Genetic markers of toxicity preparation and uses

Details Genetic markers of toxicity preparation and uses Genetic markers of toxicity preparation and uses

1999-12-08

2003-01-21

Sisson, B. L. (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C536S023100, C536S024300, C514S001000, C514S014800

Reexamination Certificate

active

06509153

ABSTRACT:

This application claims priority from prior foreign application no. 99 11405, filed on Sep. 13, 1999 in France.
The present invention is related to the technical areas of biotechnology, medicine, biology and biochemistry. Its applications concern the fields of human, animal and plant health. More particularly, the invention sets forth new methods for determining the potential toxicity of test compounds, as well as the kits and tools for the implementation of these methods. The invention also describes methods for obtaining nucleic acid sequences that can be used as genetic markers of toxicity. The invention is of special utility in the pharmaceutical industry for analysis of the toxicity profile of compounds involved in drug development and/or in pharmaceutical compositions.
Toxicity is the major reason for abandoning candidate therapeutic molecules during preclinical and clinical development. To our knowledge, at the present time there are no tests by which to rapidly determine the toxicity profile of a compound in man. Yet the regulatory authorities require that new candidate drugs undergo toxicity, mutagenicity, carcinogenicity and teratogenicity testing in animals as well as clinical trials in man. These tests are long and costly and are only partially satisfactory. For example, animal toxicity is far from being a reflection of human toxicity. Furthermore, the small number of patients enrolled in clinical trials does not systematically allow identification of toxicities associated with a small, specific population. The development, perfection and use of such tests should make it possible to identify and remove toxic compounds from the development process as far upstream as possible. In this manner new drugs could be marketed sooner and at a lesser cost to drug companies and, in turn, to health care organizations and consumers. In addition, such tests might also make it possible to detect some toxicities which currently come to light only during the post-marketing period.
The tests which are currently available do not provide sufficient characterization of toxicity markers or the potential toxicity of compounds. Some tests in bacteria, such as the Ames test, or in yeast, such as the test described in U.S. Pat. No. 4,997,757, evaluate the mutagenic potential of compounds. These tests can only detect damage at the level of the DNA. Yet many drugs exert toxic effects without being mutagens and cannot therefore be flagged by tests such as these. Other tests, such as that described in application WO 94/17208, make use of certain known eukaryotic gene promoters or response elements from these promoters, induced under different conditions of stress, to characterize drug toxicity. However, the small number of such genetic markers and the process being measured (promoter activity) do not allow prediction of the potential toxicity of the compounds. What's more, these tests are difficult to implement because they involve the culturing of transformed cell lines comprising one or more genetic constructions.
The present invention now describes rapid, effective methods by which to determine the potential toxicity of test compounds, as well as the tools and kits for the implementation of such methods. In the context of the invention, the term “toxicity” refers to any adverse and/or side effect of a compound on the metabolism of a cell or a tissue such as, in particular, its mutagenic, carcinogenic or teratogenic potential, and more generally any alteration in metabolism that can result in a harmful effect of the compound on the cell or the tissue. The present invention is based more specifically on genomics and on the development of genetic markers of toxicity that can be used to predict the toxic potential of any type of compound on any type of cell. The present invention also sets forth new methods for obtaining nucleic acid sequences that allow determination of the toxicity of compounds (eg., genetic markers of toxicity), particularly compounds entering into drug development and/or pharmaceutical compositions.
The present invention is based in part on the demonstration that genetic markers can be created and used to evaluate the toxicity of test compounds. In particular, and in an advantageous manner, these markers can be used regardless of the toxic compound being tested, and regardless of the type of cell in which the test compound is being studied. Such markers, as well as the supports, kits and methods of the invention advantageously lead to the rapid generation of toxicity profiles that are particularly thorough and reliable. Furthermore, these markers, supports, kits and methods of the invention also make it possible to determine and assign a toxicity index to the test compounds.
In particular, the present invention demonstrates that there exist genetic events that are common to situations of toxicity and to cellular metabolic pathways induced in the absence of toxic compounds. Such genetic events can therefore be induced and then used as markers of toxicity. As will be described in detail herein, such markers can further be selected or modified for the constitution of improved banks allowing a more highly predictive diagnosis of the toxicity of a compound. More specifically, the present invention now shows that genetic markers induced in a cell in a situation where cell signalling pathways are deregulated, particularly a cell in which cell viability and/or proliferation are deregulated (for example, in a situation of apoptosis), can be efficiently used to characterize the toxicity profile of test compounds. In an advantageous manner, the invention also shows that these markers can be used in toxicity tests irrespectively of the type of compound and the type of cell used. The invention also provides for the constitution of differential banks of nucleic acids characteristic of deregulated cell signalling pathway(s), particularly situations in which cell viability and/or proliferation are deregulated, and demonstrates that these banks can be used for a reliable, highly sensitive evaluation of the toxicity profile of compounds.
One subject of the invention is therefore based more specifically on the use of genetic markers characteristic of situation(s) of deregulation of cell signalling pathway(s) (situation(s) of “deregulation”) to characterize the toxicity profile of test compounds. The invention more preferably concerns the use of genetic markers induced in a situation of deregulation to characterize the toxicity profile of test compounds. The invention more preferably concerns the use of nucleic acid clones corresponding to genetic events, such as transcriptional and/or splicing events, that are characteristic of situations of deregulation, as genetic markers of toxicity.
The invention also concerns methods for analysis of the potential toxicity of a test compound, comprising at least one hybridization step between a) a sample of nucleic acids from cells treated with this compound and b) a preparation (for example, a bank) of nucleic acids corresponding to genetic events characteristic of situation(s) of deregulation, the hybridization profile indicating the toxic potential of the test compound.
The invention further concerns the kits for the implementation of these methods, as well as the compositions and nucleic acid banks comprising genetic markers of deregulation(s) of cell signalling pathways, and also the methods by which to generate such markers.
The present invention is therefore based in particular on the identification and development of genetic markers of toxicity that can be used for predictive toxicity testing of test compounds, or as targets for the study of the mechanisms of cell death or the research and development of therapeutic agents.
1. IDENTIFICATION AND DEVELOPMENT OF GENETIC MARKERS OF TOXICITY
Cell viability and differentiation are regulated by the balance that exists between mitosis and apoptosis. In the body, tissue homeostasis is also regulated by this equilibrium between cell proliferation and cell death. Alterations in this equilibrium form the

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