Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-02-28
2001-05-08
LeGuyader, John L. (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S025320, C536S025400, C536S025600
Reexamination Certificate
active
06228589
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to methods for detecting and monitoring phenotypic changes in in vitro and in vivo systems for assessing and/or determining the toxicity of chemical compounds, and more particularly, the invention relates to a method for detecting and monitoring changes in gene expression patterns in in vitro and in vivo systems for determining the toxicity of drug candidates.
BACKGROUND
The ability to rapidly and conveniently assess the toxicity of new compounds is extremely important. Thousands of new compounds are synthesized every year, and many are introduced to the environment through the development of new commercial products and processes, often with little knowledge of their short term and long term health effects. In the development of new drugs, the cost of assessing the safety and efficacy of candidate compounds is becoming astronomical. It is estimated that the pharmaceutical industry spends an average of about 300 million dollars to bring a new pharmaceutical compound to market, e.g. Biotechnology. 13: 226-228 (1995). A large fraction of these costs are due to the failure of candidate compounds in the later stages of the developmental process. That is, as the assessment of a candidate drug progresses from the identification of a compound as a drug candidate—for example, through relatively inexpensive binding assays or in vitro screening assays, to pharmacokinetic studies, to toxicity studies, to efficacy studies in model systems. to preliminary clinical studies, and so on, the costs of the associated tests and analyses increases tremendously. Consequently, it may cost several tens of millions of dollars to determine that a once promising candidate compound possesses a side effect or cross reactivity that renders it commercially infeasible to develop further. A great challenge of pharmaceutical development is to remove from further consideration as early as possible those compounds that are likely to fail in the later stages of drug testing.
Drug development programs are clearly structured with this objective in mind, however, rapidly escalating costs have created a need to develop even more stringent and less expensive screens in the early stages to identify false leads as soon as possible. Toxicity assessment is an area where such improvements may be made. for both drug development and for assessing the environmental, health, and safety effects of new compounds in general.
Typically the toxicity of a compound is determined by administering the compound to one or more species of test animal under controlled conditions and by monitoring the effects on a wide range of parameters. The parameters include such things as blood chemistry, weight gain or loss, a variety of behavioral patterns, muscle tone, body temperature, respiration rate, lethality, and the like, which collectively provide a measure of the state of health of the test animal. The degree of deviation of such parameters from their normal ranges gives a measure of the toxicity of a compound. Such tests may be designed to assess the acute, prolonged, or chronic toxicity of a compound. In general, acute tests involve administration of the test chemical on one occasion. The period of observation of the test animals may be as short as a few hours, although it is usually at least 24 hours and in some cases it may be as long as a week or more. In general, prolonged tests involve administration of the test chemical on multiple occasions. The test chemical may be administered one or more times each day, irregularly as when it is incorporated in the diet, at specific times such as during pregnancy, or in some cases regularly but only at weekly intervals. Also, in the prolonged test the experiment is usually conducted for not less than 90 days in the rat or mouse or a year in the dog. In contrast to the acute and prolonged types of test, the chronic toxicity tests are those in which the test chemical is administered for a substantial portion of the lifetime of the test animal. In the case of the mouse or rat, this is a period of 2 to 3 years. In the case of the dog, it is for 5 to 7 years.
Significant costs are incurred in establishing and maintaining large cohorts of test animals for such assays, especially the larger animals in chronic toxicity assays. Moreover, because of species specific effects, passing such toxicity tests does not ensure that a compound is free of toxic effects when used in humans. Such tests do, however, provide a standardized set of information for judging the safety of new compounds, and they provide a database for giving preliminary assessments of related compounds. An important area for improving toxicity determination would be the identification of new observables which are predictive of the outcome of the expensive and tedious animal assays.
In other medical fields, there has been significant interest in applying recent advances in biotechnology, particularly in DNA sequencing, to the identification and study of differentially expressed genes in healthy and diseased organisms, e.g. Adams et al, Science, 252: 1651-1656 (1991); Matsubara et al, Gene, 135: 265-274 (1993); Rosenberg et al, International patent application, PCT/US95/01863. The objectives of such applications include increasing our knowledge of disease processes, identifying genes that play important roles in the disease process, and providing diagnostic and therapeutic approaches that exploit the expressed genes or their products. While such approaches are attractive, those based on exhaustive, or even sampled, sequencing of expressed genes are still beset by the enormous effort required: It is estimated that 30-35 thousand different genes are expressed in a typical mammalian tissue in any given state, e.g. Ausubel et al, Editors, Current Protocols, 5.8.1-5.8.4 (John Wiley & Sons, New York, 1992). Determining the sequences of even a small sample of that number of gene products is a major enterprise, requiring industrial-scale resources. Thus, the routine application of massive sequencing of expressed genes is still beyond current commercial technology.
The availability of new assays for assessing the toxicity of compounds, such as candidate drugs, that would provide more comprehensive and precise information about the state of health of a test animal would be highly desirable. Such additional assays would preferably be less expensive, more rapid, and more convenient than current testing procedures, and would at the same time provide enough information to make early judgments regarding the safety of new compounds.
SUMMARY OF THE INVENTION
An object of the invention is to provide a new approach to toxicity assessment based on an examination of gene expression patterns, or profiles, in in vitro or in vivo test systems.
Another object of the invention is to provide a database on which to base decisions concerning the toxicological properties of chemicals, particularly drug candidates.
A further object of the invention is to provide a method for analyzing gene expression patterns in selected tissues of test animals.
A still further object of the invention is to provide a system for identifying genes which are differentially expressed in response to exposure to a test compound.
Another object of the invention is to provide a rapid and reliable method for correlating gene expression with short term and long term toxicity in test animals.
Another object of the invention is to identify genes whose expression is predictive of deleterious toxicity.
The invention achieves these and other objects by providing a method for massively parallel signature sequencing of genes expressed in one or more selected tissues of an organism exposed to a test compound. An important feature of the invention is the application of novel DNA sorting and sequencing methodologies that permit the formation of gene expression profiles for selected tissues by determining the sequence of portions of many thousands of different polynucleotides in parallel. Such profiles may be compared with those from tissues of control orga
Gorthey LeeAnn
LeGuyader John L.
Lynx Therapeutics, Inc.
Macevicz Stephen C.
Powers Vincent M.
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