Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-08-28
2002-06-11
Riley, Jezia (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S024300, C536S024330, C514S001000, C514S04400A, C435S006120, C435S068100
Reexamination Certificate
active
06403778
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a composition for use in screening toxicological effects of test compounds or therapeutic treatments and methods employing such compositions.
BACKGROUND OF THE INVENTION
Toxicity testing is a necessary and time-consuming part of the pharmaceutical drug development pipeline. A more rapid screen to detect toxicity of lead drug candidates may be the use of gene expression microarrays. For example, microarrays consisting of full length genes or gene fragments on a substrate may be formed. These arrays can then be tested with samples treated with the drug candidates to elucidate the gene expression pattern associated with treatment with the drug candidate. This gene pattern can be compared with gene expression patterns of compounds associated with known toxicological responses.
Benzo(a)pyrene (BP) is a known rodent and likely human carcinogen and is the prototype of a class of compounds, the polycyclic aromatic hydrocarbons (PAHs). It is metabolized by several forms of cytochrome P450 and associated enzymes to both activated and detoxified metabolites (Degawa et al. (1994) Cancer Res. 54: 4915-4919). The ultimate metabolites are the bay-region diol epoxide, benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) and the K-region diol epoxide, 9-hydroxy benzo(a)pyrene-4,5-oxide, which have been shown to form DNA adducts. BPDE-DNA adducts have been shown to persist in rat liver up to 56 days post dose with the treatment regimen of 10 mg/kg b.w. 3 times per week for 2 weeks (Qu and Stacey (1996) Carcinogenesis 17: 53-59). It has recently been shown that the BPDE-DNA adduct preferentially binds to methylated CpG sites in the p53 gene at sites where mutations are known to occur (Chen et al. (1998) Cancer Res. 58:2070-2075). Mutations in this tumor suppressor gene have been discovered in over 50% of human cancers (Greenblatt et al. (1994) Cancer Res. 54: 4855-4878).
Acetaminophen (APAP) is a well-recognized and widely-used analgesic. It is metabolized by specific cytochrome P450 isozymes with the majority of the drug undergoing detoxification by glucuronic acid, sulfate and glutathione conjugation pathways (Chen et al. (1998) Chem. Res. Toxicol. 11: 295-301). However, at large nontherapeutic doses, APAP can cause hepatic and renal failure by being metabolized to an active intermediate, N-acetyl-p-benzoquinone imine (NAPQI). NAPQI then binds to sulfhydryl groups of proteins causing their inactivation and leading to subsequent cell death (Kroger et al. (1997) Gen. Pharmacol. 28: 257-263).
Clofibrate (CLO) is an antilipidemic drug which lowers elevated levels of serum triglycerides. In rodents, chronic treatment produces hepatomegaly, an increase in hepatic peroxisomes and has been shown to be a hepatocarcinogen but not a mutagen (Lock et al. (1989) Ann. Rev. Pharmacol. Toxicol. 29:145). CLO has been shown to induce cytochrome P450 4A and reduce the levels of P450 4F (Kawashima et al. (1997) Arch. Biochem. Biophys. 347:148-154). It is also involved in transcription of b-oxidation genes as well as induction of peroxisome proliferator activated receptors (PPARs) (Kawashima supra).
The present invention provides compositions and methods for screening, preferably in a microarray format, of compounds and therapeutic treatments for toxicological effects.
SUMMARY OF THE INVENTION
In one aspect, the invention provides a method for screening a compound for a toxicological effect. The method comprises (i) selecting a plurality of polynucleotide targets, wherein said polynucleotide targets have first gene expression levels altered in tissues treated with known toxicological agents when compared with untreated tissues, (ii) treating a sample with the compound to induce second gene expression levels of a plurality of polynucleotide probes, and (iii) comparing the first and second gene expression levels to identify those compounds that induce expression levels of the polynucleotide probes that are similar to those of the polynucleotide targets and said similarity of expression levels correlates with a toxicological effect of the compound.
Preferably, the comparing comprises (i) contacting said polynucleotide targets with the polynucleotide probes under conditions effective to form hybridization complexes between said polynucleotide targets and said polynucleotide probes, and (ii) detecting the presence or absence of said hybridization complexes. In this context, similarity may mean that at least 1, preferably at least 5, more preferably 10, of the upregulated polynucleotide targets form hybridization complexes with the polynucleotide probes at least once during a time course to a greater extent than would the probes of a sample not treated with the test compound. Similarity may also mean that at least 1, preferably at least 3, of the downregulated polynucleotide target sequences form hybridization complexes with the polynucleotide probes at least once during a time course to a lesser extent than would the probes of a sample not treated with the test compound.
Preferred tissues are selected from the group consisting of liver, kidney, brain, spleen, pancreas and lung. Preferred toxicological agents are selected from the group consisting of benzo(a)pyrene, methylcholanthrene, benz(a)anthracene, 7,12-dimethylbenz(a)anthracene and their corresponding toxic metabolites. The polynucleotide targets comprise genes that are upregulated-or-down regulated at least 2 fold, preferably at least 3 fold, in tissues treated with known toxicological agents when compared with untreated tissues. Preferred polynucleotide targets are selected from the group consisting of SEQ ID NOs: 1-47, or fragments thereof, some of whose expression is upregulated and others of whose expression is downregulated. Even more preferable are SEQ ID NOs: 2, 8, 10, 13, 19, 26, 31, 33, 35, 37, 39, and 42 which are upregulated and SEQ ID Nos: 11, 25, 27, 28, and 45 which are downregulated. In one embodiment, the polynucleotide targets are hybridizable array elements of a microarray.
Alternatively, the invention provides methods for screening a therapeutic treatment for a toxicological effect or for screening a sample for a toxicological response to a compound or therapeutic treatment.
In another aspect, the invention provides methods for preventing a toxicological response by administering complementary nucleotide sequences against one or more selected upregulated polynucleotide target or a ribozyme that specifically cleaves such sequences. Alternatively, a toxicological response may be prevented by administering sense nucleotide sequences for one or more selected down regulated polynculeotide targets.
DESCRIPTION OF THE SEQUENCE LISTING
A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
The Sequence Listing contains the sequences of exemplary polynucleotide targets of the invention, SEQ ID NOs: 1-47.
DESCRIPTION OF THE INVENTION
Definitions
The term “microarray” refers to an ordered arrangement of hybridizable array elements. The array elements are arranged so that there are preferably at least one or more different array elements, more preferably at least 10 array elements, and most preferably at least 100 array elements, and even more preferably 10,000, on a 1 cm
2
substrate surface. Furthermore, the hybridization signal from each of the array elements is individually distinguishable. In a preferred embodiment, the array elements comprise polynucleotide sequences.
A “polynucleotide” refers to a chain of nucleotides. Preferably, the chain has from about 5 to 10,000 nucleotides, more preferably from about 50 to 3,500 nucleotides. The term “polynucleotide target” refers to a polynucleotide sequence capable of hybridizing with a “polynucleotide probe” to form a polynucleotide target/probe complex
Cunningham Mary Jane
Panzer Scott R.
Seilhamer Jeffrey J.
Zweiger Gary B.
Incyte Genomics Inc.
Incyte Genomics, Inc.
Riley Jezia
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