WO2009073252A2

WO2009073252A2 - Methods and compositions for determining cholesterol-reducing properties of compounds

Info

Publication number: WO2009073252A2
Application number: PCT/US2008/068286
Authority: WO
Inventors: Jose A. Halperin; Huseyin Atkas
Original assignee: President And Fellows Of Harvard College
Priority date: 2007-06-29
Filing date: 2008-06-26
Publication date: 2009-06-11
Also published as: CL2008001903A1; WO2009073252A3

Abstract

The present invention relates to compositions and methods for detecting the ability of a food substance, nutritional supplement, therapeutic agent or disease preventive agent to decrease, increase or otherwise modulate cholesterol levels in a cell and/or animal.

Description

METHODS AND COMPOSITIONS FOR DETERMINING CHOLESTEROL- REDUCING PROPERTIES OF COMPOUNDS

FIELD

[001] The present invention relates to compositions and/or bioassays for evaluating the cholesterol-reducing ability of a food substance, nutritional supplement, therapeutic agent or disease preventive agent.

BACKGROUND

[002] Phytosterols or plant sterols are the plant counterparts of cholesterol in animals. Phytosterols exist in the diet in many forms, but the two most abundant ones are beta -sitosterol and campesterol. Many studies have shown that phytosterols reduce intestinal absorption of dietary cholesterol and also cell cholesterol synthesis. Human studies have shown that phytosterols have a protective cardiovascular effect most likely mediated by their cholesterol-reducing action and up-regulation of prostaglandin I₂ (PGI₂). It has also been shown that the cardiovascular protective effect of phytosterols is increased when these lipids are esterified to n-3 PUFAs.

[003] Fish oils rich in n-3 PUFAs have been shown to exert anti-cancer properties. When esterified with phytosterols, n-3 PUFAs may exert both cancer preventive and cardiovascular protective effects, making n-3 PUFAs/phytosterol combinations an attractive nutritional supplement. Industrial and/or commercial production of such a nutritional supplement would require quality control bioassays to ensure that the expected biological activities are retained in the commercial preparations, and that production batches contain comparable biological activities.

[004] Elevated levels of low density lipoprotein (LDL) are a well-established risk factor for cardiovascular disease (Tyroler (1985) Am. J. Prev. Med. 1 :18) and are associated with an impairment of endothelial dependent vasodilator capacity. Endothelial- dependent vasodilation is mediated by the combined effects of nitric oxide, prostacyclin (prostaglandin I₂ (PGI₂)), and endothelial-derived hyperpolarizing factor (Vogel (1999) ORL J. Otorhinolaryngol. Relat. Spec. 61 :259). In the vasculature, PGI₂ is synthesized by the inducible form of the cyclooxygenase enzyme, cyclooxygenase-2 (COX-2) (Creminon et al. (1995) Biochim. Biophys. Acta. 1254:341 ; Camacho et al. (1998) Circ. Res. 83:353). PGI₂ plays important roles in vascular homeostasis, vasodilation, and platelet quiescence (Fitzgerald et al. (1986) New Engl. J. Med. 315:983; Hammon et al. (1986) Circulation 73:224; Oates et al. (1988) New Engl. J. Med. 319(11): 689-698; Cheng et al. (2002) Science 296:539). N-3 PUFAs improve cardiovascular health by opposing the deleterious effects of LDL, likely by reducing the intracellular cholesterol.

[005] Recent studies have identified the sterol regulatory element binding protein (SREBP), a lipid-dependent transcription factor, as mediator of the effects of cholesterol and fatty acids on hepatic gene expression (Brown et al. (1986) Science 232:34; Horton et al. (2002) Cold Spring Harb. Symp. Quant. Biol 67:491 ; Horton et al. (2002) J. Clin. Invest. 109:1125). Numerous transcriptional targets of SREBP have also been identified in extra-hepatic tissues (Shimano (2001) Prog. Lipid Res. 40:439). Intracellular cholesterol concentrations are maintained through two interrelated pathways: LDL receptor (LDLR)-mediated endocytosis of plasma lipoprotein (Brown et al. (1986) Science 232:34) and de novo synthesis via the mevalonate pathway (Goldstein et al. (1990) Nature 343:425). Under conditions of cholesterol deprivation. SREBPs increase the expression of the LDLR and the enzymes of the mevalonate pathway (Briggs et al. (1993) J. Biol. Chem. 268:14490; Hua et al. (1993) Proc. Natl. Acad. ScL U.S.A. 90:11603; Wang et al. (1993) J. Biol. Chem. 268:14497; Yokoyama et al. (1993) Cell 75:187).

[006] SREBPs are transcription factors sequestered in the endoplasmic reticulum (ER) complexed with the SREBP cleavage-activating protein (SCAP) (Sakai et al. (1996) Cell 85:1037; Sakai et al. (1997) J. Biol. Chem. 272:20213). Under low-cholesterol conditions, the SCAP transports SREBP to the Golgi apparatus, where it is cleaved by two specific proteases. The site 1 protease (SlP) and site 2 protease (S2P), releasing the transcriptionally active N-terminal domain of SREBP. Id. This bHLH-zip portion migrates to the nucleus and transactivates gene expression by binding sterol regulatory elements (SREs) in target genes (Shimomura et al. (1998) J. Biol. Chem. 273(52):35299; Horton et al. (2002) Cold Spring Harb. Symp. Quant. Biol. 67:491; Horton et al. (2002) J. Clin. Invest. 109:1 125).

[007] Cholesterol and fatty acids exert a negative feedback mechanism on their own synthesis by inhibiting the translocation of SREBP to the Golgi, and thus prevent SREBP activation (Korn et al. (1998) J. Clin. Invest. 102:2050). Interestingly, LDL- cholesterol decreases PGI₂ production in a dose-dependent manner. Consistently, both LDL and free cholesterol alters endothelial COX-2 gene expression.

[008] Total cholesterol deprivation increases COX-2 mRNA and protein, whereas high concentrations of cholesterol has the opposite effect. It has recently been demonstrated that endothelial cells express functional SREBP, and that endothelial COX-2 is directly regulated by SREBP at the transcriptional level. An SRE located at -422 bp from the transcription start site of COX-2 mediates the SREBP-dependent activation of the human COX-2 promoter.

SUMMARY

[009] Certain exemplary embodiments described herein are based in part on the discovery of stable, endothelial cell lines that can be utilized for monitoring the activity of agents that cause reduced activation of SRE-containing genes, such as, for example, the COX-2 promoter.

[010] hi certain exemplary embodiments, a cell line including one or more cells stably expressing a nucleic acid sequence encoding a sterol regulatory promoter, wherein the nucleic acid sequence encoding a sterol regulatory promoter is operably linked to a nucleic acid sequence encoding a detectable label is provided. In certain aspects, the sterol regulatory promoter is a COX-2 promoter or a sterol regulatory hepatic promoter. In certain aspects, the detectable label is selected from one or more of an enzyme, a fluorescent marker, a chemi luminescent marker or a bioluminescent marker such as, for example, luciferase. In certain aspects, the one or more cells are cholesterol-responsive.

[Oil] In certain aspects, cholesterol deprivation (e.g., incubation in low cholesterol media) induces expression of the detectable label. In certain aspects, the one or more cells are sterol regulatory element binding protein (SREBP)-responsive. In certain aspects, a compound that activates SREBP (e.g., an HMG-CoA reductase inhibitor such as, e.g., lovastatin) induces expression of the detectable label. In certain aspects, the one or more cells comprise one or more endothelial cells such as, for example, HUVEC, HMVEC, BAEC, HGEC, HLMEC or BBMC. In certain aspects, the one or more endothelial cells are selected from one or both of HUVEC and BAEC. In certain aspects, the sterol regulatory promoter comprises at least one sterol regulatory element (SRE) that can optionally bind one or both of SREBP-Ia and SREBP-2.

[012] In certain exemplary embodiments, a method of determining whether a compound reduces cholesterol is provided. The method includes the steps of providing a first sample of one or more cells stably expressing a nucleic acid sequence encoding a sterol regulatory promoter, wherein the nucleic acid sequence encoding a sterol regulatory promoter is operably linked to a nucleic acid sequence encoding a first detectable label, providing a second sample of one or more cells stably expressing a nucleic acid sequence encoding a sterol regulatory promoter, wherein the nucleic acid sequence encoding a sterol regulatory promoter is operably linked to a nucleic acid sequence encoding a second detectable label under a condition that induces expression of the second detectable label, contacting the first sample with a compound, detecting expression levels the first and second detectable labels, and comparing the expression levels the first and second detectable labels, wherein the expression level of the first detectable label is about 50% or greater than the expression level of the second detectable label if the compound reduces cholesterol.

[013] In certain aspects, the condition that induces expression of the second detectable label is incubation in low cholesterol media or incubation in the presence of lovastatin. In certain aspects, the expression level of the first detectable label is between about 50% and about 150%, between about 80% and about 120%, between about 90% and about 1 10%, or between about 95% and about 105% of the expression level of the second detectable label if the compound reduces cholesterol. In certain aspects, the compound comprises at least one phytosterol and/or at least one phytosterol ester (such as, e.g., a phytosterol ester that includes one or more n-3 polyunsaturated fatty acids). In certain aspects, the first detectable label and the second detectable label are different, and in other aspects, the first detectable label and the second detectable label are the same.

[014] In certain exemplary embodiments, a method of determining potency of a compound to reduce cholesterol is provided. The method includes the steps of providing a first sample of one or more cells stably expressing a nucleic acid sequence encoding a sterol regulatory promoter, wherein the nucleic acid sequence encoding a sterol regulatory promoter is operably linked to a nucleic acid sequence encoding a first detectable label, providing a second sample of one or more cells stably expressing a nucleic acid sequence encoding a sterol regulatory promoter, wherein the nucleic acid sequence encoding a sterol regulatory promoter is operably linked to a nucleic acid sequence encoding a second detectable label under a condition that induces expression of the second detectable label, contacting the first sample with a compound, detecting expression levels the first and second detectable labels, and comparing the expression levels the first and second detectable labels, wherein the expression level of the first detectable label is about 50% or greater than the expression level of the second detectable label if the compound is potent for reducing cholesterol.

[015] In certain aspects, the condition that induces expression of the second detectable label is incubation in low cholesterol media or in the presence of lovastatin. In certain aspects, the expression level of the first detectable label is between about 50% and about 150%, between about 80% and about 120%, between about 90% and about 1 10% or between about 95% and about 105% of the expression level of the second detectable label if the compound is potent for reducing cholesterol. In certain aspects, the compound comprises at least one phytosterol and/or at least one phytosterol ester (such as, e.g., a phytosterol ester that includes one or more n-3 polyunsaturated fatty acids). In certain aspects, the first detectable label and the second detectable label are different, and in other aspects, the first detectable label and the second detectable label are the same.

[016] In certain exemplary embodiments, a method of determining batch homogeneity of a plurality of compounds for reducing cholesterol is provided. The method includes the steps of providing a sample of one or more cells stably expressing a nucleic acid sequence encoding a sterol regulatory promoter for each of a plurality of compounds, wherein the nucleic acid sequence encoding a sterol regulatory promoter is operably linked to a nucleic acid sequence encoding a first detectable label, providing a second sample of one or more cells stably expressing a nucleic acid sequence encoding a sterol regulatory promoter, wherein the nucleic acid sequence encoding a sterol regulatory promoter is operably linked to a nucleic acid sequence encoding a second detectable label under a condition that induces expression of the second detectable label, contacting each first sample for each of a plurality of compounds with a compound, detecting expression levels the first and second detectable labels, and comparing the expression levels of the first detectable label for each of the first samples and the expression level of the second detectable label to determine batch homogeneity, wherein the expression of each of the first detectable labels is between about 50% and about 150% of the expression level of the second detectable label if the batch is homogeneous.

[017] In certain aspects, the expression of each of the first detectable labels is between about 80% and about 120%, between about 90% and about 1 10% or between about 95% and about 115% of the activity of the second detectable label if the batch is homogeneous. In certain aspects, the compound comprises at least one phytosterol and/or at least one phytosterol ester (such as, e.g., a phytosterol ester that includes one or more n-3 polyunsaturated fatty acids). In certain aspects, the plurality of compounds are obtained from a single plant source and/or are obtained from a single industry source. In certain aspects, the first detectable label and the second detectable label are different, and in other aspects, the first detectable label and the second detectable label are the same.

[018] In certain exemplary embodiments, a method of determining potency of a plurality of compounds to reduce cholesterol is provided. The method includes the steps of providing a sample of one or more cells stably expressing a nucleic acid sequence encoding a sterol regulatory promoter for each of a plurality of compounds, wherein the nucleic acid sequence encoding a sterol regulatory promoter is operably linked to a nucleic acid sequence encoding a first detectable label, providing a second sample of one or more cells stably expressing a nucleic acid sequence encoding a sterol regulatory promoter, wherein the nucleic acid sequence encoding a sterol regulatory promoter is operably linked to a nucleic acid sequence encoding a second detectable label under a condition that induces expression of the second detectable label, contacting each first sample for each of a plurality of compounds with a compound, detecting expression levels the first and second detectable labels, and comparing the expression levels of the first detectable label for each of the first samples and the expression levels of the second detectable label to determine batch homogeneity, wherein the expression of each of the first detectable labels is between about 50% and about 150% of the level of the second detectable label if the batch is homogeneous.

[019] In certain aspects, the expression of each of the first detectable labels is between about 80% and about 120%, between about 90% and about 110% or between about 95% and about 1 15% of the activity of the second detectable label if the batch is potent in reducing cholesterol. In certain aspects, the compound comprises at least one phytosterol and/or at least one phytosterol ester (such as, e.g., a phytosterol ester that includes one or more n-3 polyunsaturated fatty acids). In certain aspects, the plurality of compounds are obtained from a single plant source from different geographical regions and/or from different industry sources. In certain aspects, the first detectable label and the second detectable label are different, and in other aspects, the first detectable label and the second detectable label are the same. DETAILED DESCRIPTION

[020] Certain exemplary embodiments described herein are based in part on the discovery of stable, endothelial cell lines that can be utilized for monitoring the activity of agents that cause reduced activation of SRE-containing genes, such as, for example, the COX-2 promoter. Certain exemplary embodiments described herein are based in part on the discovery of bioassays that may be used to quantitate the ability of a compound, e.g., a nutriceutical, to inhibit, upregulate or otherwise modulate cholesterol levels in an organism or a cell.

[021] In certain embodiments, methods (also referred to herein as a "screening assays") for identifying and characterizing nutriceutical compounds that act as cholesterol modulators, e.g., candidate or test compounds or agents (e.g., peptides, cyclic peptides, peptidomimetics, small molecules, small organic molecules, or other drugs) which have a stimulatory or inhibitory effect on cholesterol levels in a cell and/or animal are provided.

[022] The term "nutriceutical," as used herein, is a combination of "nutritional" and '"pharmaceutical," and refers to an ingestible substance that has one or more beneficial effects on an organism such as a human. The term nutriceutical can also refer to one or more compounds which are present in an ingestible substance. Ingestible substances include, but not limited to dietary supplements, foods, beverages and the like.

[023] Nutriceuticals include phytosterol compounds. The term "phytosterol compound," as used herein refers to a group of steroid alcohol phytochemicals naturally occurring in plants. The most abundant phytosterols include beta-sitosterol, campesterol and stigmasterol. Phytosterols are typically obtained from vegetable oils (e.g., rice bran, corn, wheat germ, flax seed, cottonseed, soybean, peanut, olive, coconut, palm and the like), nuts (e.g., cashew, almond, pecan, pistachio, walnut and the like) and/or legumes (e.g., pea, kidney bean, broad bean and the like). [024] Phytosterol compounds may be obtained from plants using a variety of methods including, but not limited to, distillation, expression, solvent extraction and the like. Phytosterol compounds may be derived from many different portions of a plant such as berries, seeds, bark, wood, rhizomes, leaves, resin, flowers, peel, root and the like.

[025] In certain exemplary embodiments, phytosterol compounds include one or more plant sterols that have been esterified to one or more n-3 polyunsaturated fatty acids (PUFAs) (e.g., phytosterol esters). Methods of esterification of phytosterols with fatty acids are well known in the art (See, e.g., Ewart et al. (2002) J. Nutr. 132:1149; Villeneuve et al. (2005) Em. Microb. Tech. 37:150).

[026] In certain exemplary embodiments, nutriceuticals include n-3 PUFA compounds, alone or in combination with phytosterol compounds. As used herein, the term "n-3 PUFA" is intended to include, but is not limited to, omega-3 fatty acids such as those found in oil from oily fish such as mackerel, salmon, sardines and the like, or vegetable sources such as the seeds of chia, perilla, flax, walnut, purslane, ligonberry, seabuckthorn, hemp, and the like, and fruits from plants such as the acai palm. Omega-3 fatty acids include, but are not limited to, α-linoleic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), stearidonic acid, eicosatetraenoic acid, docosapentaenoic acid and the like. Omega-3 fatty acids are commercially available, and methods for their extraction are well-known in the art (See Omega-3 Fatty Acids: Chemistry, Nutrition, and Health Effects (ACS Symposium), Finley (2001) American Chemical Society).

[027] In certain exemplary embodiments, methods of determining the potency of a composition to modulate (e.g., reduce) cholesterol in an animal are provided. The term "potency," as used herein, is intended to include, but is not limited to, the effectiveness of a compound, e.g., a nutriceutical, to reduce, inhibit, stimulate, upregulate or otherwise modulate cholesterol. The potency of a composition can be defined as the ability of the composition to reduce, inhibit, upregulate or otherwise modulate cholesterol levels in a sample (e.g., a biological sample) relative to a standard (e.g., a cell expressing a detectable label under conditions that induce expression of the label as described further herein).

[028] As used herein, a "biological sample" refers to a sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., plasma, serum, sputum, urine), cell sample (e.g., a cheek scraping), or tissue (e.g., a biopsy). As used herein, the term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Biological samples may be of any biological tissue or fluid or cells. Typical biological samples include, but are not limited to, sputum, lymph, blood, blood cells (e.g., white cells), fat cells, cervical cells, cheek cells, throat cells, mammary cells, muscle cells, skin cells, liver cells, spinal cells, bone marrow cells, tissue (e.g., muscle tissue, cervical tissue, skin tissue, spinal tissue, liver tissue and the like) fine needle biopsy samples, urine, cerebrospinal fluid, peritoneal fluid and pleural fluid, or cells therefrom. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes.

[029] A biological sample may be obtained from a mammal, including, but not limited to horses, cows, sheep, pigs, goats, rabbits, guinea pigs, rats, mice, gerbils, non-human primates and humans. Biological samples may also include cells from microorganisms (e.g., bacterial cells, viral cells, yeast cells and the like) and portions thereof. As used herein, the term "biological fluid" is intended to include any fluid taken from a biological organism. Biological fluids include, but are not limited to, sputum, lymph, blood, urine, tears, breast milk, nipple aspirate fluid, seminal fluid, vaginal secretions, cerebrospinal fluid, peritoneal fluid, pleural fluid, pus, ascites and the like.

[030] A biological sample may be a single cell or many cells. A biological sample may comprise a single cell type or a combination of two or more cell types. A biological sample further includes a collection of cells that perform a similar function such as those found, for example, in a tissue. Accordingly, certain aspects of the invention are directed to biological samples containing one or more tissues. As used herein, a tissue includes, but is not limited to, epithelial tissue (e.g., skin, the lining of glands, bowel, skin and organs such as the liver, lung, kidney), endothelium (e.g., the lining of blood and lymphatic vessels), mesothelium (e.g., the lining of pleural, peritoneal and pericardial spaces), mesenchyme (e.g., cells filling the spaces between the organs, including fat, muscle, bone, cartilage and tendon cells), blood cells (e.g., red and white blood cells), neurons, germ cells (e.g., spermatozoa, oocytes), placenta, stem cells and the like. A tissue sample includes microscopic samples as well as macroscopic samples.

[031] In certain exemplary embodiments, assays in which the cholesterol inhibiting or modulation activity of a composition is compared to a standard using one or more of the bioassays described herein are provided. A composition may have an activity level that is 0.001%, 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1 10%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000% or greater than the activity of the standard.

[032] In certain aspects, a composition may have a cholesterol inhibiting or modulating activity level that is between about 1% and 200%, between about 5% and 195%, between about 10% and 190%, between about 20% and 180%, between about 30% and 170%, between about 40% and 160%, between about 50% and 150%, between about 60% and 140%, between about 65% and 135%, between about 70% and 130%, between about 75% and 125%, between about 80% and 120%, between about 85% and 115%, between about 90% and 110%, between about 91% and 109%, between about 92% and 108%, between about 93% and 107%, between about 94% and 106%, between about 95% and 105%, between about 96% and 104%, between about 97% and 103%, between about 98% and 102%, or between about 99% and 101%, of the activity of the standard. [033] A nutriceutical or composition including a nutriceutical of the present invention may be diluted or concentrated to decrease or increase its cholesterol reduction activity, cholesterol up-regulation activity or cholesterol modulation activity relative to the standard, respectively.

[034] In certain exemplary embodiments, assays in which batch homogeneity of compositions is determined by comparing the relative activity of two or more (e.g., 10, 100, 1000, 10,000 1,000,000 or more) compositions using one or more of the bioassays described herein are provided. As used herein, the term "batch homogeneity" is intended to refer, but is not limited to, the relative cholesterol reducing or modulation activity of two or more compositions in a batch. As used herein, the term "batch" refers, but is not limited to, a group of two or more compositions. A batch includes compositions prepared together or compositions from two or more sources (e.g., geographical, plant, animal, commercial, and/or synthetic sources).

[035] In certain exemplary embodiments, a standard is a compound or composition having a cholesterol inhibiting, stimulating or modulating activity as determined by one or more of the bioassays described herein. Standards may be obtained from a variety of sources such as the sources of omega-3 fatty acids described herein. Standards may be synthesized in the laboratory or obtained from commercial sources. A standard may be diluted or concentrated to decrease or increase its cholesterol inhibiting, stimulating or modulating activity, respectively. Exemplary standards include, but are not limited to, HMG-CoA reductase inhibitors (e.g., statins) such as, for example, atorvastatin (Lipitor), fluvastatin (Lescol), fluvastatin Extended-Release (Lescol XL), lovastatin (Mevacor), lovastatin Extended Release (Altocor, Altoprev), pravastatin (Pravachol), rosuvastatin (Crestor), simvastatin (Zocor) and the like, and low cholesterol media. HMG-CoA reductase inhibitors are well-known in the art and are commercially available (e.g., from pharmaceutical companies such as, for example, Pfizer, AstraZeneca, Merck and the like). Low cholesterol media is cell culture media that has less cholesterol than typical control media. Low cholesterol media is well-known in the art (See, e.g., Porter et al. (1996) Science 274:255; Brown et al. (1999) Proc. Natl. Acad. Set U.S.A. 96:11041; DeBose-Boyd et al. (1999) Cell 99:703; Nohturfft et al. (1999) Proc. Natl. Acad ScL U.S.A. 96:1 1235).

[036] In certain exemplary embodiments, the nutriceuticals disclosed herein can be used in the treatment of disorders associated with aberrant cholesterol levels, such as, for example, hypercholesterolemia, atherosclerosis, cardiovascular disease, coronary artery disease, angina pectoris, myocardial infarction, transient ischemic attacks, strokes, peripheral vascular disease, high blood pressure and the like. Physiological processes influenced by cholesterol levels include, but are not limited to, vascular homeostasis, vasodilation, platelet quiescence, intracellular cholesterol levels, overall cardiovascular health and the like. One or more physiological processes can be altered by one or more of the nutriceutical compositions described herein.

[037] In certain exemplary embodiments, the nutriceuticals disclosed herein can be used in the treatment of disorders associated with aberrant cellular proliferation such as cellular proliferative disorders, (e.g., cancer). Treatment of cellular proliferative disorders is intended to include inhibition of proliferation including rapid proliferation. As used herein, the term "cellular proliferative disorder" includes disorders characterized by undesirable or inappropriate proliferation of one or more subset(s) of cells in a multicellular organism. The term "cancer" refers to various types of malignant neoplasms, most of which can invade surrounding tissues, and may metastasize to different sites (see, for example, PDR Medical Dictionary 1st edition (1995)). The terms "neoplasm" and "tumor" refer to an abnormal tissue that grows by cellular proliferation more rapidly than normal and continues to grow after the stimuli that initiated proliferation is removed (see, for example, PDR Medical Dictionary 1st edition (1995)). Such abnormal tissue shows partial or complete lack of structural organization and functional coordination with the normal tissue which may be either benign (i.e., benign tumor) or malignant (i.e., malignant tumor).

[038] The language "treatment of cellular proliferative disorders" is intended to include the prevention of the growth of neoplasms in a subject or a reduction in the growth of pre-existing neoplasms in a subject. The inhibition also can be the inhibition of the metastasis of a neoplasm from one site to another. Examples of the types of neoplasms intended to be encompassed by the present invention include but are not limited to those neoplasms associated with cancers of the breast, skin, bone, prostate, ovaries, uterus, cervix, liver, lung, brain, larynx, gallbladder, pancreas, rectum, parathyroid, thyroid, adrenal gland, immune system, neural tissue, head and neck, colon, stomach, bronchi, and/or kidneys.

[039] As used herein, the term "cell line" is intended to refer to established cell lines, transformed cell lines, primary cell lines and the like. Cells of such cell lines can be any prokaryotic or eukaryotic cells.

[040] In certain exemplary embodiments, cells are endothelial cells. As used herein, the term "endothelial cell" refers to a cell derived from the endothelium of an organism. Endothelial cells line the entire circulatory system (e.g., heart, arteries, veins, capillaries, lymph nodes and the like). Endothelial cells may be derived from a variety of locations within an organism including, but not limited to, the abdomen, gastrointestinal system, heart, spleen, lungs, axillary lymph node, embryonic yolk sack, aorta, brain and the like. Endothelial cell lines are well-known in the art and include, but are not limited to: murine SVEC4-10, murine SVEC4-10EE2, murine SVEC4-10EHR1, murine 2F2B, murine 2Hl 1, murine IPlB, and murine 1P2-E4, (American Type Culture Collection, Manassas, VA); Human dermal neonatal microvascular endothelial cells (HMVEC) and EGM2-MV (Cambrex, East Rutherford, NJ); bovine brain endothelial cells (BBMC), human lung microvascular endothelial cells (HLMEC), human glomerular endothelial cells (HGEC) (Polyplus- Transfection Inc., New York, NY); bovine aortic endothelial cells (BAEC), human umbilical vein endothelial cells (HUVEC) and the like. Endothelial cell lines are described in Walter- Yohrling et al. (2004) Clin. Cancer Res. 10:2179.

[041] In certain exemplary embodiments, vectors, such as expression vectors, containing, for example, a nucleic acid sequence encoding a sterol regulatory promoter operably linked to a nucleic acid encoding a detectable label, are provided. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[042] The expression vectors described herein include a nucleic acid in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).

[043] The term "regulatory sequence" is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors described herein can be introduced into host cells to thereby produce proteins or peptides, including detectable labels, encoded by nucleic acids as described herein.

[044] In certain exemplary embodiments, a regulatory sequence is a sterol regulatory element (SRE) to which SREBPs bind and act as transcription factors. SREs and their consensus sequences are well-known in the art and are described in Horton et al. (2003) Proc. Natl. Acad. ScL U.S.A. 100:1125 and Yokoyama et al. (1993) Cell 75:187.

[045] In certain exemplary embodiments, a nucleic acid sequence described herein is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and ρMT2PC (Kaufman et al. (1987) EMBO J. 6:187). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and Simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 15-17 of Sambrook, J., Russell, D., and Russell D.W. Molecular Cloning: A Laboratory Manual. 3d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 2001. In other embodiments, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type, e.g., in an endothelial cell. [046] Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced, containing sequences which allow it to homologously recombine into a specific site of the host cell's genome. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. A host cell can be any prokaryotic or eukaryotic cell.

[047] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (supra) and other laboratory manuals.

[048] For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a detectable translation product or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die). [049] In certain exemplary embodiments, one, two or more detectable labels are used with the cell based assays described herein. Examples of detectable markers include various radioactive moieties, enzymes, prosthetic groups, fluorescent markers, chemiluminescent markers, bioluminescent markers and the like. Examples of fluorescent proteins include, but are not limited to, yellow fluorescent protein (YFP), green fluorescence protein (GFP), cyan fluorescence protein (CFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin and the like. Examples of bioluminescent markers include, but are not limited to, luciferase (e.g., bacterial, firefly, click beetle, sea pansy (Renilla) and the like), luciferin, aequorin and the like. Examples of enzyme systems having visually detectable signals include, but are not limited to, galactosidases, glucorinidases, phosphatases, peroxidases, cholinesterases and the like. Identifiable markers also include radioactive compounds such as ¹²⁵1, ³⁵S, ¹⁴C, or ³H. Identifiable markers are commercially available from a variety of sources.

[050] In certain exemplary embodiments, pharmaceutically acceptable compositions and/or methods of administering a compound to an individual by providing pharmaceutically acceptable compositions are provided. In one embodiment, pharmaceutically acceptable compositions comprise a therapeutically effective amount of one or more of the compounds (e.g., nutriceuticals) described herein, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. The pharmaceutical compositions described herein may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; or (4) intravaginally or intrarectally, for example, as a pessary, cream or foam. In one embodiment, the therapeutic compound is administered orally. The compounds of the invention can be formulated as pharmaceutical compositions for administration to a subject, e.g., a mammal, including a human.

[051] The compounds described herein can be administered to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo. As used herein, the term "biologically compatible form suitable for administration in vivo" refers to a compound to be administered in which any toxic effects are outweighed by the therapeutic effects of the compound. The term "subject" is intended to include living organisms such as mammals. Examples of subjects include humans, monkeys, pigs, dogs, cats, rabbits, ferrets, mice, rats, frogs, toads and transgenic species thereof. Administration of a therapeutically active amount of the therapeutic compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example, a therapeutically active amount of a compound of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of antibody to elicit a desired response in the individual. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[052] The active compound (e.g., nutriceutical) may be administered in a convenient manner such as by injection (subcutaneous, intravenous, and the like), oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the active compound may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound.

[053] A compound (e.g., nutriceutical) described herein can be administered to a subject in an appropriate carrier or diluent, co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes. The term "pharmaceutically acceptable carrier," as used herein, is intended to include diluents such as saline and aqueous buffer solutions. To administer a compound of the invention by other than parenteral administration, it may be necessary tc coat the compound with, or co-administer the compound with a material to prevent its inactivation. Liposomes include water-in- oil-in-water emulsions as well as conventional liposomes (Strejan et al. (1984) J. Neuroimmunol 7:27). The active compound may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[054] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The pharmaceutically acceptable carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[055] Sterile injectable solutions can be prepared by incorporating active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient (e.g., antibody) plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[056] When the active compound is suitably protected, as described above, the composition may be orally administered, for example, with an inert diluent or an assimilable edible carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[057] It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (1) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (2) the limitations inherent in the art of compounding such an active compound for the therapeutic treatment of individuals.

[058] This invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference. EXAMPLE I

Assays

Proof of principle

[059] Low cholesterol media-induced expression of firefly luciferase operably linked to a COX-2 promoter compared to a negative control promoter operably linked to renilla luciferase is described further herein.

Lovastatin Increased Endothelial PGI? Production and Induced COX-2 Expression

[060] The SREBP transcriptional pathway can be activated by pharmacological inhibition of cholesterol synthesis using, for example, HMG-CoA reductase inhibitors such as lovastatin. Lovastatin increased the luciferase activity in bovine aortic endothelial cells (BAECs) transfected with the plasmid p459COX-2Luc up to ten-fold in a dose- dependent manner. In contrast, cells transfected with a plasmid containing the - 327COX-2 promoter lacking the SRE were unaffected by lovastatin treatment, similar to reports in the art (Smith et al. (2005) J. Lipid Res. 46:862), Lovastatin treatment also increased endothelial COX-2 mRNA production in a dose-dependent manner.

[061] There are three fundamental characteristics that identify genes as cholesterol- and SREBP-responsive (Shimano (2001) Prog. Lipid Res. 40:439). First, the gene in question should exhibit an inverse regulatory relationship with cholesterol. Although it was previously shown that endothelial COX-2 protein and mRNA expression is induced by cholesterol depletion (Smith et al. (2002) Arterioscler. Thromb. Vase. Biol. 22:983), the present study extends these observations by demonstrating that cholesterol deprivation activates the human COX-2 promoter in stably transfected endothelial cells. In accord with this mechanism of gene regulation, it was determined that overexpression of either constitutively active SREBP-Ia or SREBP-2 increased COX-2 promoter activity. Furthermore, targeted mutation or deletion of the COX-2 SRE decreased the ability of SREBP to increase COX-2 promoter activity. These functional data are further supported by experiments demonstrating that cholesterol deprivation increased endothelial PGI₂ production. [062] Second, the gene in question must have a sequence that corresponds to the canonical SREs as previously described (Yokoyama et al. (1993) Cell 75: 187; Horton et al. (2003) Proc. Natl Acad. ScL U.S.A. 100:12027). Indeed, sequence analysis identified one highly conserved SRE at -422 bp upstream from the transcription start site in the human COX-2 promoter. This sequence was fully capable of forming specific complexes with both SREBP-Ia and SREBP-2, and was an effective competitor of SREBP binding to the LDLR SRE.

[063] Third, compounds known to activate SREBP must induce the gene. The competitive inhibitor of HMG-CoA reductase, lovastatin, indirectly activates SREBP by reducing intracellular cholesterol synthesis. In the study presented herein, lovastatin induced a dose-dependent increase in COX-2 promoter-dependent luciferase activity in cells transfected with the -459COX-2 promoter that contained the SRE. Consistent with this observation, a 2-fold induction of COX-2-dependent PGI₂ production was observed in HUVECs treated with various concentrations of lovastatin. These data are fully consistent with SREBP-dependent transactivation of the LDLR promoter and other cholesterol-regulated genes (Yokoyama et al., supra).

[064] The cholesterol-sensitive SREBP is activated when intracellular stores of cholesterol are depleted, linking PGI₂ production and cholesterol homeostasis. SREBP- dependent transactivation of the COX-2 gene increases COX-2 mRNA and protein expression, which, in turn, promotes PGI₂ production. PGL; facilitates cholesterol ester hydrolysis, thereby increasing intracellular free cholesterol, and thus reducing the synthesis of cholesterol. Thus, the induction of PGI₂ production under low- cholesterol conditions provides a complementary mechanism for regulating intracellular cholesterol concentrations.

[065] In summary, two independent assays are described herein that utilize SREBP- dependent regulation of COX-2 and SRE promoters by cholesterol to measure intracellular cholesterol levels. EXAMPLE II Materials and Methods

Plasmids

[066] The SRE-Luc plasmid in which transcription of firefly luciferase is dependent on SREBP activity has been described by Dr. Joseph Goldstein (Shimomura et al. (1998) J. Biol. Chem. 273:35299). Similarly, human COX-2 promoter constructs have been described by Dr. Steve Prescott and Dr. Hyroishi Inoue (Inoue et al. (1994) FEBS Lett. 350:51 ; Meade et al. (1999) J. Biol. Chem. 274:8328). A promoterless firefly expression plasmid was obtained from Promega Corporation (Madison, WI) and modified the plasmids by inserting sterol regulatory hepatic promoters (Shimomura et al., supra) or a COX-2 promoter (Smith et al. (2005) supra).

Transfections and Luciferase Assay

[067] Bovine aortic endothelial cells (BAECs) at 70-80% confluence were transfected using LIPOFECTAMINE™ PLUS™ (Invitrogen, Carlsbad, CA) with 0.6 g of the COX-2 pGL-2 construct and 0.2 g of the plasmid pCMV-RLuc, which contains the renilla luciferase gene under control of the CMV promoter. COX-2 promoter constructs were co-transfected with pBABE plasmid which provides resistance to puromycin antibiotic. The activity of luciferases was determined using a dual luciferase assay, and firefly-luciferase activity was corrected for renilla-luciferase activity.

Claims

What is claimed:

1. A cell line comprising: one or more cells stably expressing a nucleic acid sequence encoding a sterol regulatory promoter, wherein the nucleic acid sequence encoding a sterol regulatory promoter is operably linked to a nucleic acid sequence encoding a detectable label.

2. The cell line of claim 1, wherein the sterol regulatory promoter is a COX-2 promoter.

3. The cell line of claim 1, wherein the sterol regulatory promoter is a sterol regulatory' hepatic promoter.

4. The cell line of claim 1, wherein the detectable label is selected from the group consisting of an enzyme, a fluorescent marker, a chemiluminescent marker and a bioluminescent marker.

5. The cell line of claim 4, wherein the bioluminescent marker is luciferase.

6. The cell line of claim 1, wherein the one or more cells are cholesterol- responsive.

7. The cell line of claim 6, wherein cholesterol deprivation induces expression of the detectable label.

8. The cell line of claim 7, wherein incubation in low cholesterol media induces expression of the detectable label.

9. The cell line of claim 1, wherein the one or more cells are sterol regulatory element binding protein (SREBP)-responsive.

10. The cell line of claim 9, wherein a compound that activates SREBP induces expression of the detectable label.

1 1. The cell line of claim 10, wherein the compound is an HMG-CoA reductase inhibitor.

12. The cell line of claim 11, wherein the HMG-CoA reductase inhibitor is lovastatin.

13. The cell line of claim 1, wherein the one or more cells comprise one or more endothelial cells.

14. The cell line of claim 13. wherein the one or more endothelial cells are selected from the group consisting of HUVEC, HMVEC, BAEC, HGEC, HLMEC and BBMC.

15. The cell line of claim 14, wherein the one or more endothelial cells are selected from one or both of HUVEC and BAEC.

16. The cell line of claim 1, wherein the sterol regulatory promoter comprises at least one sterol regulatory element (SRE).

17. The cell line of claim 16, wherein the SRE can bind one or both of SREBP-Ia and SREBP-2.

18. A method of determining whether a compound reduces cholesterol comprising: providing a first sample of one or more cells stably expressing a nucleic acid sequence encoding a sterol regulatory promoter, wherein the nucleic acid sequence encoding a sterol regulatory promoter is operably linked to a nucleic acid sequence encoding a first detectable label; providing a second sample of one or more cells stably expressing a nucleic acid sequence encoding a sterol regulatory promoter, wherein the nucleic acid sequence encoding a sterol regulatory promoter is operably linked to a nucleic acid sequence encoding a second detectable label under a condition that induces expression of the second detectable label; contacting the first sample with a compound; detecting expression levels the first and second detectable labels; and comparing the expression levels the first and second detectable labels, wherein the expression level of the first detectable label is about 50% or greater than the expression level of the second detectable label if the compound reduces cholesterol.

19. The method of claim 18, wherein the condition that induces expression of the second detectable label is incubation in low cholesterol media.

20. The method of claim 18, wherein the condition that induces expression of the second detectable label is incubation in the presence of lovastatin.

21. The method of claim 18, wherein the expression level of the first detectable label is between about 50% and about 150% of the expression level of the second detectable label if the compound reduces cholesterol.

22. The method of claim 18, wherein the expression level of the first detectable label is between about 80% and about 120% of the expression level of the second detectable label if the compound reduces cholesterol.

23. The method of claim 18, wherein the expression level of the first detectable label is between about 90% and about 110% of the expression level of the second detectable label if the compound reduces cholesterol.

24. The method of claim 18, wherein the expression level of the first detectable label is between about 95% and about 105% of the expression level of the second detectable label if the compound reduces cholesterol.

25. The method of claim 18, wherein the compound comprises at least one phytosterol.

26. The method of claim 18, wherein the compound comprises at least one phytosterol ester.

27. The method of claim 26, wherein the at least one phytosterol ester includes one or more n-3 polyunsaturated fatty acids.

28. A method of determining potency of a compound to reduce cholesterol comprising: providing a first sample of one or more cells stably expressing a nucleic acid sequence encoding a sterol regulatory promoter, wherein the nucleic acid sequence encoding a sterol regulatory promoter is operably linked to a nucleic acid sequence encoding a first detectable label; providing a second sample of one or more cells stably expressing a nucleic acid sequence encoding a sterol regulatory promoter, wherein the nucleic acid sequence encoding a sterol regulatory promoter is operably linked to a nucleic acid sequence encoding a second detectable label under a condition that induces expression of the second detectable label; contacting the first sample with a compound; detecting expression levels the first and second detectable labels; and comparing the expression levels the first and second detectable labels, wherein the expression level of the first detectable label is about 50% or greater than the expression level of the second detectable label if the compound is potent for reducing cholesterol.

29. The method of claim 28, wherein the condition that induces expression of the second detectable label is incubation in low cholesterol media.

30. The method of claim 28, wherein the condition that induces expression of the second detectable label is incubation in the presence of lovastatin.

31. The method of claim 28, wherein the expression level of the first detectable label is between about 50% and about 150% of the expression level of the second detectable label if the compound is potent for reducing cholesterol.

32. The method of claim 28, wherein the expression level of the first detectable label is between about 80% and about 120% of the expression level of the second detectable label if the compound is potent for reducing cholesterol.

33. The method of claim 28, wherein the expression level of the first detectable label is between about 90% and about 110% of the expression level of the second detectable label if the compound is potent for reducing cholesterol.

34. The method of claim 28, wherein the expression level of the first detectable label is between about 95% and about 105% of the expression level of the second detectable label if the compound is potent for reducing cholesterol.

35. The method of claim 28, wherein the compound comprises at least one phytosterol.

36. The method of claim 28, wherein the compound comprises at least one phytosterol ester.

37. The method of claim 36, wherein the at least one phytosterol ester includes one or more n-3 polyunsaturated fatty acids.

38. A method of determining batch homogeneity of a plurality of compounds for reducing cholesterol comprising the steps of: providing a sample of one or more cells stably expressing a nucleic acid sequence encoding a sterol regulatory promoter for each of a plurality of compounds, wherein the nucleic acid sequence encoding a sterol regulatory promoter is operably linked to a nucleic acid sequence encoding a first detectable label; providing a second sample of one or more cells stably expressing a nucleic acid sequence encoding a sterol regulatory promoter, wherein the nucleic acid sequence encoding a sterol regulatory promoter is operably linked to a nucleic acid sequence encoding a second detectable label under a condition that induces expression of the second detectable label; contacting each first sample for each of a plurality of compounds with a compound; detecting expression levels the first and second detectable labels; and comparing the expression levels of the first detectable label for each of the first samples and the expression level of the second detectable label to determine batch homogeneity, wherein the expression of each of the first detectable labels is between about 50% and about 150% of the expression level of the second detectable label if the batch is homogeneous.

39. The method of claim 38, wherein the expression of each of the first detectable labels is between about 80% and about 120% of the activity of the second detectable label if the batch is homogeneous.

40. The method of claim 38, wherein the expression of each of the first detectable labels is between about 90% and about 1 10% of the activity of the second detectable label if the batch is homogeneous.

41. The method of claim 38, wherein the expression of each of the first detectable labels is between about 95% and about 115% of the activity of the second detectable label if the batch is homogeneous.

42. The method of claim 38, wherein the compound comprises at least one phytosterol.

43. The method of claim 38, wherein the compound comprises at least one phytosterol ester.

44. The method of claim 43, wherein the at least one phytosterol ester includes one or more n-3 polyunsaturated fatty acids.

45. The method of claim 38, wherein the plurality of compounds are obtained from a single plant source.

46. The method of claim 38, wherein the plurality of compounds are obtained from a single industry source.

47. A method of determining potency of a plurality of compounds to reduce cholesterol comprising the steps of: providing a sample of one or more cells stably expressing a nucleic acid sequence encoding a sterol regulatory promoter for each of a plurality of compounds, wherein the nucleic acid sequence encoding a sterol regulatory promoter is operably linked to a nucleic acid sequence encoding a first detectable label; providing a second sample of one or more cells stably expressing a nucleic acid sequence encoding a sterol regulatory promoter, wherein the nucleic acid sequence encoding a sterol regulatory promoter is operably linked to a nucleic acid sequence encoding a second detectable label under a condition that induces expression of the second detectable label; contacting each first sample for each of a plurality of compounds with a compound; detecting expression levels the first and second detectable labels; and comparing the expression levels of the first detectable label for each of the first samples and the expression levels of the second detectable label to determine batch homogeneity, wherein the expression of each of the first detectable labels is between about 50% and about 150% of the level of the second detectable label if the batch is homogeneous.

48. The method of claim 47, wherein the expression of each of the first detectable labels is between about 80% and about 120% of the activity of the second detectable label if the batch is potent in reducing cholesterol.

49. The method of claim 47, wherein the expression of each of the first detectable labels is between about 90% and about 110% of the activity of the second detectable label if the batch is potent in reducing cholesterol.

50. The method of claim 47, wherein the expression of each of the first detectable labels is between about 95% and about 115% of the activity of the second detectable label if the batch is potent in reducing cholesterol.

51. The method of claim 47, wherein the compound comprises at least one phytosterol.

52. The method of claim 47, wherein the compound comprises at least one phytosterol ester.

53. The method of claim 52, wherein the at least one phytosterol ester includes one or more n-3 polyunsaturated fatty acids.

54. The method of claim 47, wherein the plurality of compounds are obtained from a single plant source from different geographical regions.

55. The method of claim 47, wherein the plurality of compounds are obtained from a single plant source from different industry sources.

56. The method of claim 47, wherein the first detectable label and the second detectable label are different.

57. The method of claim 47, wherein the first detectable label and the second detectable label are the same.