Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-07-30
2004-04-27
Horlick, Kenneth R. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S320100, C435S252800, C435S174000, C435S183000, C382S129000, C382S133000, C382S153000, C382S173000, C382S286000, C382S291000, C702S019000, C702S022000, C536S022100
Reexamination Certificate
active
06727066
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a composition comprising a plurality of cDNAs which are differentially expressed in treated human C3A liver cell cultures and which may be used entirely or in part to diagnose, to stage, to treat, or to monitor the progression or treatment of liver disorders such as hyperlipidemia.
BACKGROUND OF THE INVENTION
Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, arrays provide a platform for examining which genes are tissue specific, carrying out housekeeping functions, parts of a signaling cascade, or specifically related to a particular genetic predisposition, condition, disease, or disorder.
The potential application of gene expression profiling is particularly relevant to improving diagnosis, prognosis, and treatment of disease. For example, both the levels and sequences expressed in tissues from subjects with hyperlipidemia may be compared with the levels and sequences expressed in normal tissue.
Toxicity testing is a mandatory and time-consuming part of drug development programs in the pharmaceutical industry. A more rapid screen to determine the effects upon metabolism and to detect toxicity of lead drug candidates may be the use of gene expression microarrays. For example, microarrays of various kinds may be produced using full length genes or gene fragments. These arrays can then be used to test samples treated with the drug candidates to elucidate the gene expression pattern associated with drug treatment. This gene pattern can be compared with gene expression patterns associated with compounds which produce known metabolic and toxicological responses.
The human C3A cell line is a clonal derivative of HepG2/C3 (hepatoma cell line, isolated from a 15-year-old male with liver tumor), which was selected for strong contact inhibition of growth. The use of a clonal population enhances the reproducibility of the cells. C3A cells have many characteristics of primary human hepatocytes in culture: i) expression of insulin receptor and insulin-like growth factor II receptor; ii) secretion of a high ratio of serum albumin compared with &agr;-fetoprotein iii) convertion of ammonia to urea and glutamine; iv) metabolize aromatic amino acids; and v) are able to proliferate in glucose-free and insulin-free medium. The C3A cell line is now well established as an in vitro model of the mature human liver (Mickelson et al. (1995) Hepatology 22:866-875; Nagendra et al. (1997) Am J Physiol 272:G408-416).
Clofibrate is an hypolidemic drug which lowers elevated levels of serum triglycerides. In rodents, chronic treatment produces hepatomegaly and an increase in hepatic peroxisomes (peroxisome proliferation). Peroxisome proliferators (PPs) are a class of drugs which activate the PP-activated receptor in rodent liver, leading to enzyme induction, stimulation of S-phase, and a suppression of apoptosis (Hasmall and Roberts (1999) Pharmacol. Ther. 82:63-70). PPs include the fibrate class of hypolidemic drugs, phenobarbitone, thiazolidinediones, certain non-steroidal anti-inflarnmatory drugs, and naturally-occuring fatty acid-derived molecules (Gelman et al. (1999) Cell. Mol. Life Sci. 55:932-943). Clofibrate has been shown to increase levels of cytochrome P450 4A. It is also involved in transcription of &bgr;-oxidation genes as well as induction of PP-activated receptors (Kawashima et al. (1997) Arch. Biochem. Biophys. 347:148-154). Peroxisome proliferation that is induced by both clofibrate and the chemically-related compound fenofibrate is mediated by a common inhibitory effect on mitochondrial membrane depolarization (Zhou and Wallace (1999) Toxicol. Sci. 48:82-89).
Captopril is an antihypertensive known as an angiotensin converting enzyme (ACE) inhibitor. ACE is a target for treatment of myocardial infarction and hypotension. ACE inhibitors can be classified into three broad groups based on chemical structure: i) sulfhydryl-containing ACE inhibitors, structurally related to captopril (e.g., fentiapril, pivalo-pril, zofenopril, alacepril); ii) dicarboxyl-containing ACE inhibitors, structurally related to enalapril (e.g., lisinopril, benazepril, quinapril, moexipril, ramipril, spirapril, perindopril, indolapril, pentopril, indala-pril, cilazapril); and iii) phosphorus-containing ACE inhibitors, structurally related to fosinopril. Many ACE inhibitors are ester-containing prodrugs that are 100 to 1000 times less potent as ACE inhibitors than their active metabolites, but have a much better oral bioavailability than the active molecules. Approximately 16 different ACE inhibitors are used world-wide. In general, ACE inhibitors differ with respect to potency; whether ACE inhibition is due to the drug itself or to activation of a prodrug; and pharmacokinetic properties. With the notable exceptions of fosinopril and spirapril (which display balanced elimination by the liver and kidneys), ACE inhibitors are cleared predominantly by the kidneys. Drugs that interfere with the renin-angiotensin system play a prominent role in the treatment cardiovascular disease and have been used as a therapy for a number of diseases including hypotension, left ventricular systolic dysfunction, myocardial infarction, progressive renal impairment, and scleroderma renal crisis.
Enalapril is a prodrug that is not highly active and, as such, it must be hydrolyzed by esterases in the liver to produce the active parent dicarboxylic acid, enalaprilat. Enalaprilat is a highly potent inhibitor of ACE with a Ki of 0.2 nM but differs from captopril in that it is an analog of a tripeptide rather than a dipeptide. Enalapril is rapidly absorbed when given orally and has an oral bioavailability of about 60% (not reduced by food). Although peak concentrations of plasma enalapril occur within an hour, enalaprilat concentrations do not peak until three to four hours. Enalapril has a half-life of only 1.3 hours. However, because it binds tightly to ACE, enalaprilat has a plasma half-life of about 11 hours. Nearly all the drug is eliminated by the kidneys either as intact enalapril or enalaprilat.
Dexamethasone and its derivatives, dexamethasone sodium phosphate and dexamethasone acetate, are synthetic glucocorticoids used as anti-inflammatory or immunosuppressive agents. Dexamethasone has little to no mineralocorticoid activity and is usually selected for management of cerebral edema because of its superior ability to penetrate the central nervous sytem. Glucocorticoids are naturally occurring hormones that prevent or suppress inflammation and immune responses when administered at pharmacological doses. Responses can include inhibition of leukocyte infiltration at the site of inflammation, interference in the function of mediators of inflammatory response, and suppression of humoral immune responses. The anti-inflammatory actions of corticosteroids are thought to involve phospholipase A
2
inhibitory proteins, collectively called lipocortins. The numerous adverse effects related to corticosteroid use usually depend on the dose administered and the duration of therapy. Proposed mechanisms of action include decreased IgE synthesis, increased number of &bgr;-adrenergic receptors on leukocytes, and decreased arachidonic acid metabolism. During an immediate allergic reaction, such as in chronic bronchial asthma, allergens bridge the IgE antibodies on the surface of mast cells, which triggers these cells to release chemotactic substances. Mast cell influx and activation, therefore, is partially responsible for the inflammation and hyperirritability of the oral mucosa in asthmatic patients. This inflammation can be retarded by administration of adrenocorticoids. As with other corticosteroids, the effects upon liver metabolism and hormone clearance mechanisms are important to understand the pharmacodynamics
Horlick Kenneth R.
Incyte Corporation
Incyte Corporation
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