Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1999-12-10
2004-12-28
Woitach, Joseph (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S004000, C800S008000, C800S009000, C800S013000, C435S325000
Reexamination Certificate
active
06835866
ABSTRACT:
BACKGROUND OF THE INVENTION
A. Field of the Invention
The present invention relates generally to the fields of biochemistry and molecular biology. More particularly, it relates to lipid metabolism and the regulation of lipid metabolism. In a specific embodiment, it relates to inhibition of intestinal absorption of cholesterol by ligands that bind to the retinoid X nuclear hormone receptor (RXR).
B. Description of Related Art
Sterols are structural lipids present in the membranes of most eukaryotic cells. These lipids are rigid and characterized by a four ring hydrocarbon steroid nucleus. Sterols are required not only to impart membrane fluidity, but also serve as the precursors for a variety of products with specific biological activities. For example, cholesterol, an amphipathic sterol with a polar hydroxyl head group and nonpolar hydrocarbon body (the steroid nucleus), is the major sterol found in animal tissues. Cholesterol is an essential molecule, playing a critical role in the structural integrity of cell membranes, a precursor for steroid hormones and serves as a precursor for bile acids. Cholesterol is synthesized in the liver from isoprene precursors and further obtained via dietary intake.
Although cholesterol is a requisite molecule, high levels of blood cholesterol or hypercholesterolemia has been implicated in atherosclerosis, heart attack, and stroke (Schultheis, 1990; Mitchell, 1990). Hypercholesterolemia, if not controlled, is one of several conditions that can lead to coronary artery disease. Coronary artery disease is the leading cause of death in the United States, accounting for approximately 600,000 deaths per year. Thus, the need exists for methods of treatment that can reduce cholesterol levels and methods to screen patients at risk for high cholesterol.
Possible targets for treatment are transcription factors involved in cholesterol metabolism. One such set of factors, nuclear receptors, are ligand-activated transcription factors that govern aspects of every major developmental and metabolic pathway (reviewed in Kastner et al., 1995; Mangelsdorf et al., 1995). For example, the LXRs were first identified as “orphan” members of the nuclear receptor superfamily whose ligands and functions are unknown (Willy and Mangelsdorf, 1998). The LXRs have recently been shown to be activated by a specific class of naturally occurring, oxidized derivatives of cholesterol, including 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 24,25(S)-epoxycholesterol (Janowski et al., 1996; Lehmann et al., 1997). Oxysterols are concentrated in tissues where cholesterol metabolism and LXR expression are high, such as liver, brain, and placenta (Lavy et al., 1977; Spencer et al., 1985; Lütjohann et al., 1996).
LXRs function as heterodimers with the retinoid X receptors (RXRs), and thus, the RXR/LXR complex can be activated by both RXR ligands (i.e., rexinoids) and oxysterols (Teboul et al., 1995; Willy et al., 1995; Janowski et al., 1996). Two LXR proteins (&agr; and &bgr;) are known to exist in mammals. The expression of LXR&agr; is restricted, with highest levels in the liver (hence, the name liver X receptor) and lower but significant levels in kidney, intestine, spleen, and adrenals (Apfel et al., 1994; Willy et al., 1995). LXR&bgr; expression is more widespread and has been found in nearly every tissue examined (Shinar et al., 1994; Song et al., 1994).
The pattern of expression of LXRs and their oxysterol ligands first suggested that these receptors may have a role in cholesterol metabolism. Cholesterol has two essential metabolic fates in mammals: conversion into steroid hormones or bile acids. Since steroid hormone synthesis is known to be governed by the orphan nuclear receptor steroidogenic factor-1 (SF-1) (Parker and Schimmer, 1997), it is possible that LXRs are involved in bile acid synthesis (Janowski et al., 1996). A likely target for any bile acid inducer is cholesterol 7&agr;-hydroxylase (Cyp7a), the rate-limiting enzyme in the classical bile acid synthesis pathway (Janowski et al., 1996; Lehmann et at., 1997). Experiments by the inventors and others have shown that the Cyp7a promoter contains a functional LXR response element that can be activated by RXR/LXR heterodimers in an oxysterol- and retinoid-dependent manner (Lehmann et (al., 1997). The formation of bile acids is one of two major pathways for the catabolism and excretion of cholesterol in mammals (Russell and Setchell, 1992). Perturbations in this pathway may lead to a variety of disorders, including cholesterol gallstones, atherosclerosis, and some lipid storage diseases (Akiyoshi et al., 1986; Turley and Dietschy, 1988; Carey and Duane, 1994). Together, these observations have raised an interesting possibility that LXRs may function as transcriptional control points in bile acid metabolism.
The RXR protein of RXR homo- and heterodimers has been observed to be regulated by 9-cis retinoic acid, which binds to the carboxy-teminus of RXR (Mangelsdorf and Evans, 1995). RXR can form heterodimers with numerous other proteins in the nuclear receptor superfamily, including LXR. Depending on the receptor protein that dimerizes with RXR, and the ligands present, the resulting effects of the heterodimer on transcription can vary. Synthetic retinoids have been found to selectively bind and activate RXRs (U.S. Pat. No. 5,780,676 and U.S. Pat. No. 5,455,265).
The potential to modulate lipid concentrations in vivo, by targeting proteins of the nuclear hormone receptor superfamily with specific ligands would be particularly useful in the treatment of various diseases related to lipid metabolism. For example, high blood cholesterol levels are associated with coronary disease. Lowering dietary cholesterol intake can significantly reduce cholesterol levels in most people. However, lowering dietary intake of cholesterol often is not enough, as certain individuals sustain high cholesterol blood levels due to inefficient endogenous cholesterol homeostasis. Thus, the ability to reduce blood cholesterol levels would prove to be extremely beneficial to these individuals. Currently, there are various drugs that are administered to treat hypercholesterolemia and other abnormal blood lipid levels. For example, cholestyramine and colestipol are resins that bind bile acids in the intestinal tract, causing the liver to increase its production of bile acids and thus lower the cholesterol levels, by converting cholesterol into bile acid. However, the efficacy of these drugs is low, they are unpleasant to take, and result in constipation and bloating. Nicotinic acid, gemfibrizol, probucol, and lovastatin also are drugs used to lower blood lipid levels, but each has undesirable side effects associated with it. For example, probucol lowers total cholesterol levels, but also results in the undesirable lowering of HDL cholesterol.
Given the high incidence of coronary artery disease in the United States and its association with high cholesterol, there is a high demand for a treatment that can lower cholesterol levels without adverse side effects. Furthermore, a treatment that can lower LDL cholesterol levels, without affecting total lipid levels is highly desirable.
SUMMARY OF THE INVENTION
It is, therefore, a goal of the present invention, to provide both compositions and methods that are related to cholesterol metabolism and abnormalities therein. More particularly, the invention is intended to provide tool for the identification of compositions for use in treating cholesterol-related pathologies, as well as therapeutic methods of use.
Thus, in a first embodiment, the present invention provides a non-human transgenic mammal, the cells of which comprise at least one non-functional endogenous LXR&agr; allele. The transgenic mammal may further comprise two non-functional endogenous LXR&agr; alleles. The mammal of the present invention may a mouse, rat, hamster, guinea pig, rabbit, cow, and sheep, or other suitable organism.
In particular embodiments, either one or both non-functional LXR&agr; allele may contain (a) an interruption in the
Dietschy John M.
Mangelsdorf David J.
Repa Joyce J.
Turley Stephen D.
Board of Regents The University of Texas Systems
Fulbright & Jaworski
Woitach Joseph
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