WO2008104084A1 - Linoleoyl-containing phospholipids and methods for their use - Google Patents

Abstract

The invention provides compositions and methods for raising plasma apolipoprotein A-I levels in a mammal, for treating or preventing dyslipidemia, atherosclerosis and coronary artery disease. In embodiments, the compositions comprise linoleoyl-containing phosphatidic acid, phosphatidylinositol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, or diphosphatidylglycerol, or any combination thereof, and the method comprises administering these linoleoyl-containing phospholipids to a mammal.

Description

LINOLEOYL-CONTAINING PHOSPHOLIPIDS AND METHODS FOR THEIR USE

FIELD OF THE INVENTION

The present invention relates generally to the field of medicine and more particularly to materials and methods for the treatment and prevention of coronary artery disease.

More specifically, the present invention relates to certain linoleoyl-containing phospholipids and methods for their use in raising the plasma level of apolipoprotein A-I (apo A-I) in a mammal. These phospholipids and methods find utility for example in treating or preventing coronary artery disease (CAD) in mammals.

BACKGROUND

Coronary artery disease (CAD) is the leading cause of mortality and morbidity in the USA and most Western countries. As such, CAD is a world-wide health concern.

Most CAD is due to atherosclerosis (a condition characterized by subintimal thickening due to deposition of atheromas) of the large and medium-sized arteries of the heart .

The risk of developing atheroscelorisis or CAD is increased in patients who have dyslipi'demia. It is known that the risk of developing atherosclerosis or CAD is increased in patients having elevated plasma levels of low-density lipoprotein cholesterol (LDL-C) , and very- low-density lipoprotein cholesterol (VLDL-C) . Cholesterol-lowering medications are known and include a class of HMG CoA reductase inhibitors known as "statins" . It is also known that, conversely, high plasma levels of high-density lipoprotein cholesterol (HDL-C) , which contain apolipoprotein A-I (apo A-I) as a primary protein consitutuent , are protective against atherosclerosis and CAD. However, the drugs currently available to raise plasma HDL-C levels have drawbacks, such as adverse side-effects and limited efficacy. As a result, dyslipidemia tends to be under-treated in the majority of patients at risk for developing atherosclerosis and CAD.

For example, niacin (nicotinic acid) is used clinically to increase HDL-C levels. However, patients find the adverse effects of niacin intolerable. Niacin has been associated with flushed skin, itching, hot flashes, gastrointestinal distress, glucose intolerance, hyperuricemia, and hepatotoxicity . Also, niacin therapy may be counterindicated or inappropriate in patients suffering from other medical conditions. Physicians therefore typically reserve niacin treatment for patients with higher risk of cardiovascular events, usually in combination with statins, or in patients who do not reach their lipoprotein profile targets with statin monotherapy.

Fibrates (fibric acid derivatives, such as (clofibrate, gemfibrozil, ciprofibrate, bezafibrate and fenofibrate) are also used clinically to raise plasma HDL-C levels. Fibrates induce the synthesis of the major HDL-C lipoproteins, apo A-I and apo A-II and reduce total cholesterol, LDL cholesterol, triglyceride-rich VLDL, and total triglycerides. These actions are believed to be due, at least in part, to the ability of fibrates to modify the transcription of genes encoding for proteins that control lipoprotein metabolism. For example, a key target of fibrates is peroxisome proliferator-activated receptor-alpha (PPAR-alpha), a transcription factor. Fibrates activate PPAR-alpha, which then down-regulates hepatic apolipoprotein C-III and increases lipase gene expression, key players in triglyceride metabolism, and upregulates apo A-I gene transcription. However, fibrates have some unwelcome side effects and drug interactions. Common side effects of fibrates include unpleasant gastrointestinal problems (including constipation and nausea) . Fibrates interact with blood thinning medications (e.g. warfarin), increasing their blood thinning effect. Also, fibrates are typically used as an adjunctive with other cholesterol-lowering medications (i.e. statins), but such adjunctive therapy with fibrates has been reported to increase the risk of rhabdomyolysis, which is a serious and potentially life-threatening condition.

Moreover, therapies utilitizing niacin and fibrate have not been very effective at raising apo A-I levels, and their ability to elevate HDL-C is variable (see for example Meyers , C . D . and Kashyap,M.L. (2004) Curr.Opin. Cardiol . vol. 19: 366-373; Davidson, M. H . and Toth,P.P. (2004) Prog.Cardiovasc.Dis. vol. 47: 73-104).

A new class of drug, the cholesterol ester transfer protein (CETP) inhibitors, have been shown in preliminary studies to raise high-density lipoprotein cholesterol (HDL-C) levels. However, clinical trials on a leading drug candidate in this class, torcetrapib, were recently halted due to a significant increase in mortality in patients taking torcetrapib in combination with the statin "Lipitor^®" as compared to patients taking Lipitor^® alone.

Much work has also been done to develop drugs to raise HDL-C levels by upregulating ATP-binding-cassette (ABC) transport molecules, as there are several lines of research that suggest that upregulation of ABC transporters would raise plasma levels of HDL-C. The ABC transporters appear to act as phospholipid and cholesterol translocases, promoting the transfer of lipids to nascent HDL-C and thereby causing the maturation of HDL-C (Oram, J. F. 2002. ATP-binding cassette transporter Al and cholesterol trafficking.

Curr.Opin.Lipidol. 13:373-381). In addition, mutations in a particular ABC transporter, ABCAl, have been shown to be associated with low HDL levels (Marcil ,M. , Brooks-Wilson, A. , Clee,S.M., Roomp,K., Zhang, L. H., Yu, L., Collins, J.A. , van Dam, M., Molhuizen,H.O. , Loubster,0. et al. 1999. Mutations in the ABCAl gene in familial HDL deficiency with defective cholesterol efflux [see comments] Lancet 354:1341-1346; Oram, J. F. 2002. ATP-binding cassette transporter Al and cholesterol trafficking.

Curr.Opin.Lipidol. 13:373-381; Fielding, C. J. and Fielding, P. E. 2001. Cellular cholesterol efflux. Biochim.Biophys.Acta 1533:175-189). In addition fibrates, which are known to raise plasma levels of HDL-C (as discussed in more detail above) , are known to upregulate ABC transporter expression.

Consequently, liver X receptor (LXR) agonists, which are known to upregulate ABCAl expression, have also been investigated as potential HDL-C raising drugs. However, to date, attempts to upregulate ABCAl expression with LXR agonists have not resulted in significant increases in plasma levels of HDL-C. The reasons for this result are not known, but it is interesting to note that LXR agonists have recently been shown to inhibit apo A-I synthesis and secretion in human liver-derived cells (Huuskonen, J. et al. 2006. Liver X Receptor Inhibits the Synthesis and Secretion of Apolipoprotein Al by Human Liver Derived Cells. Biochemistry 45 ; 15068-15074) . Lipostabil^® (a product of Sanofi-Aventis) is a blend of purified phospholipids from soybean (comprising about 70% phosphatidylcholine (PC) , and about 30% phosphatidylinositol (PI) and phosphatidylethanolamine (PE) ) and polyunsaturated fatty acids. In human trials, this composition has been shown to increase the amount of cholesterol taken up from LDL and increase the storage capacity of HDL modified in vitro to incorporate polyenephosphatidylcholine (PPC) (Atherosclerosis. 1981; 39 (4) : 527-42) . Lipostabil has also been shown to increase serum levels of HDL-C (Int J Clin Pharmacol Ther. 1994; 32(2):53-6. Ter Arkh. 2000; 72(8):57-8. J Lipid Res . 1988; 29 (11) : 1405-15) as well as apo A-I (Sb Lek. 1995; 96 (1) : 43-8. ) . However, none of these publications investigate which component or components of Lipostabil are responsible for the observed HDL-C increasing activity. Furthermore, Lipostabil is a nutraceutical extracted from plant products, and as such, its composition has not been fully characterized nor has its safety been fully tested. The ill-defined nature of neutraceuticals limits their medical utility, as their components can have undesirable side effects or interactions with conventional medicines and doctors are therefore reluctant to recommend their use to patients.

In related work, a soybean lecithin extract (a mixture of lipids whose predominant component was phosphatidylinositol) has been shown to raise plasma HDL-C levels (Burgess et al . J. Lipid Res. 2005; 46 (2) : 350-355) . Purified negatively charged phospholipids (e.g. phosphatidylinositol) have also been used to raise plasma HDL-C levels (US Patent 6,828,306; and WO 2006/125304) . These references all teach that negatively charged phospholipids, as a class of compounds, can raise plasma HDL-C levels. However, ensuing research suggests that large oral doses of these conventional phosphatidylinositol -containing compositions (e.g. about 5.6 g/day) are required to achieve clinically significant increases in plasma HDL-C (-20%) and apo A-I (-10%) . Such large dosage amounts are somewhat impractical for long-term daily therapeutic regimens. Moreover, research (Wilson et al. Athersclerosis 1998 140:147-153) has suggested that the lineolate content of soy lecithin does not explain its anti-atherogenic properties.

Thus, there is a need for alternative drugs and methods of raising plasma levels of HDL-C and/or its constituent apo A-I .

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for raising the plasma level of apolipoprotein A-I in a mammal, the method comprising administering an effective amount of a phospholipid or a salt thereof, wherein said phospholipid is :

(a) phosphatidic acid;

(b) phosphatidylinositol;

(c) phosphatidylcholine;

(d) phosphatidylserine,-

(e) phosphatidylethanolamine; or

(f) diphosphatidylglycerol ;

and wherein said phospholipid comprises at least one linoleoyl radical.

In another aspect, the present invention provides a pharmaceutical composition for use in raising the plasma level of apolipoprotein A-I in a mammal, wherein said pharmaceutical composition comprises a phospholipid as defined above, or a salt thereof.

In another aspect, the present invention provides use of a phospholipid as defined above, or a salt thereof, in the preparation of a medicament for raising the plasma level of apolipoprotein A-I in a mammal.

In another aspect, the present invention provides use of a phospholipid as defined above, or a salt thereof, for raising the plasma level of apolipoprotein A-I in a mammal.

In another aspect, the present invention provides a commercial package comprising a phospholipid as defined above, or a salt thereof, together with instructions for use in raising the plasma level of apolipoprotein A-I in a mammal .

In another aspect, the invention provides a combination for raising plasma levels of apolipoprotein A-I (apo A-I) in a mammal, comprising:

(a) a PPAR-alpha agonist; and

(b) an ABC transporter antagonist;

wherein the PPAR-alpha agonist and the ABC transporter antagonist are present in amounts that render the combination thereof effective for raising plasma levels of apo A-I in said mammal, and wherein the PPAR-alpha agonist and the ABC transporter antagonist are formulated for simultaneous or sequential administration. In embodiments, the PPAR-alpha agonist is a phospholipid or a salt thereof, wherein said phospholipid is:

(a) phosphatidic acid; (b) phosphatidylinositol ;

(c) phosphatidylcholine;

(d) phosphatidylserine;

(e) phosphatidylethanolamine; or

(f) diphosphatidylglycerol ;

and wherein said phospholipid comprises at least one linoleoyl radical. In embodiments, the ABC transporter antagonist is glyburide .

In another aspect, the invention provides a method for raising the plasma level of apolipoprotein A-I in a mammal, comprising administering the combination of a PPAR-alpha agonist and an ABC transporter antagonist, as described above .

In another aspect, the invention provides use of the combination of a PPAR-alpha agonist and an ABC transporter antagonist, as described above, in the preparation of a medicament for raising the plasma level of apolipoprotein A-I in a mammal.

In another aspect, the invention provides use of the combination of a PPAR-alpha agonist and an ABC transporter antagonist, as described above, for raising the plasma level of apolipoprotein A-I in a mammal.

In another aspect, the invention provides a commercial package comprising the combination of a PPAR-alpha agonist and an ABC transporter antagonist, as described above, together with instructions for use in raising the plasma level of apolipoprotein A-I in a mammal. In embodiments, the phospholipid defined above is in admixture with an intestinal absorption enhancer, to improve bioavailability.

In embodiments, the phospholipid defined above enhances secretion of apolipoprotein A-I from cells of said mammal.

In embodiments, the plasma apolipoprotein A-I level in a mammal is being raised to treat or prevent dyslipidemia or atheroscelorosis in said mammal.

In embodiments, the phospholipid is used in combination with another normo-lipidemic or anti-atherogenic agent, such as a statin (e.g. atorvastatin, also known as Lipitor^®) .

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Effects of inhibitors of PPAR-alpha (peroxisome proliferator-activated receptor-alpha) and PPAR-gamma (peroxisome proliferator-activated receptor-gamma) on secretion of apolipoprotein A-I from HepG2 cells as induced by (A) phosphatidylinositol (PI) and (B) dilinoleoylphospha- tidylcholine (DLPC) . HepG2 cells were grown to 80% confluence, and serum-starved quiescent cells were treated for 24 hours with: (A) dimethylsulfoxide (DMSO) control;

10 mg/ml PI; 10 micromolar MK886; 10 micromolar GW9662; 10 mg/ml PI and 10 micromolar MK886; or a combination of 10 mg/ml PI and 10 micromolar of GW9662; or (B) DMSO control; 10 mg/ml DLCP; 10 micromolar MK886; 10 micromolar GW9662; 10 mg/ml DLCP and 10 micromolar MK886; or combination of 10 mg/ml DLCP and 10 micromolar GW9662. Secreted Apo A-I was measured. Values are means ± standard deviation of at least four independent experiments.

Figure 2: Effects of clofibrate (CIo) and PI on PPAR-alpha expression in HepG2 cells, compared to vehicle control (Ctrl) . HepG2 cells were grown to 80% confluence and serum- starved quiescent cells were incubated for 24 hours with: vehicle control; 10 micromolar clofibrate (CIo); or 1 microgram, 5 microgram, or 10 microgram soy PI. PPAR-alpha expression was detected by immunoblot analysis. Values are means ± standard deviation of at least four independent experiments .

Figure 3 : Effects of insulin and PI on phosphorylation of ERK1/2 (extracellular-signal regulated kinase 1/2) in HepG2 cells. HepG2 cells were grown to 80% confluence and serum- starved quiescent cells were incubated with: control [No treatment]; insulin [100 nano molar for 5 min.]; or 10 mg/ml PI for 5 minutes, 10 minutes, 15 minutes, 30 minutes, or 60 minutes. ERKl/2 phosphorylation was detected by immunoblot analysis. Values are means ± standard deviation for at least four independent experiments.

Figure 4 : Effect of glyburide (a potent ABC transporter inhibitor) on PI- and DLPC-induced apoA-I secretion in HepG2 cells. HepG2 cells were grown to 80% confluence and serum- starved quiescent cells were incubated for 24 hours with:

(A) DMSO (control); 10 mg/ml PI; 50 micromolar glyburide; or a combination of 10 mg/ml PI and 50 micromolar glyburide; or

(B) DMSO (control) ; 10 mg/ml DLPC; 50 micromolar glyburide; or a combination of 10 mg/ml DLPC and 50 micromolar glyburide. Secreted Apo A-I was measured. Values are means ± standard deviation for at least four independent experiments .

Figure 5: Effect of PI on expression of ABCGl in HepG2 cells. HepG2 cells were grown to 80% confluence and serum- starved quiescent cells were incubated for 24 hours with: control [no treatment]; 10 mg/ml PI; or 10 μM 9-cis-retinoic acid [this retinoid X receptor (RXR) inducer is used to increase ABC transporter expression] . ABCGl expression was detected by immunoblot analysis using an antibody specific for ABCGl. Values are means ± standard deviation for at least three independent experiments.

Figure 6 : Effects of glyburide and clofibrate on secretion of apoA-I from HepG2 cells. HepG2 cells were grown to 80% confluence and serum-starved quiescent cells were incubated for 24 hours with: DMSO (control) ; 25 micromolar clofibrate; 50 micromolar glyburide; or a combination of 25 micromolar clofibrate and 50 micromolar glyburide. Values are means ± standard deviation for four independent experiments .

Figure 7: Proposed mechanistic view by which linoleoyl - containing phospholipids have stimulatory effects on apoA-I secretion and inhibitory effects on ABC transporter expression. Linoleoyl -containing PI and PC may act through MAPK and PPAR-alpha pathways to stimulate apoA-I synthesis and secretion. Linoleoyl -containing phospholipids may also augment apoA-I secretion by decreasing the expression and function of ABC transporters such as ABCGl.

Figure 8 : Graphical representation of synergistic effect of linoleic acid on levels of hepatic apo A-I secretion in presence of linoleic acid enriched phospholipids.

Figure 9: Graphical representation of inhibition of (A) ABCAl and (B) ABCGl protein expression in hepatic cells by phosphatidylinositol and dilinoleoylphosphatidylcholine . DETAILED DESCRIPTION

Phospholipids (PLs) having certain chemical features were found to be particularly effective at stimulating apo A-I secretion in hepatoma cells (HepG2) in vitro. Specifically, linoleoyl-containing phosphatidic acid (PA) , phosphatidylinositol (PI) , phosphatidylcholine (PC) , phosphatidylserine (PS) , phosphatidylethanolamine (PE) , and diphosphatidylglycerol (DPG, also known as "cardiolipin" ) were found to be particularly effective at stimulating apo A-I secretion in hepatoma cells (HepG2) in vitro, as compared to a variety of other species of phospholipids. Both mono and dilinoleoyl -phospholipids were found to be effective. Based on these results, one skilled in the art would predict that phospholipids having one or more linoleic acid side chains may be advantageous for raising the plasma levels of apo A-I (and hence HDL-C) in mammals, for example to treat or prevent dyslipidemia or atherosclerosis in a mammal .

Linoleate containing glycerides have demonstrated a similar tendency to enhance hepatic apo A-I secretion.

Linoleic acid, which alone does not enhance apo A-I secretion, has been shown to have a synergistic effect on hepatic apo A-I secretion when administered with a linoleoyl containing phospholipid.

PPAR-alpha agonist (such as fibrates, discussed above) are known to stimulate apo A-I gene transcription. Herein, PPAR-alpha antagonists (e.g. MK886) were found to inhibit the ability of linoleoyl-containing PI and PC to induce apo A-I secretion in HepG2 cells, which suggests that linoleoyl-containing phospholipids can stimulate secretion of apo A-I from HepG2 cells by activating PPAR-alpha (see Figures IA and IB) . Linoleoyl -containing phospholipids (e.g. PI) were also found to increase PPAR-alpha expression in HepG2 cells (see Figure 2). Linoleoyl -containing phospholipids (e.g. PI) were also found to phosphorylate ERK1/2 (extracellular-signal regulated kinase 1/2), which is a MAP (mitogen-activated protein) kinase that is known to stimulate PPAR-alpha (see Figure 3) .

It has also been found that ABC transporter antagonists (e.g. glyburide) slightly increase apo A-I secretion from HepG2 cells in vitro when used alone (see Figures 4A, 4B and 6) . Further, it has been found that ABC transporter antagonists (e.g. glyburide) can increase the effect of PPAR-alpha agonists (e.g. linoleoyl -containing PI and clofibrate) on apo A-I secretion from HepG2 cells in vitro (see Figures 4A, 4B and 6) . These results are surprising and directly contrary to the commonly held view (discussed above) that upregulation of ABC transporter activity would increase plasma HDL-C levels. It has further been found that linoleoyl-containing PLs (e.g. PI) can downregulate ABC transporter expression, which may explain why linoleoyl- containing PLs (e.g. PI and PC) were found to be more effective than fibrates (e.g. clofibrate) at stimulating apo A-I secretion in HepG2 cells (see Figures IA, 5 and 6) . Inhibition of ABC transporter expression may be due to the linoleic acid component of linoleoyl-containing PLs.

Linoleic acid has been previously shown to inhibit ABC transporter expression (Wang et al . J Lipid Res. 2007 May 48 (5) : 1062-8; Uehara et al . Atherosclerosis. 2007 Mar 191 (1) :11-21) .

Applicant has shown that glyburide (Glibemclamide) , a potent ABCA-I inhibitor, can stimulate the apo A-I secretory activity of the PPAR-alpha agonists, LA-enriched phospholipids and clofibrate, in HepG2 cells. It would be expected that glyburide would also augment apo A-I secretion by other PPAR-alpha agonists, such as the fibrate drugs Ciprofibrate, Bezafibrate, Fenofibrate and Gemfibrozil. It would also be expected that other ABCA-I inhibitors such as Cyclosporin A, Probucol and BLT-4, and other Sulfonylureas (Glipizide, Gliclazide, Glimepiride, and Chlorpropamide) would be expected to stimulate PPAR-alpha induced apo A-I secretion.

Thus, in some cases, a combination of PPAR-alpha agonist activity and ABC transporter antagonist activity may be used to raise plasma levels of apo A-I, and may be more effective than use of either activity alone. Combined PPAR-alpha agonist activity and ABC transporter antagonist activity can be achieved either by using a single drug that has both desired activities, or a combination of drugs that provide both desired activities, in amounts effective to raise plasma levels of apo A-I. For example, in embodiments, a PPAR-alpha agonist (such as a fibrate or a linoleoyl- containing PL) can be used in combination with an ABC transporter antagonist (such as glyburide) to raise plasma levels of apo A-I (and hence HDL-C) .

The results presented herein may provide insight into the mechanism of action of linoleoyl -containing PLs (see Figure 7) and other HDL-C raising drugs. Moreover, using the insights and technology advanced herein, other drugs (including but not limited to phospholipids) and drug combinations can be identified that are capable of raising plasma levels of apo A-I, and the invention is therefore not limited to the expressly disclosed embodiments. More particularly, using the insights and technology advanced herein, other phospholipids can be identified that are capable of raising plasma levels of apo A-I, and the invention is therefore not limited to expressly disclosed phospholipids .

Thus, phospholipids having the ability to stimulate secretion of apo A-I from HepG2 cells can be used therapeutically, either alone, or in any combination of two or more such lipids, to raise plasma levels of apo A-I in mammals. Furthermore, these phospholipid (s) may be used in combination with either or both of one or more additional PPAR-alpha agonists (e.g. fibrates) and one or more additional ABC transporter antagonists (e.g. glyburide) to raise plasma levels of apo A-I in mammals.

Apo A-I is the primary protein constituent of HDL-C. Accordingly, any agent that raises plasma apo A-I levels should provide a concomitant increase in the plasma HDL-C levels. However, it should be noted that plasma HDL-C levels can be raised by other factors, such as an increase in the amount of cholesterol carried by high-density lipoproteins (HDL) .

A. Phospholipids

Thus, in embodiments, the present invention can be practiced using phospholipids having the following general structure (formula I) :

wherein each of R¹ and R² is independently a saturated or unsaturated Cio-2₆ hydrocarbyl group, provided that at least one of R^3--C(O)- and R²-C(O)- is 9-cis-12-cis-octadecadienoyl (linoleoyl) ; and

R³ is O or a linear or branched, unsubstituted or substituted Ci_-I0 hydrocarbyloxy group, or (as in the case of DPG) a phosphatidylglycerol radical, having the formula:

wherein each of R⁴ and R⁵ is independently a saturated or unsaturated Ci_O-₂₆ hydrocarbyl group.

R³ may be Ci-_I0 alkoxy or may be a cycloalkyloxy. R³ may be branched or unbranched and may contain substituents such as hydroxyl , alkoxy, amino, amine and azanium salt. Examples of suitable values for R³ include oxy radicals of serine, inositol, ethanolamine and choline.

Linoleoyl -containing sphingomyelin can also be used for practising the present invention.

R¹, R², R⁴ and R⁵ can be saturated or unsaturated C₁₀-2₆ hydrocarbyl groups. In many cases, these R groups will have between 12 and 24 carbon atoms and often between 16 to 20 carbon atoms. These R groups can be substituted, provided that the substitutions do not interfere with the utility of the compound. These R groups can contain 0, 1, 2, 3, or 4 unsaturations (carbon-carbon double bonds) . Unsaturations can be in a cis or trans configuration, but in many cases the cis configuration will be preferred.

In some cases, it is preferred that the R^2-C(O)- radical, which is at the C2 position of the phospholipid, is linoleoyl. Similarly, in some cases, it is preferred that R⁵-C(O)- is linoleoyl.

As shown in formula I, each of R¹, R², R⁴ and R⁵ is covalently bound to -C(O)- to form an acyl radical. Herein, acyl radicals are referred to by trivial names (terms of art, in common usage) or chemical names (either by full chemical name or by any shorthand commonly used in the art, which specifies the number of carbon atoms and unsaturations, e.g. "18:2" means "18 carbon atoms and 2 unsaturations") . The correspondence between the trivial names and the chemical names for several acyl radicals are shown below:

tetradecanoyl [myristoyl (14:0)],

9-cis-tetradecenoyl [myristoleoyl (14:1)],

hexadecanoyl [palmitoyl (16:0)],

9-cis-hexadecenoyl [palmitoleoyl (16:1)],

9-trans-hexadecenoyl (palmitoleoyl 16:1),

octadecanoyl [stearoyl (18:0)],

6-cis-octadecenoyl [petroselinoyl (18:1)],

9-cis-octadecenoyl [oleoyl (18:1)],

9-trans-octadecadienoyl [elaidoyl (18 : 1) ] ,

9-cis-12-cis-octadecadienoyl [linoleoyl (18:2)], 9-cis-12-cis-15-cis-octadecatrienoyl [linolenoyl (18 :3) ] ,

11-cis-eicosenoyl [eicosenoyl (20:1)],

5-cis-8-cis-ll-cis-14-cis-eicosatetraenoyl [arachidonoyl (20:4)],

13-cis-docosenoyl [erucoyl (22:1)], and

15-cis-tetracosenoyl [nervonyl (24:1)].

Note that the acyl radical component of a phospholipid may be referred to differently elsewhere in the art. For example, acyl radicals are sometimes referred to generically as "fatty acids" or "fatty acid chains", or specifically in terms of the acid from which the acyl radical is derived (e.g. a linoleoyl radical may be referred to in the lipid arts as the "linoeic acid" or "linoleic acid chain" component of a lipid) .

Herein, the "-PO₃R³" components of the phospholipid are sometimes collectively referred to as a "head group", consistent with terminology in use in the lipid arts.

When R³ is O or a Ci-_I0 hydroxycarbyloxy group, the phospholipid contains two acyl radicals and therefore can contain one or two linoleoyl radicals. When R³ is a phosphatidylglycerol radical (as defined above) , the phospholipid contains four acyl radicals and can therefore contain one, two, three or four linoleoyl radicals. Mention is made of the following preferred species of phospholipid for use in the invention:

• 1-palmitoyl -2 -linoleoyl-phosphatidylinositol ;

• 1-palmitoyl -2 -linoleoyl -phosphatidylcholine; • 1-oleoyl -2 -linoleoyl -phosphatidylcholine;

• dilinoleoyl -phosphatidylcholine;

• dilinoleoyl -phosphatidylinositol ;

• dilinoleoyl-phosphatidic acid;

• dilinoleoyl -phosphatidylethanolamine;

• dilinoleoyl -phosphatidylserine;

• 1 -stearoyl-2 -1inoleoyl -phosphatiydlcholine ; and

• tetralinoleoyl-diphosphatidylglycerol .

Phospholipids for use in the present invention can be naturally-occurring phospholipids that have been obtained from a natural source or prepared using Standard chemistry. Non-naturally occurring phospholipids of formula I can also be prepared by chemical synthesis, and these phospholipids can be used, or are even preferred in some cases, for practicing the present invention.

Phospholipids for use in the present invention can be derived from any plant source (such as edible oil seed) or from animal sources. Mention is made of soybean, safflower, sunflower and canola as suitable plant sources for linoleoyl-containing phosphatidylinositol and phosphatidylcholine. Mention is made of animal heart tissue as a suitable animal source of linoleoyl-containing DPG.

Linoleoyl-containing phospholipids and extracts enriched for linoleoyl-containing phospholipids may be prepared by methods known in the art. (See, for example, Aneja et al . , "A General Synthesis of Glycerophospholipids, " Biochim Biophys . Acta 1970; 218:102-111; Oro,J. "Chemical synthesis of lipids and the origin of life" (1995) Journal of Biological Physics, 20 (1-4), 135-147; Adlerereutz,D. , Budde,H., Wehtje,E. "Synthesis of phosphatidylcholine with defined fatty acid in the sn-1 position by lipase-catalyzed esterification and transesterification reaction" (2002) Biotechnology and Bioengineering, 78 (4), 403-411; Kim et al . "Phospholipase Al-catalyzed synthesis of phospholipids enriched in n - 3 polyunsaturated fatty acid residues" Enzyme and Microbial Technology 2007 40 (5) : 1130-1135.)

Table 1 (below) illustrates the acyl radical composition of different phospholipid species isolated from plant and animal tissues, where "PE" is phosphatidylethanolamine, "PA" is phosphatidic acid, and "PG" is phosphatidylglycerol .

Table 1: Acyl radical composition of several naturally occurring phospholipids

Acyl radical

16:0 18:0 20:0 16:1 18:1 18:2 18:3

M 20:1 20:2 20:3 20:4 22:6 Other

Notably :

• l-palmitoyl-2-linoleoyl-phosphatidylinositol is the predominant phosphatidylinositol species in soybean and can be extracted therefrom;

• l-palmitoyl-2-linoleoyl-phosphatidylcholine is the predominant phosphatidylcholine species in soybean and can be extracted therefrom; and

• tetralinoleoyl cardiolipin is the predominant cardiolipin species from bovine heart and can be extracted therefrom.

Phospholipids for use in the present invention can be purified or isolated or substantially pure. A compound is "substantially pure" when it is separated from the components that naturally accompany it. Typically, a compound is substantially pure when it is at least 60%, more generally at least 75%, 80%, 85%, 90% or 95%, by weight, of the total material in a sample. A substantially pure phospholipid can be obtained by extraction from a natural source or by chemical synthesis. A phospholipid that is chemically synthesized will generally be substantially free from its naturally associated components. Purity can be measured using any appropriate method such as HPLC, thin layer chromatography, mass spectrometry, etc.

However, it is not essential for a phospholipid to be purified prior to use in the present invention, provided that the phospholipid is not associated with components that interfere substantially with its utility. The skilled person will appreciate that a natural source or partially-purified source of a phospholipid can be used in the invention, and that the phospholipid component can constitute a small percentage (for example 10-20%, but preferably at least 30%, 40%, 50% or more) of the total material obtained from such a source.

B. Glycerides

Linoleate containing glycerides have also been demonstrated to enhance apo A-I secretion (see Example 5) .

Monolinoleoyl, dilinoleoyl and trilinoleoyl glycerides have exhibited this effect.

C. Therapeutic formulations

In embodiments of the present invention, linoleoyl -containing phospholipids or glycerides, or any combination thereof, are used as active agent in an amount effective for raising plasma levels of apo A-I in a mammal.

In embodiments, the phospholipid or glyceride is combined with an intestinal absorption enhancer (IAE) to improve bioavailability (for example as described in WO 2006/125304, herein incorporated by reference) . Examples of IAEs include: a bile acid or salt thereof; a surfactant or salt thereof; and a medium chain fatty acid or salt thereof. Mention is made of sodium lauryl sulfate.

In embodiments, the phospholipid or glyceride is used in combination therapy with one or more additional normo-lipidemic or anti -atherogenic agents, such as fibrates, niacin, ezetimibe, bile acid sequestrants, and statins (for example as described in USSN 11/434157, herein incorporated by reference) . Mention is made of the following statins: atorvastatin, lovastatin and mevinolin (US Patent No. 4231938), pravastatin sodium (US Patent No. 4346227), fluvastatin (US patent Nos . 4739073 and 5354772) , atorvastatin (US Patent No. 5273995) , itavastatin (European Patent No. 0304063) , mevastatin (US Patent No. 3983140) , rosuvastatin, velostatin, and synvinolin, and simvastatin (US Patent Nos . 4448784 and 4450171) , and their pharmaceutically acceptable salts and ester derivatives.

In embodiments, one or more PPAR-alpha agonists (e.g. linoleoyl -containing phospholipids) are used in combination therapy with one or more ABC transporter antagonists (e.g. glyburide) , as active agents, wherein the PPAR-alpha agonist and the ABC transporter antagonist are used in amounts that render the combination thereof effective for raising plasma levels of apo A-I in a mammal. Suitable PPAR-alpha agonists also include fibrates (see above) .

In many cases, oral administration will be the preferred route of administration of the formulations of the invention. However, alternative routes of administration (e.g. rectal, buccal, and intravenous administration) will be preferred in some cases.

Accordingly, the active agent (e.g. phospholipid) can be formulated by incorporating it into a pharmaceutical composition (for oral, rectal, intravenous or buccal administration) , or into a supplement (such as a nutritional supplement or neutraceutical) , a food product, a beverage, or the like, as known in the art. Such formulations can be used to raise plasma apo A-I levels, e.g. for preventing or treating dyslipidemia, atherosclerosis, CAD or related conditions .

A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as a clinically significant increase in plasma apo A-I levels or a reduction in CAD-related or atherosclerosis-related disease progression. A therapeutically effective amount of the active agent can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the particular active agent to elicit a desired response in the individual . Dosage regimens can be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects.

A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing or inhibiting the rate of CAD-related or atherosclerosis-related disease onset or progression. A prophylactically effective amount can be determined as described above for the therapeutically effective amount. For any particular subject, specific dosage regimens can be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions.

Thus, typically the active agent (e.g. phospholipid), or combination of active agents, is administered in an amount effective either to achieve improvement in at least one clinical sign and/or symptom of a disease caused at least in part by insufficient plasma levels of apo A-I and/or HDL-C (i.e. by raising plasma levels of apo A-I and/or HDL-C) or to delay onset of or progression of such signs or symptoms of disease. Cure is not required, nor is it required that the improvement or delay be achievable in a single dose.

In embodiments, treatment is sufficient to increase plasma apo A-I levels by at least 10% (or at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) of the initial plasma level of apo A-I for the patient. Plasma apo A-I levels can be measured using routine techniques.

The composition can be administered at regular intervals (e.g. daily, weekly, biweekly etc.) . For example, the composition can be administered daily for a period of at least two months (e.g. at least three to six months or for at least one, two, five, ten, twenty, twenty five or more years) . In some cases, such as treatment of stroke or ischemia, acute treatment can be beneficial and may require different dosages than daily or long-term treatment.

The safety and efficacy of new formulations of the invention can be tested using routine in vivo and in vitro techniques. For example, the new formulations can be tested (for their ability to modulate plasma apo A-I levels) in vivo in an animal model (such as rats, pigs, mice, primates, etc.) e.g. as described herein in the Examples below, followed by tests in humans. In vitro models (such as described herein and in e.g. Cano-Cebrian et al . Current Drug Delivery, 2005, Volume 2, pp.9-22) can also be useful for evaluating the safety and efficacy of new formulations of the invention. In vitro studies are convenient in some respects (low cost, high throughput) and therefore can be useful in initial screening, but safety and efficacy of candidate formulations are ultimately confirmed in vivo. Data obtained from in vitro assays and animal studies can be used in formulating a range of dosages for use in humans.

Typically, a unit dose of phospholipid or glyceride comprises between about 0.1 mg to about 300 mg of phospholipid per kg of body weight of the mammal being treated. Effective doses can vary according to a number of factors (see above) , and dosage regimens can be adjusted to provide the optimum therapeutic or prophylactic response. Doses will also vary according to the efficacy of the particular phospholipid or glyceride being administered.

For example, for a unit dose of phosphatidylinositol formulated for oral administration (without an intestinal absorption enhancer) can comprise between about 0.1 mg to about 300 mg, about 10 mg to about 200 mg, 100 mg to 150 mg, or about 120 mg of phosphatidylinositol per kg of body weight .

By combining phospholipid or glyceride with an effective IAE, either lower doses can be used or higher efficacy can be achieved for the same dose, when orally administered. For example, where phosphatidylinositol is combined with an intestinal absorption enhancer (IAE) , a unit dose can comprise between about 0.05 mg to about 100 mg, about 1 mg to about 50 mg, about 5 mg to about 20 mg, or about 15 mg phosphatidylinositol per kg of body weight.

By using a phospholipid or glyceride in combination with an ABC transporter inhibitor (e.g. glyburide) , either lower doses of phospholipid or glyceride can be used or higher efficacy can be achieved for the same dose, when orally administered. For example, where phosphatidylinositol is used in combination with an ABC transporter inhibitor (e.g. glyburide), a unit dose can comprise between about 0.05 mg to about 100 mg, about 1 mg to about 50 mg, about 5 mg to about 20 mg, or about 15 mg phosphatidylinositol per kg of body weight .

Pharmaceutical compositions of the present invention can include a pharmacologically acceptable excipient or carrier. As used herein "pharmaceutically acceptable carrier" or "excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible and suitable for oral administration. 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 pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The 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. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Moreover, the present formulations can be administered in a time release formulation, for example in a composition which includes a slow release polymer. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG) . Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.

Specific examples of orally administrable pharmaceutical compositions include dry-filled capsules consisting of gelatin, and also soft sealed capsules consisting of gelatin and a plasticizer, such as glycerol or sorbitol. The dry- filled capsules can contain the active ingredient in the form of granules, for example in admixture with fillers, such as lactose, binders, such as starches, and/or glidants, such as talc or magnesium stearate, and optionally stabilisers. In soft capsules, the active ingredient is preferably dissolved or suspended in suitable liquids, such as fatty oils, paraffin oil or liquid polyethylene glycols, to which stabilisers can also be added.

The invention also provides corresponding methods of medical treatment. For example, the invention provides methods of medical treatment in which a therapeutically effective amount of the phospholipid or glyceride is in a pharmacologically acceptable formulation for administering orally (or rectally or buccally) to a mammal subject in need thereof. Where the phospholipid or glyceride is used in combination with an IAE, an ABC transporter inhibitor (e.g. glyburide) , or with additional normo-lipidemic or anti -atherogenic agents, the combination can be administered simultaneously, or as separate dosages or dosage forms. In many cases, phospholipids/glycerides and IAEs will be in admixture for simultaneous administration, whereas phospholipids/glycerides and other anti-atherogenic or normo-lipidemic agents (such as statins) or an ABC transporter inhibitor (e.g. glyburide) will be formulated for administration separately. Such methods can include monitoring the subject (e.g. for plasma apo A-I levels or another sign or symptom of the condition or disorder) before, during or after treatment.

All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication, patent, or patent application is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

The present invention is further described in the following non- limiting examples.

EXAMPLES

Example 1 :

The effect of varying the head group and acyl radical components of phospholipids on ability to stimulate apo A-I secretion from cells in vitro in a hepatoma cell line, HepG2 (obtained from the American Tissue Type Culture collection (Rockville, MD) ) , was tested.

HepG2 cells were seeded in 12 well plates and grown to about 85% confluency in Dulbecco ' s Modified Eagle's Medium (DMEM) high glucose (obtained from Gibco ) containing 10% fetal bovine serum (obtained from Gibco ) , penicillin (100 U/ml

(obtained from Gibco) and streptomycin (100 μg/ml ; obtained from Gibco ) .

Phospholipids were obtained from Avanti Polar Lipids^" . Phospholipid vesicles were prepared by sonication as follows: Each test phospholipid in chloroform were dispensed into 12 x 75mm glass tubes using a Hamilton syringe and dried down thoroughly under nitrogen. One (1) ml of double distilled (d.d.) water was added to the tube, and the sample was then sonicated under nitrogen for 1 minute at constant duty cycle. The phospholipids were then incubated at 37⁰C for 30 minutes and then sonicated for an additional 5 minutes at 95% duty cycle. The preparations were filter sterilized through a 0.2 micron filter, assayed for phosphorus content to determine phospholipid concentration and then added to cells.

The experiments described below with various test compounds were carried out in high glucose DMEM without other additives. However, similar results were obtained in experiments carried out in regular DMEM (data not shown) .

Confluent monolayers were washed twice with high glucose DMEM and then incubated at 37⁰C in high glucose DMEM containing 11.7 nmoles of the test phospholipid in vesicle form in d.d. water, using d.d. water as a control.

The cells were then incubated with the test compounds for

24 hours at 37°C. The medium was removed from the cells and analyzed for apolipoprotein A-I content by sandwich ELISA using a monoclonal anti -human apo A-I antibody (obtained from Cedarlane , catalogue number H45402M) as the capture antibody and a horseradish peroxidase conjugated goat anti-human apo A-I antibody (obtained from Cedarlane , catalogue number A-1K45252P) as the detection antibody. The cell monolayer was solubilized in 0.2 N NaOH and then assayed for protein content using a bicinchoninic (BCA) assay (obtained from Pierce , catalog number 23227) . The results are expressed as fold increase for each test compound (phospholipid) when compared to the vehicle control, double distilled water.

A. Effects of varying the acyl radical composition of PI on apo A-I levels in cell media

Table 2 shows the effects of soy PI (derived from soybean; the predominant species is l-palmitoyl-2-linoleoyl- phosphatidylinositol) , bovine liver PI (a mixture of phospholipids, where the Cl position is occupied by 18:0 and the C2 position is occupied by 18:1 (14.5%), 18:2 (8.8%), 20:3 (9.2%) and 20:4 (13.4%)), and dioleoyl PI (which has two 18:1 acyl radicals) on apo A-I levels in cell media. Incubations with soy derived PI produced a noticeably greater increase in the levels of apo A-I in the HepG2 extracellular medium as compared to bovine PI and dioleoyl PI.

Table 2. Effect of various species of PI on apo A-I levels in cell media

B. Effect of various soy-derived phospholipids on apo A-I levels in cell media

Table 3 shows the effects of various soy derived phospholipids (PI, PA, PC, PS, and PE) on apo A-I levels in cell media. In each of the phospholipids, the predominant acyl group at the C2 position of the glycerol backbone is linoleoyl (an 18:2 acyl radical) . The acyl group at the Cl position varies.

Soy PI and soy PC increased the levels of apo A-I in cell media by 1.8 fold and 2.3 fold respectively. Incubations with soy PA, soy PS and soy PE resulted in smaller increases in the levels of apo A-I in the cell medium.

Table 3. Effect of soy-derived phospholipids on apo A-I levels in cell media

C. Effects of varying the acyl radical component of PC on ability to increase apo A-I levels in cell media

Table 4 compares the effects of soy PC (which predominantly has linoleoyl at the C2 position; the acyl at the Cl position varies) and several synthetic PCs with differing acyl radical compositions, namely:

• dioleoyl-PC (DOPC, which has two 18:1 acyl radicals);

• palmitoyl-oleoyl-PC (POPC, which has one 16:0 acyl radical and one 18:1 acyl radical);

• dilinoleoyl-PC (DLPC, which has two 18:2 acyl radicals);

• dilinolenoyl-PC (DLnPC, which has two 18:3 acyl radicals)

• palmitoyl-linoleoyl-PC (PLPC, which has an 16:0 acyl radical and a 18:2 acyl radical);

• dimyristoyl-PC (DMPC, which has two 14:0 acyl radicals); and

• diarachidonyl-PC (DAPC, which has two 20:4 acyl radicals.

Soy PC, DLPC, and PLPC, all of which contain linoleoyl acyl radicals, produced substantial increases in apo A-I levels in cell media as compared to d.d. water controls (vehicle) . DLnPC (which has two 18:3 acyl radicals) and DAPC (20:4, 20:4) produced smaller increases in cell medium apo A-I levels. DOPC (18:1, 18:1), POPC (16:0, 18:1), and DMPC (14:0, 14:0) did not produce any increase in apo A-I levels in cell media. Table 4 Effect of varying the acyl radical composition of phosphatidylcholine on ability to increase apo A-I levels in cell media

D. Linoleoyl-rich cardiolipin stimulates apo A-I secretion in cells

Table 5 compares the effects of bovine heart cardiolipin and synthetic tetraoleoyl cardiolipin on apo A-I levels in HepG2 cell medium. Heart cardiolipin has 4 acyl radicals per molecule that contain 87% linoleoyl (18:2) acyl radicals and therefore, the predominant species is tetralinoleoyl . Heart cardiolipin increased apo A-I levels in the cell medium by about 3 fold versus d.d. water (vehicle) control. In contrast, tetraoleoyl cardiolipin (which has mostly or entirely 18:1 acyl radicals), was without effect. Table 5. Effect of heart cardiolipin and synthetic tetraoleoyl cardiolipin on apo A-I levels cell media of HepG2 cells.

Test compound Apo A- I level in cell medium (fold increase VS . control)

d.d. water (control) 1.00 ± 0 05

Heart cardiolipin 2.97 ± 0 17

Tetraoleoyl cardiolipin 0.88 ± 0 05

Example 2 :

The effects of free linoleic acid (LA) , soy PI and DLPC on apo A-I secretion were compared. This experiment was carried out as described above in Example 1, with the exception that phospholipids or pure linoleic acid were solubilized in dimethylsulfoxide (DMSO) and then administered to cells. The final DMSO concentration was 1% in the tissue culture medium.

The results of this experiment are shown in Table 6. Free linoleic acid has no effect on apo A-I secretion.

Table 6. Effects of linoleic acid and linoleoyl -containing phospholipids on medium apo A-I levels in HepG2 cells

Test compound Apo A- I level in cell medium (fold increase vs. control)

DMSO 1.00 + 0.07

11.7 nmoles linoleic acid 1.09 + 0.07

11.7 nmoles soy PI 2.07 + 0.19

23.4 nmoles linoleic acid 0.94 + 0.12

11.7 nmoles DLPC 4.32 + 0.65

Thus, the phospholipids appear to be more effective when administered to cells in the form of micelles in admixture with DMSO as compared to vesicles in d.d. water. This difference in efficacy may be due to an increase in stability of micelles in DMSO versus vesicles in d.d. water.

Example 3 :

The effects of equimolar amounts of DLPC (dilinoleoyl- phosphatidylcholine) and several other synthetic dilinoleoyl (2x 18:2) phospholipids on apo A-I secretion were compared. The results of this experiment are shown in Table 7.

DLPA (phosphatidic acid) increased media apo A-I by 1.3 -fold, DLPE (phosphatidylethanolamine) by 1.5 fold, and DLPS (phosphatidylserine) by 1.4-fold. DLPG

(phosphatidylglycerol) did not increase media apo A-I levels . Table 7: Effect of other lineolate containing phospholipids on apo A-I levels

Example 4 :

The effects of free linoleic acid (LA) and linoleic acid enriched phospholipids were compared. The phospholipids tested were soy phosphatidylinositol (PI) and dilinoleoylposphatidylcholine (DLPC) . HepG2 cells were incubated with various pure lipids. Apo A-I secretion was measured. Aqueous vesicular mixtures of PI and DLPC and/or linoleic acid (LA) (12μM) were added to the cells and incubated for 24 h. Apo A-I was quantified in the media by ELISA.

The results are depicted in Figure 8, wherein apo A-I secretion is presented relative to total cell protein values and expressed as mean + SEM of at least 4 independent experiments. These results demonstrate that free linoleic acid augments the effect of the linoleic acid enriched phospholipids in increasing apo A-I secretion in hepatic cells. When linoleic acid enriched phospholipid stimulated HepG2 cells were pretreated with linoleic acid (24μM) , the inductions in apo A-I secretion by PI and DPLC were augmented (i.e. there was a synergistic effect) by about 50% and 25% respectively.

Since unsaturated fatty acids, such as linoleic acid, are known inhibitors of ABC transporter expression, the effect of PI and DPLC were also tested on ABCAl and ABCGl protein expression in HepG2 cells. Hep2G cells were incubated with phosphatidylinositol (PI) , dilinoleoylphosphatidylcholine (DLPC) (12μM) or phosphate buffered saline (PBS) as a control for 24 h. ABC transporter expression and β-actin were analyzed by Western blot. Figure 9 represents a densitometry analysis of ABCAl and ABCGl, relative to total β-actin control. Values are expressed as mean ±SEM of at least 4 independent experiments .

As shown in Figures 9A and 9B, PI and DPLC reduced hepatic ABCAl and ABCGl protein expression. PI reduced ABCAl and ABCGl protein levels by 50% and 74% respectively. DLPC reduced ABCAl and ABCGl protein levels by 32% and 55% respectively. These results establish that PI and DLPC inhibit ABCAl and ABCGl expression in HepG2 cells.

Example 5

The effects of DLPC (in PBS) and other linoleate containing compounds, all solubilized in DMSO, on apo A-I secretion have been compared. The results of this experiment are shown in Table 8.

The assay shows that equimolar amounts of ethyl linoleate, methyl linoleate, cholesteryl linoleate and monolinoleoyl- glycerol (24 μM) have minimal effect on apo A-I secretion. DLPC (12 μM) , dilinoleoyl -glycerol (12 μM) and glycerol trilinoleate (8 μM) all increase apo A-I secretion from HepG2 cells by 2.1 , 1.3 and 1.2 -fold, respectively. This demonstrates that lineolate containing mono-, di-, and tri-glycerides also affect apo A-I secretion.

Table 8: Effect of linoleate containing compounds on medium apo A-I levels in HepG2 cell media.

Media Addition Medium apo A-I (fold over control)

DMSO 1.00 ± 0.07

DLPC 2.10 + 0.40

Ethyl linoleate 1.00 ± 0.12

Methyl linoleate 1.05 ± 0.14

Cholesteryl linoleate 0.97 + 0.12

Monolinoleoyl -rac-glycerol 1.11 ± 0.14

Dilinoleoyl-rac-glycerol 1.33 + 0.17

Glyceryl Trilinoleate 1.21 + 0.17

Example 6 :

Various compounds were tested for their effects on apolipoprotein A-I (apo A-I) secretion, PPAR-alpha expression, ERKl/2 phosphorylation and/or ABC transporter expression in vitro, as described below.

The experiments were carried out in vitro, using cultured hepatoma cells (HepG2 ; obtained from the American Tissue Type Culture collection, Rockville, MD) , seeded in 6 or 12 well plates and grown to about 85% confluence in high glucose DMEM (purchased from Gibco) containing 10% fetal bovine serum (Gibco), and penicillin (100 U/tnl; purchased from Gibco) and streptomycin (100 μg/ml ; purchased from Gibco) . Soy phosphatidylinositol (PI) and synthetic dilinoleoylphosphatidylcholine (DLPC) were purchased from Avanti Polar Lipids.

MK886, a PPAR-alpha antagonist, was purchased from Cayman Chemical Co., Ann Arbor, Michigan, USA. GW9662, a

PPAR-gamma antagonist, was purchased from Cayman Chemical Co., Ann Arbor, Michigan, USA. Clofibrate, a PPAR-alpha agonist, was purchased from Cayman Chemical Co., Ann Arbor, Michigan, USA. Glyburide (N-p- [2- (5-Chloro- 2-methoxybenzamido) ethyl] benzenesulfonyl-N' -cyclohexylurea) , a potent inhibitor of ABCAl transporter expression, was purchased from Sigma Chemical. Insulin was purchased from Sigma Chemical.

Experiments with control and test compounds were carried out in high glucose DMEM without other additives. For PI experiments, ddH₂O was a vehicle control and when noted, it was DMSO (maximum 1% at final concentration) . Cells were grown to desired confluence and serum-starved quiescent cells were treated with or without the drugs of interest as indicated and then incubated at 37°C in DMEM containing test phospholipid, PI or DLPC in vesicle form for required time and concentration as shown.

Phospholipid vesicles were prepared by sonication as follows. Phospholipids in chloroform were dispensed into 12 x 75mm glass tubes using a Hamilton syringe and dried down thoroughly under nitrogen and 1 ml of double distilled water (ddH₂O) was added and the sample was then sonicated under nitrogen for 1 minute at constant duty cycle. The phospholipids were then incubated at 37°C for 30 minutes and then sonicated for an additional 5 minutes at 95% duty cycle. The preparations were filter sterilized through a 0.2 micron filter, assayed for phosphorus content to determine phospholipid concentration and then added to cells .

To measure apolipoprotein A-I (apo A-I) secretion following the incubation period, equal amounts of cell media were removed from the cells and analyzed for apo A-I content by sandwich ELISA using a monoclonal anti-human apo A-I antibody (purchased from Biodesign International, Saco, ME, catalogue number H45402M) as the capture antibody and a horseradish peroxidase conjugated goat anti -human apo A-I antibody (purchased from Biodesign International, Saco, ME catalogue number K45252P) as the detection antibody. The cell monolayer was solubilized in 0.2 N NaOH and then assayed for total cell protein using a bicinchoninic (BCA) assay (purchased from Pierce, catalog number 23227) . The amount of apo A-I measured was first normalized against total cell protein, and then compared to vehicle control, so that the measure of apo A-I secretion is expressed as fold increase versus the vehicle control in the experiments described below. Means ± standard deviation were calculated for at least four independent experiments.

For the Western blot analysis experiments, HepG2 cells were grown to desired confluence and serum-starved quiescent cells were treated with or without drugs for the indicated time and concentration in presence or absence of phospholipids. Cell lysates were prepared using lysis buffer [NaF 1 mmol/L, NaCl 5 mmol/L, EDTA 1 mmol/L, NP40 1 mmol/L (Roche Diagnostics, Indianapolis, IN), HEPES 10 mmol/L, pepstatin A 1 mg/mL, leupeptin 1 mg/mL, aprotinin 1 mg/mL, Na3VO4 1 mmol/L, PMSF 1 mmol/L] obtained from Sigma Chemical Co., and equal amount of protein (25μg) was separated for each sample on 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and then transferred on nitrocellulose membranes. Specific protein expression was detected by immunoblot analysis using specific primary and secondary antibody as required and indicated below. Data were quantified by densitometric scanning of immunoblots and expressed as a percentile increase above vehicle control (Ctrl) . The results presented are the means ± SE of at least four independent experiments .

Experiment A: PPAR-alpha inhibition down-regulates PI- and DLPC-induced secretion of apo A-I from HepG2 cells into culture media

HepG2 cells were grown to 80% confluence, and serum-starved quiescent cells were treated for 24 hours with:

(A) DMSO control; 10 mg/ml PI; 10 micromolar MK886; 10 micromolar GW9662; 10 mg/ml PI and 10 micromolar MK886; or a combination of 10 mg/ml PI and 10 micromolar GW9662; or

(B) DMSO control; 10 mg/ml DLCP; 10 micromolar MK886;

10 micromolar GW9662; 10 mg/ml DLCP and 10 micromolar MK886; or combination of 10 mg/ml DLCP and 10 micromolar GW9662.

At the end of the incubation time, the cell media were analyzed to measure secreted apo A-I as described above. The results are shown in Figures IA and IB (values are means ± standard deviation of at least four independent experiments) .

In these experiments, MK886 was found to reduce PI- and DLPC-induced apo A-I secretion, whereas GW9662 had little effect on PI- and DLPC-induced apo A-I secretion as compared to controls. Experiment B: Phosphatidylinositol increases PPAR-alpha expression in Hepatic Cells

HepG2 cells were grown to 80% confluence and serum-starved quiescent cells were incubated for 24 hours with: vehicle control; 10 micromolar clofibrate (CIo); or 1 microgram/ml (1.2 micromolar), 5 microgram (6 micromolar), or 10 microgram (12 micromolar) soy PI. Cell lysates were prepared and equal amounts [25 microgram] of protein were separated for each sample on 12% SDS-PAGE then transferred on nitrocellulose membranes. PPAR-alpha expression was detected by immunoblot analysis using a PPAR-alpha specific antibody (purchased from Santa Cruz Biotechnology, CA, USA, catalogue number scl985) , as described above. Data were quantified by densitometric scanning of immunoblots and expressed as a % increase above vehicle control (Ctrl) , as described above. The mean ± standard deviations of at least four independent experiments were calculated. Results are shown in Figure 2.

In this experiment, clofibrate and PI were both found to increase PPAR-alpha expression as compared to vehicle control .

Experiment C: PI induces ERKl/2 phosphorylation in a time- dependent manner in HepG2 cells

HepG2 cells were grown to 80% confluence and serum-starved quiescent cells were incubated with: control [no treatment] ; insulin [100 nano molar for 5 min.] ; or 10 mg/ml PI for 5 minutes, 10 minutes, 15 minutes, 30 minutes, or 60 minutes. Cell lysates were prepared and samples of equal amounts of protein were separated on 12% SDS-PAGE. ERKl/2 phosphorylation was detected by immunoblot analysis using an antibody specific for ERKl/2 phosphorylated at p4l/p42 (purchased from Cell Signalling Technology, Danvers, MA, USA, catalogue number 9101) . Data were quantified by densitometric scanning of immunoblots and expressed as a % increase as compared to vehicle control . The results are shown in Figure 3.

In this experiment, PI was found to induce ERKl/2 phosphorylation in a time-dependent manner. Insulin at 100 nano molar concentrations increased ERKl/2 phosphorylation fully at 5 min and then declined for 10-15 min. and reappeared at 30 minute for sustained activation up to 2h in CHO, HepG2 and 3T3L1. An almost similar pattern has been shown in regard to PKB/Akt phosphorylation in vitro.

Experiment D :

HepG2 cells were grown to 80% confluence and serum-starved quiescent cells were incubated for 24 hours with:

(A) DMSO (control); 10 mg/ml PI; 50 micromolar glyburide; or a combination of 10 mg/ml PI and 50 micromolar glyburide; or

(B) DMSO (control) ; 10 mg/ml DLPC; 50 micromolar glyburide; or a combination of 10 mg/ml DLPC and 50 micromolar glyburide .

At the end of the incubation time, the cell media were analyzed to measure secreted apo A-I as described above. The results (means ± standard deviation) of at least four independent experiments are shown in Figures 4A and 4B.

In these experiments, glyburide demonstrated a synergistic effect on PI- and DLPC-induced apo A-I secretion. Experiment E :

HepG2 cells were grown to 80% confluence and serum-starved quiescent cells were incubated for 24 hours with: control [no treatment]; 10 mg/ml PI; or 10 μM 9-cis-RA [9-cis- retinoic acid] , [this retinoid X receptor (RXR) inducer is used to increase ABC transporter expression] . Cell lysates were prepared and samples of equal amounts of protein were separated on 12% SDS-PAGE. ABCGl expression was detected by immunoblot analysis using an antibody specific for ABCGl [SantaCruz Biotech, SanDiego, CA, catalogue number sc-11150] . Data were quantified by densitometric scanning of immunoblots and expressed as a % increase as compared to actin control. The results are shown in Figure 5 (values are mean ± standard deviation for at least three independent experiments) .

In this experiment, PI downregulated expression of ABCGl.

Experiment F :

HepG2 cells were grown to 80% confluence and serum-starved quiescent cells were incubated for 24 hours with: DMSO (control); 25 micromolar clofibrate; 50 micromolar glyburide; or a combination of 25 micromolar clofibrate and 50 micromolar glyburide. At the end of the incubation time, the cell media were analyzed to measure secreted apo A-I as described above. The results (means ± standard deviation of four independent experiments are shown in Figure 6.

In these experiments, clofibrate alone did not produce a significant change in apo A-I secretion. However, clofibrate and glyburide had a synergistic effect on apo A-I secretion when used in combination.

Claims

CLAIMS :

1. A method for raising the plasma level of apolipoprotein A-I in a mammal, the method comprising administering an effective amount of a phospholipid or a salt thereof, wherein said phospholipid is:

(a) phosphatidic acid;

(b) phosphatidylinositol;

(c) phosphatidylcholine;

(d) phosphatidylserine;

(e) phosphatidylethanolamine; or

(f) diphosphatidylglycerol;

and wherein said phospholipid comprises at least one linoleoyl radical.

2. The method of claim 1, wherein said phospholipid is phosphatidylinositol.

3. The method of claim 2, wherein said phosphatidylinositol comprises one linoleoyl radical and one palmitoyl radical .

4. The method of claim 2, wherein said phosphatidylinositol comprises two linoleoyl radicals.

5. The method of claim 1, wherein said phospholipid is phosphatidylcholine.

6. The method of claim 5, wherein said phosphatidylcholine comprises one linoleoyl radical and one palmitoyl radical.

7. The method of claim 5, wherein said phosphatidylcholine comprises two linoleoyl radicals.

8. The method of claim 2 or claim 5, wherein said phosphatidylinositol is purified from a plant source.

9. The method of claim 8, wherein said plant is soy.

10. The method of claim 1, wherein said phospholipid is diphosphatidylglycerol .

11. The method of claim 10, wherein said diphosphatidylglycerol comprises at least two linoleoyl radicals.

12. The method of claim 10, wherein said diphosphatidylglycerol comprises four linoleoyl radicals.

13. The method of claim 10, wherein said diphosphatidylglycerol is purified from bovine heart.

14. The method of claim 1 wherein said phospholipid is phosphatidic acid.

15. The method of claim 14, wherein said phosphatidic acid comprises two linoleoyl radicals.

16. The method of claim 1 wherein said phospholipid is phosphatidylserine .

17. The method of claim 16, wherein said phosphatidylserine comprises two linoleoyl radicals.

18. The method of claim 1 wherein said phospholipid is phosphatidylethanolamine .

19. The method of claim 18, wherein said phosphatidylethanolamine comprises two linoleoyl radicals.

20. The method of any one of claims 1 to 19, wherein said phospholipid enhances secretion of apolipoprotein A-I from cells of said mammal .

21. The method of any one of claims 1 to 20, wherein said phospholipid is administered in a daily dose of about

0.1 mg to about 300 mg of said phospholipid per kg of body weight of said mammal .

22. The method of any one of claims 1 to 20, wherein said phospholipid is administered orally in a daily dose of about 0.05 mg to about 100 mg of said phospholipid per kg of body weight of said mammal, and wherein said phospholipid is in admixture with an intestinal absorption enhancer.

23. The method of claim 22, wherein said intestinal absorption enhancer is selected from the group consisting of: a bile acid or salt thereof; a surfactant or salt thereof; and a medium chain fatty acid or salt thereof.

24. The method of claim 23, wherein said intestinal absorption enhancer is sodium lauryl sulfate.

25. The method of any one of claims 22 to 24, which comprises per unit dose: about 0.1 mg to about 100 mg of said phospholipid; and about 0.05 mg to about 100 mg of said intestinal absorption enhancer, per kg of body weight of said mammal .

26. The method of any one of claims 1 to 25, which is for treating or preventing dyslipidemia or atheroscelorosis in said mammal .

27. The method of claim 26, wherein said phospholipid is used in combination with one or more additional normo-lipidemic or anti -atherogenic agents.

28. The method of claim 27, wherein said normo-lipidemic agent is a statin.

29. The method of any one of claims 1 to 28, wherein said phospholipid has a linoleoyl radical at the C2 position.

30. A pharmaceutical composition for use in raising the plasma level of apolipoprotein A-I in a mammal, wherein said pharmaceutical composition comprises a phospholipid or a salt thereof, wherein said phospholipid is:

(a) phosphatidic acid;

(b) phosphatidylinositol;

(c) phosphatidylcholine;

(d) phosphatidylserine;

(e) phosphatidylethanolamine; or

(f) diphosphatidylglycerol;

and wherein said phospholipid comprises at least one linoleoyl radical.

31. Use of a phospholipid, or a salt thereof, in the preparation of a medicament for raising the plasma level of apolipoprotein A-I in a mammal, wherein said phospholipid is :

(a) phosphatidic acid;

(b) phosphatidylinositol;

(c) phosphatidylcholine;

(d) phosphatidylserine; (e) phosphatidylethanolamine; or

(f) diphosphatidylglycerol;

and wherein said phospholipid comprises at least one linoleoyl radical.

32. Use of a phospholipid, or a salt thereof, for raising the plasma level of apolipoprotein A-I in a mammal, wherein said phospholipid is:

(a) phosphatidic acid;

(b) phosphatidylinositol;

(c) phosphatidylcholine;

(d) phosphatidylserine;

(e) phosphatidylethanolamine; or

(f) diphosphatidylglycerol ;

and wherein said phospholipid comprises at least one linoleoyl radical.

33. A commercial package comprising a phospholipid, or a salt thereof, together with instructions for use in raising the plasma level of apolipoprotein A-I in a mammal, wherein said phospholipid is:

(a) phosphatidic acid;

(b) phosphatidylinositol;

(c) phosphatidylcholine;

(d) phosphatidylserine;

(e) phosphatidylethanolamine; or (f) diphosphatidylglycerol ;

and wherein said phospholipid comprises at least one linoleoyl radical.

34. A combination for raising plasma levels of apolipoprotein A-I (apo A-I) in a mammal, comprising:

(c) a PPAR-alpha agonist; and

(d) an ABC transporter antagonist;

wherein the PPAR-alpha agonist and the ABC transporter antagonist are present in amounts that render the combination thereof effective for raising plasma levels of apo A-I in said mammal, and wherein the PPAR-alpha agonist and the ABC transporter antagonist are formulated for simultaneous or sequential administration.

35. The combination of claim 34 wherein the PPAR-alpha agonist is a phospholipid or a salt thereof, wherein said phospholipid is:

(a) phosphatidic acid;

(b) phosphatidylinositol ;

(c) phosphatidylcholine;

(d) phosphatidylserine;

(e) phosphatidylethanolamine; or

(f) diphosphatidylglycerol;

and wherein said phospholipid comprises at least one linoleoyl radical.

36. The combination of claim 35 wherein said phospholipid is phosphatidylinositol .

37. The combination of claim 36, wherein said phosphatidylinositol comprises one linoleoyl radical and one palmitoyl radical.

38. The combination of claim 36, wherein said phosphatidylinositol comprises two linoleoyl radicals.

39. The combination of claim 35, wherein said phospholipid is phosphatidylcholine.

40. The combination of claim 39, wherein said phosphatidylcholine comprises one linoleoyl radical and one palmitoyl radical .

41. The combination of claim 39, wherein said phosphatidylcholine comprises two linoleoyl radicals.

42. The combination of claim 36 or claim 39, wherein said phosphatidylcholine is purified from a plant source.

43. The combination of claim 42, wherein said plant is soy.

44. The combination of claim 35, wherein said phospholipid is diphosphatidylglycerol .

45. The combination of claim 44, wherein said diphosphatidylglycerol comprises at least two linoleoyl radicals .

46. The combination of claim 44, wherein said diphosphatidylglycerol comprises four linoleoyl radicals.

47. The combination of claim 44, wherein said diphosphatidylglycerol is purified from bovine heart.

48. The combination of claim 35 wherein said phospholipid is phosphatidic acid.

49. The combination of claim 48, wherein said phosphatidic acid comprises two linoleoyl radicals.

50. The combination of claim 35 wherein said phospholipid is phosphatidylserine .

51. The combination of claim 50, wherein said phosphatidylserine comprises two linoleoyl radicals.

52. The combination of claim 35 wherein said phospholipid is phosphatidylethanolamine .

53. The combination of claim 52, wherein said phosphatidylethanolamine comprises two linoleoyl radicals.

54. The combination of any one of claims 35 to 53, wherein said phospholipid enhances secretion of apolipoprotein A-I from cells of said mammal.

55. The combination of any one of claims 35 to 54, wherein said phospholipid is administered in a daily dose of about 0.1 mg to about 300 mg of said phospholipid per kg of body weight of said mammal.

56. The combination of any one of claims 35 to 54, wherein said phospholipid is administered orally in a daily dose of about 0.05 mg to about 100 mg of said phospholipid per kg of body weight of said mammal, and wherein said phospholipid is in admixture with an intestinal absorption enhancer.

57. The combination of claim 56, wherein said intestinal absorption enhancer is selected from the group consisting of: a bile acid or salt thereof; a surfactant or salt thereof; and a medium chain fatty acid or salt thereof.

58. The combination of claim 57, wherein said intestinal absorption enhancer is sodium lauryl sulfate.

59. The combination of any one of claims 56 to 58, which comprises per unit dose: about 0.1 mg to about 100 mg of said phospholipid; and about 0.05 mg to about 100 mg of said intestinal absorption enhancer, per kg of body weight of said mammal .

60. The combination of any one of claims 35 to 59, wherein said ABC transporter antagonist is glyburide .

61. The combination of any one of claims 35 to 60, wherein said PPAR-alpha agonist and said ABC transporter antagonist are in admixture and formulated in a single dosage unit form, for administration simultaneously.

62. The combination of any one of claims 35 to 60, wherein said PPAR-alpha agonist and said ABC transporter antagonist are formulated in divided dosages, for administration separately.

63. The combination of any one of claims 35 to 62, wherein said PPAR-alpha agonist and said ABC transporter antagonist are formulated for oral administration.

64. The combination of any one of claims 35 to 63, which is for treating or preventing dyslipidemia or atheroscelorosis in said mammal.

65. A method for raising the plasma level of apolipoprotein A-I in a mammal, comprising administering the combination of any one of claims 35 to 64 to said mammal.

66. Use of the combination of any one of claims 35 to 64 in the preparation of a medicament for raising the plasma level of apolipoprotein A-I in a mammal.

67. Use of the combination of any one of claims 35 to 64 for raising the plasma level of apolipoprotein A-I in a mammal .

68. A commercial package comprising the combination of any one of claims 35 to 64, together with instructions for use in raising the plasma level of apolipoprotein A-I in a mammal.