activity was effective for therapeutic treatment of a patient having the protease activity by using the result of the measurement. The present invention was achieved on the basis of these findings.
The present invention thus provides a method for judging effectiveness of a drug having protease inhibitory activity, which comprises the steps of:
(1) bringing a biosample into contact with a thin membrane containing a protease substrate and formed on a surface of a support, wherein said biosample is isolated or collected from a patient with a disease in which participation of a protease is suspected;
(2) detecting a trace of digestion formed on the thin membrane by action of a protease; and
(3) judging that a drug having protease inhibitory activity is effective for the patient when the trace of digestion is formed on the thin membrane. In the aforementioned method, when a distinct trace of digestion are not formed on the thin membrane, it is judged that administration of a drug having protease inhibitory activity is not effective for treatment of the patient.
According to another aspect of the present invention, there is provided a method for judging propriety of administration of a drug having protease inhibitory activity to a patient with a disease in which participation of a protease is suspected, which comprises the following steps of:
(1) bringing a biosample isolated or collected from the patient into contact with a thin membrane containing a protease substrate and formed on a surface of a support;
(2) detecting a trace of digestion formed on the thin membrane by action of protease; and
(3) judging that a drug having protease inhibitory activity should be administered to the patient when the trace of digestion is formed on the thin membrane.
In the aforementioned methods, participation of a protease can be more accurately determined by bringing a sample substantially the same as that used in the step (1) into contact with a thin membrane containing a protease substrate and a protease inhibitor and formed on a surface of a support and then comparing traces of digestion on the above membrane with the traces of digestion on the thin membrane obtained in the step (1), and thus more accurate judgment can be realized. In this embodiment, it is more preferable to use two or more thin membranes each containing a different protease inhibitor. Furthermore, effectiveness of a drug for treatment of the patient can be more accurately judged by bringing the collected sample into contact with a protease inhibitor beforehand for treatment with the inhibitor, then performing the steps (1) and (2), followed by comparing the result with that obtained from a non-treated sample.
According to preferred embodiments of the aforementioned methods, there are provided the aforementioned method, wherein the biosample is cancer tissue, lymph node, periodontal disease tissue, gingival crevicular exudate, destructive morbid tissue or extract (e.g., synovial fluid of rheumatic morbidity, exudate of alveolar pyorrhea tissue), pleural effusion, ascites, cerebrospinal fluid, bronchial washing fluid, mammary gland abnormal secretion fluid, intraovarian retention fluid, sputum or urine; the aforementioned method, wherein the protease is a matrix metalloproteinase or a serine protease; the aforementioned method, wherein the thin membrane to be used is a gelatin thin membrane, casein thin membrane or collagen thin membrane formed on the surface of the support; the aforementioned method, wherein the thin membrane to be used is a fluorescence-labeled gelatin thin membrane, fluorescence-labeled casein thin membrane or fluorescence-labeled collagen thin membrane formed on the surface of the support; the aforementioned methods wherein the traces of digestion on the thin membrane are detected by staining the membrane with a dye; the aforementioned method wherein the traces of digestion on the thin membrane are detected by a fluorescence microscope; the aforementioned method wherein the traces of digestion on the thin membrane are detected by visual inspection using a microscope; and the aforementioned method wherein quantification or numeration of the traces of digestion is carried out by using an image processing apparatus. Furthermore, the present invention also provides a drug having protease inhibitory activity which is judged to be effective for a patient by the aforementioned method; and a thin membrane used for the
Kaise Hiroshi
Kusama Mikihiro
Nemori Ryoichi
Fuji Photo Film Co., LTD
Leary Louise N.
Sughrue & Mion, PLLC
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