Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-08-13
2001-03-13
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S004000, C435S008000, C435S091100, C435S471000, C435S476000
Reexamination Certificate
active
06200760
ABSTRACT:
TECHNICAL FIELD
This invention is directed to a method of screening agents for potential as drugs or sources of drugs, and especially for screening agents for potential as drugs for disorders caused or mediated by the expression of genes involved in inflammation and/or carcinogenesis.
BACKGROUND OF THE INVENTION
There is strong evidence that cyclooxygenase-2 (COX-2) is important in carcinogenesis and that inhibition of COX-2 protects against tumor formation. Moreover, COX-2 is known to be associated with inflammatory responses incident to a number of disorders, and major pharmaceutical companies are actively involved in the development of selective COX-2 inhibitors to treat inflammation.
There is also strong evidence that matrix metalloproteinases are associated with carcinogenesis and inflammation and are involved in tumor metastasis. The term “matrix metalloproteinase” is referred to hereinafter as “MMP”.
One of the limitations of drug discovery is the time and expense involved in identifying candidate compounds. There is, at present, no rapid and easy method for identifying sources of novel anti-cancer compounds and/or anti-inflammatory compounds.
SUMMARY OF THE INVENTION
It has been discovered herein that determining whether an agent suppresses the stimulation of a gene promoter that has been implicated in carcinogenesis, or inflammation, is an effective tool for screening agents as candidates for preventing or treating cancer and/or inflammation.
The invention herein relying on this discovery, is directed at a method for screening agents as candidates for prophylaxis or treatment of cancer or inflammation. The method relies on cells that are transfected with a construct containing transcriptional promoter element(s) that have been implicated in carcinogenesis or inflammation ligated to a reporter gene. Determination in the method, of inhibition of activation of said transcriptional promoter element(s) by agent which is being screened, indicates the agent is a candidate as a drug or source of a drug for prophylaxis or treatment of cancer or inflammation. Thus, a method herein is directed to screening agents as candidates for prophylaxis or treatment of cancer or inflammation and comprises (a) providing cells that are transfected with a construct containing transcriptional promoter element(s) that have been implicated in carcinogenesis or inflammation ligated to a reporter gene, and (b) determining whether putative agent inhibits activation of said transcriptional promoter element(s) with a determination of inhibition indicating that the putative agent is a candidate as a drug or as a source of a drug for prophylaxis or treatment of cancer or inflammation. The method has particular application to screening agents as candidates for drugs for prophylaxis or treatment of mammalian (e.g., human) disorders caused or mediated by cyclooxygenase-2. In this case, putative therapeutic agents are investigated for their ability to suppress stimulation of cyclooxygenase-2 promoter activity by exogenous stimulus. The method also has particular application to screening agents as candidates for drugs for prophylaxis or treatment of mammalian (e.g., human) disorders caused or medicated by MMPs. In this case, putative therapeutic agents are investigated for their ability to suppress stimulation of MMP promoter activity by exogenous stimulus.
There are a plurality of transcriptional promoter elements in cyclooxygenase-2 and in other genes, e.g., the Phorbol Ester Responsive Element (TRE), Nuclear Factor-&kgr;B(NF-&kgr;B) and a cyclic AMP response element (CRE), that have been implicated in carcinogenesis and in inflammatory response. In one embodiment of the invention herein, a battery of screenings are carried out on a putative agent utilizing in each case cells transfected with a different construct and/or transfected cells activated with a different activator. In the method of this embodiment, both specificity or sensitivity (where the putative agent inhibits one or less than all the transcriptional promoter elements) and breadth of activity are determined. The more different transcriptional promoter elements a putative agent inhibits and the more different activators a putative agent suppresses, the greater the likelihood is that the agent is an effective drug or the source of an effective drug for the prevention or treatment of cancer and/or inflammation. This embodiment increases the predictive value of the method herein.
As used herein, the term “transcriptional promoter element(s) that have been implicated in carcinogenesis or inflammation” means element(s) that mediate the transactivation function of transcription factors that control expression of the COX-2 gene or members of the MMP gene family.
As used herein the term “MMP gene family” and “MMP” includes genes that express collagenase(s), genes that express gelatinase(s), genes that express elastin, genes that express stromelysins, and other genes that express matrix metalloproteinases.
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Dannenberg Andrew J.
Pasco David S.
Subbaramaiah Kotha J.
Cornell Research Foundation Inc.
Myers Carla J.
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