Method for in Vivo Measurement of Reverse Cholesterol

Transport

This application claims priority under 35 U. S. C. §119 (e) to U.S. Provisional Patent Application No. 61/074,787, filed on June 23, 2008. The foregoing application is incorporated by reference herein.

Field of the Invention

The present invention relates to the measurement of reverse cholesterol transport.

Background of the Invention

Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated by reference herein as though set forth in full.

Reverse cholesterol transport (RCT) was first introduced in 1968 to describe the process of how extrahepatic cholesterol is returned to the liver for elimination (Glomset, J.A. (1968) J. Lipid Res., 9:155- 67) . Inefficient RCT is believed to play a key role in the development of atherosclerosis (hardening of the arterial walls) . Its association with atherosclerosis involves cholesterol efflux from macrophages, foam cells that are laden with cholesterol and deposit within the arterial wall, to the liver for excretion (Cuchel et al . (2006) Circulation, 113:2548-55). LDL cholesterol (bad cholesterol) is engulfed by macrophages forming what are known as foam cells. These foam cells in turn deposit in arterial walls, culminating in atherosclerosis development and associated cardiovascular infarcts.

RCT-mediated transport of HDL (good) cholesterol is believed to enable hepatic elimination of LDL cholesterol from foam cells via a regulated co-transport mechanism. Thus, a major focus in atherosclerosis research is identifying molecules that promote RCT.

RCT is a complex process involving multiple pathways and acceptor particles. A major area of interest in therapeutic approaches to atherosclerosis involves developing pharmacological agents to enhance RCT (Lewis et al. (2005) Cir Res., 96:1221-32).

Therapies currently under development focus on several targets of the RCT pathway including: upregulation of apolipoprotien A-I, high density lipoprotein cholesterol (HDL-C) , adenosine triphosphate-binding cassette protein (ABC) Al, and ABCGl; inhibition of cholesteryl ester transfer protein; and synthetic agonists of the nuclear receptors liver X receptor and peroxisome proliferators- activated receptors (PPAR) -α, -δ, and-γ. Therefore, assessing the effects of therapeutic agents on RCT is critical. Currently, there is no available method to assess RCT in humans. Attempts to measure RCT, including administration of tritiated water and quantification of bile and fecal sterol excretion, have detected single steps of the RCT pathway, but have failed to measure net "reverse cholesterol" flux from extrahepatic tissues to the liver or on fecal sterol excretion (Cuchel et al. (2006) Circulation, 113:2548- 55) . There is a recognized need for the development of novel biomarkers and kinetic methods to assess the effects of pharmacological agents on RCT in vivo (Lewis et al. (2005) Cir Res., 96:1221-32).

Summary of the Invention

In accordance with one aspect of the instant invention, methods for measuring reverse cholesterol transport in a mammal are provided. In a specific embodiment, the methods comprise the steps of a) administering at least one sterol to a mammal, b) administering at least one cholesterol absorption inhibitor to the mammal, c) obtaining at least one biological sample from the mammal; and d) determining the amount of the sterol administered in step a) in the biological sample obtained in step c) . The decrease in the administered sterol over time correlates to the reverse cholesterol transport in the subject. In a particular embodiment, the sterol administered to the mammal is not synthesized by the mammal. In yet another embodiment, the sterol is a phytosterol.

In accordance with another aspect of the instant invention, methods for determining the ability of a test compound to modulate reverse cholesterol transport in a mammal are provided. In a particular embodiment, the methods comprise generating a first elimination curve of a sterol administered to a mammal in the above methods for measuring reverse cholesterol transport, and repeating the methods for measuring reverse cholesterol transport after administering a test compound to the mammal, thereby generating a second elimination curve. Differences between the first and second elimination curves are indicative of the test compound' s ability to modulate reverse cholesterol transport.

Brief Description of the Drawing

Figures IA and IB are graphs of the ratio of sterols present in plasma and hair, respectively, of

mice placed on a 1% phytosterol diet for three weeks and then placed back on a chow diet at day 0.

Figures 2A and 2B are graphs of the ratio of campesterol present in plasma and skin, respectively, of humans after removal from phytosterol diet.

Detailed Description of the Invention

The instant invention provides compositions and methods for measuring reverse cholesterol transport in a mammal. The methods generally comprise a) administering at least one sterol to the mammal, wherein the administered sterol is not synthesized by the mammal; b) administering at least one cholesterol absorption inhibitor to the mammal; c) obtaining at least one biological sample from the mammal; and d) determining the amount of the sterol administered in step a) in the biological sample obtained in step c) . The modulation (in particular, decrease) of the sterol over time correlates to the reverse cholesterol transport in the mammal (e.g., the net reverse cholesterol transport kinetics). The rate of decrease (e.g., an elimination curve) can be compared to standard curves obtained from normal (healthy) individuals and/or patients with defective reverse cholesterol transport, in order to determine whether the tested subject has normal or deficient reverse cholesterol transport. In other words the method can be used as a diagnostic method for deficient reverse cholesterol transfer and associated disorders and diseases such as atherosclerosis. The methods of the instant invention use sterols which are not synthesized by the mammal to which they are administered. The sterol may be naturally occurring or synthetic. In a preferred embodiment, the sterol has similar properties to cholesterol. For example, it is

preferred that the sterol is found in the same human cells as cholesterol and utilizes the same cellular mechanisms as cholesterol with regard to uptake, transport, storage, and excretion. Because the sterols used in the instant invention are not synthesized by the subject, the amount of plasma and tissue sterol can be manipulated by its addition and removal from the diet. In a particular embodiment, the sterol is a phytosterol. In yet another embodiment, the sterol is sitosterol or, in particular, campesterol. Examples of other sterols that can be used in the methods of the instant invention include, without limitation: other naturally occurring ergosterols (not limited to plant origin) , chemically synthesized sterols such as 20 (R) -n-pentylpregn-5en-3b- ol, and detectably labeled sterols such as hexa- deuterated cholesterol or tritiated cholesterol. The administered sterol may be tagged with a detectable label (e.g., isotope, radioisotope, fluorescent compound) . Phytosterols are plant sterols (e.g., campesterol and sitosterol) and their respective stands (5 alpha- saturated derivatives) , which chemically resemble cholesterol (Ostlund, R. E. (2004) Curr. Opin. Lipid, 15:37-41). They are present in a normal diet from fruits and vegetables and are not made by humans.

Indeed, sources of phytosterols include rice bran, corn bran, corn germ, wheat germ oil, corn oil, safflower oil, oat oil, olive oil, cotton seed oil, soybean oil (e.g., soybean oil distillates), peanut oil, black tea, orange juice, Valencia, green tea, Colocsia, kale, broccoli, sesame seeds, shea oils, grapeseed oil, rapeseed oil, linseed oil, canola oil, tall oil from wood pulp and other resinous oil from wood pulp. Phytosterols are absorbed by the same mechanism as

cholesterol (i.e., micelles), but when consumed in combination with dietary cholesterol, they can displace cholesterol from micelles (Piironen et al. (2000) J. Sci. Food Agric, 80:939-66). Indeed, phytosterols can inhibit intestinal cholesterol absorption, thereby lowering blood total and low-density lipoprotein (LDL) cholesterol concentrations. Plant sterols are absorbed proportionally to cholesterol, but to a lesser extent (1-5%) (Gylling et al. (2005) Ann. Clin. Biochem., 42:254-63). As stated above, plant sterols are absorbed via micelles and are transported to the enterocyte for absorption.

Plant sterol absorption in humans has been studied. Bhattacharya and colleagues studied one person and showed that plant sterols constitute 7% of total skin surface sterols and are transferred from the plasma to the epidermal basal cells of the skin (Bhattacharyya et al. (1972) J. Clin. Invest., 51:2060-70). Kwiterovich and Jakulj have shown that, in a limited number of subjects, maximal plasma levels of plant sterols are obtained between 4 and 6 weeks (Kwiterovich et al. (2003) J. Lip. Res., 44:1143-55; Jakulj et al . (2005) J. Lip. Res., 46:2692-8). Bhattacharya showed a similar period was required to maximize the amount of skin sterol (Bhattacharyya et al. (1983) J. Invest.

Dermatol., 80:294-6). Ostlund using a tracer study determined a plasma half-life of 3-4 days for campesterol and sitosterol which is in agreement with earlier work by Bhattacharya (Bhattacharyya et al . (1983) J. Invest. Dermatol., 80:294-6; Ostlund et al.

(2002) Am. J. Physiol. Endrocrinol . Metab., 282.E911-6). Bhattacharyya and colleagues have shown that it takes about 5 weeks for radiolabled sitosterol to be cleared from the skin, but 7 weeks for plant sterols to

completely disappear from the skin when the diet is completely devoid of phytosterols .

In a particular embodiment of the instant invention, the sterol is administered to the subject in step a) until a stable amount of the sterol is present in the tissue, fluid, and/or cells from which the biological sample is to be obtained. For example, the sterol may be administered one time, preferably multiple times over the course of hour(s), day(s), week(s), or month (s). In a particular embodiment, the sterol is administered for about 2 to about 4 weeks. The sterol may be administered within a composition comprising a biologically acceptable carrier. The sterol, such as a phytosterol, may be administered via the administration of dietary fruit and vegetables.

In another specific embodiment, the administration of at least one cholesterol absorption (uptake) inhibitor to the mammal coincides approximately with the halting of the administration of the at least one sterol. For example, once the desired level of the administered sterol is achieved, the administration of the sterol may be stopped and the cholesterol absorption inhibitor may then be administered while biological sample are obtained from the subject. The cholesterol absorption inhibitor is administered to the mammal to prevent further dietary sterol absorption. Examples of cholesterol absorption inhibitors are provided hereinbelow. Typically, the beginning of the administration of the cholesterol absorption inhibitor marks the beginning of the elimination curve. While the administration of the cholesterol absorption inhibitor is preferred in the instant methods, the administration of the cholesterol absorption inhibitor is not absolutely required. If the cholesterol absorption

inhibitor is not present, the stopping of the administration of the sterol (e.g., ending the sterol containing diet) represents the beginning of the elimination curve. The biological sample obtained from the patient can be any biological tissue, cell(s), or fluid from the subject which comprises the administered sterol. Preferably, the biological sample is accessible from an individual through sampling by minimally invasive or non-invasive approaches (e.g., urine collection, blood drawing, needle aspiration, and the like) . Biological samples include, without limitation, serum, plasma, blood, urine, feces, skin tissue samples, and hair samples. The samples may be obtained at regular or irregular intervals from the patient. In a particular embodiment, at least one sample is obtained at the time the administration of cholesterol absorption inhibitor begins and at least one other sample is obtained at a later timepoint . The sample may be obtained over the course of an hour(s), day(s), week(s), or month(s). In a particular embodiment, the samples are obtained over the course of about 20 to about 40 days.

The amount of sterol present in the biological sample may be determined by any method. The sterol may be isolated/purified from the biological sample. In a particular embodiment, the sterol is measured by mass spectrometry. If the steroid is radiolabeled, then the sterol can be detected by radiography.

In accordance with another aspect of the instant invention, the methods of measuring reverse cholesterol transfer described hereinabove are performed on a mammal to generate a first profile of the elimination of the administered sterol (e.g., an elimination curve) from the subject (i.e., a baseline curve is generated). The

methods are repeated on the mammal wherein at least one test compound has been administered to the subject. In a particular embodiment, the at least test compound is administered after the administration of the sterol. The repeated method leads to the production of a second elimination curve. The first and second elimination curves can then be compared. A faster and/or greater elimination of the administered sterol in the second elimination curve indicates the test compound increases reverse cholesterol transport. A slower and/or decreased elimination of the administered sterol in the second elimination curve indicates the test compound decreases reverse cholesterol transport. If the first and second elimination curves are the same, then test compound has no effect on reverse cholesterol transport. The instant methods encompass performing the assay on the same patient in the presence and absence of a test compound as well as performing the assay on a patient in the presence of a test compound and then comparing to a standard baseline curve (e.g., from a similar subject (s) ) .

The test compound administered to the subject can be any molecule including, but not limited to, small molecules, chemical compounds, amino acids, carbohydrates, fatty acids, peptides, polypeptides, proteins, antibodies, cytokines, hormones, sugars, lipids, nucleic acid molecules, and polynucleotides.

Test compounds determined to modulate (e.g., increase) reverse cholesterol transport by the above methods may be administered to a patient to treat atherosclerosis .

The methods of the instant invention are performed on mammalian subjects, including humans. Mammals include, but are not limited to, primate, feline,

canine, bovine, ovine, porcine, equine, rodent, lagomorph, and human subjects.

In accordance with another aspect of the instant invention, kits for the performance of the methods of the instant invention are provided. The kits may comprise at least one composition comprising the sterol to be administered to the subject in a pharmaceutically acceptable carrier and at least one at least one cholesterol absorption inhibitor in a pharmaceutically acceptable carrier. The kit may further comprise one or more of the following components: instruction material, vials, tubes, means for obtaining a biological sample from a subject (e.g., needles), and mass spectrometry reagents (e.g., buffers).

Definitions

The term "reverse cholesterol transport" refers to the net movement (e.g., efflux or transport) of extrahepatic cholesterol to the liver for elimination/excretion. The term "reverse cholesterol transport" may encompass the entire process by which cholesterol (including precursors, metabolites, and derivatives thereof) moves from cells into the bloodstream and from the bloodstream out of the body. In other words, the term "reverse cholesterol transport" may encompass the general process by which cholesterol is eventually removed from a living subject.

"Pharmaceutically acceptable" indicates approval by a regulatory agency of the Federal government or a state government. "Pharmaceutically acceptable" agents may be listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

A "carrier" refers to, for example, a diluent, adjuvant, excipient, auxilliary agent or vehicle with which an active agent of the present invention is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

The term "isolated" is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the desired activity, and that may be present, for example, due to incomplete purification, or the addition of stabilizers. The term "phytosterol" refers to sterols produced in plants. More particularly, phytosterols are sterol compounds produced by plants which are structurally very similar to cholesterol. Phytosterols may be alkylated at the Ci7 position and/or contain substitutions (e.g., methyl or ethyl substituents) at the C ₂₄ position on the sterol side chain. Phytosterols include, without limitation, plant sterols, esters of plant sterols, plant stands or stanol esters, and stanols and stanol esters derivable from plant sterols. Examples of phytosterols include, without limitation, sitosterol (e.g., alpha or beta sitosterol), stigmasterol, ergosterol, campesterol, sitostanol (e.g., alpha or beta sitostanol) , campestanol, and brassiciasterol, their

fatty acid esters, and the like. In a particular embodiment, the phytosterol is campesterol.

As used herein, the term "cholesterol absorption inhibitor" means any agent capable of capable of inhibiting the absorption of one or more sterols, including but not limited to cholesterol, preferably phytosterols, when administered in a therapeutically effective amount to a mammal or human. Cholesterol absorption inhibitors may block the movement of cholesterol from the intestinal lumen into enterocytes of the small intestinal wall, thus reducing serum cholesterol levels. Examples of cholesterol absorption inhibitors are described in U.S. Patent Nos. 5,846,966; 5,631,365; 5,767,115; 6,133,001; 5,886,171; 5,856,473; 5,756,470; 5,739,321; and 5,919,672; WO 00/63703; WO 00/60107; WO 00/38725; WO 00/34240; WO 00/20623; WO 97/45406; WO 97/16424; WO 97/16455; and WO 95/08532. In a particular embodiment, the cholesterol absorption inhibitor is ezetimibe (1- (4-fluorophenyl) -3 (R) - [3 (S) - (4-fluorophenyl) -3-hydroxypropyl) ] -4 (S) - (4- hydroxyphenyl) -2-azetidinone, described in U.S. Patent Nos. 5,767,115 and 5,846,966). Notably, U.S. Patent No. 5,767,115 also provides a method by which to identify cholesterol absorption inhibitors. Therapeutically effective amounts of cholesterol absorption inhibitors include dosages from about 0.01 mg/kg to about 30 mg/kg of body weight per day, particularly about 0.1 mg/kg to about 15 mg/kg. The cholesterol absorption inhibitor may be delivered as a single daily dose, or in divided doses two to about six times a day, or in a sustained release form.

The following example is provided to illustrate various embodiments of the present invention. It is not intended to limit the invention in any way.

EXAMPLE

Mice lacking the low density lipoprotein receptor (LDLr) and apolipoprotein B mRNA editing enzyme complex 1 (LDLr ^~/~ /apobec ^~/~ ) were fed a loading dose of phytosterols (1% in diet) for three weeks. The depletion of cholesterol, sitosterol, and campesterol from plasma and hair was then followed once the dietary source with phytosterol was removed and the mice were placed back on a chow diet (Figures IA and IB, respectively) . Upon removing the phytosterol diet the amount of both plasma campesterol and sitosterol decreased by about 80% over a three week period. Over the same time period, plasma cholesterol remained relatively constant. Similarly, the hair concentration of campesterol and sitosterol decreased over time upon their removal from the diet, while cholesterol remained relatively unaffected. The plant sterol concentration likely does not reach zero because of reabsorption of excreted sterol and the presence of residual amounts of plant sterol in mouse chow.

Because phytosterols are found in a variety of fruits and vegetables in low amounts, it may be difficult to place subjects on a diet completely devoid of plant sterols. Ezetimibe (ZETIA®; MSP Pharmaceuticals) is an approved lipid-lowering medication which inhibits intestinal uptake of dietary and biliary cholesterol at least in part by interfering with the transporter, Niemann-Picc Cl-Like 1 (NPClLl) (von Bergmann et al. (2005) Am. J. Cardiol., 4:10D-14D).

ZETIA® has been shown to partially block the absorption of plant sterols, to levels that are below the typical American diet, without affecting the absorption of fat soluble vitamins (Jakulj et al. (2005) J. Lip. Res., 46:2692-8). ZETIA® can be used in the post-loading phase to limit the amount of intestinal plant sterol that is absorbed. A study in man confirmed the utility of this method. Normal volunteers were fed a diet supplemented with plant sterol for 4 weeks at which time the supplement was removed from the diet and ezetimibe was given to prevent absorption of plant sterol. The decrease in plant sterol (as determined by mass spectrometry) from plasma and skin is shown in Figures 2A and 2B, respectively.

While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims .