HK1115823B - Charged lipoprotein complexes and their uses - Google Patents

Abstract

The present disclosure provides charged lipoprotein complexes that include as one component a negatively charged phospholipid that is expected to impart the complexes with improved therapeutic properties.

Description

Charged lipoprotein complexes and uses thereof

1. CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application No. 60/665,180 filed 3/24/2005, as 35 u.s.c. § 119(e), which is incorporated herein by reference in its entirety.

2. Field of the invention

The present disclosure provides charged lipoprotein complexes, pharmaceutical compositions comprising the complexes, and methods of using the complexes to treat or prevent a variety of conditions and disorders, including dyslipidemia and/or diseases, disorders, and/or conditions associated therewith.

3. Background of the invention

Circulating cholesterol is transported via plasma lipoprotein complex particles composed of lipids and proteins in the blood that transport lipids. Lipoprotein particles circulating in plasma have the following four major classes and are all included in the fat transport system: chylomicrons, Very Low Density Lipoproteins (VLDL), Low Density Lipoproteins (LDL), and High Density Lipoproteins (HDL). Chylomicrons constitute a short-lived product of intestinal fat absorption. VLDL and especially LDL are responsible for the delivery of cholesterol from the liver (where it is synthesized or obtained from dietary sources) to extrahepatic tissues, including the arterial wall. In contrast, HDL mediates the reverse transport of cholesterol (RCT) and removal of cholesterol lipids, particularly from extrahepatic tissues to the liver where it is stored, metabolized, eliminated or recycled. HDL also plays a critical role in inflammation, transport of oxidized lipids and interleukins.

Lipoprotein particles have a hydrophobic core comprising cholesterol (typically in the form of cholesterol esters) and triglycerides. The core is surrounded by a surface layer comprising phospholipids, unesterified cholesterol and apolipoproteins. Apolipoproteins mediate lipid transport, and a portion of apolipoproteins may interact with enzymes involved in lipid metabolism. At least ten apolipoproteins have been identified, including: ApoA-I, ApoA-II, ApoA-IV, ApoA-V, ApoB, ApoC-I, ApoC-II, ApoC-III, ApoD, ApoE, ApoJ and ApoH. Other proteins associated with lipoproteins, such as LCAT (lecithin: cholesterol acyltransferase), CETP (cholesteryl ester transfer protein), PLTP (phospholipid transfer protein) and PON (paraoxonase), have also been found.

Cardiovascular diseases, such as coronary heart disease, coronary artery disease and atherosclerosis, are associated to a large extent with elevated cholesterol concentrations in serum. For example, atherosclerosis is a slowly progressive disease characterized by the accumulation of cholesterol within the arterial wall. Compelling evidence that demonstrates the theoretical deposition of lipids in atherosclerotic lesions stems primarily from plasma LDL; thus, LDL has been generally considered as "harmful" cholesterol, in contrast, HDL serum concentration is inversely proportional to coronary heart disease. Indeed, higher serum concentrations of HDL are considered to be a negative risk factor. It is hypothesized that higher concentrations of plasma HDL not only protect against coronary artery disease, but may actually induce regression of atherosclerotic plaques (see, e.g., Badimon et al, 1992, Circulation86(suppl. III): 86-94; Dansky and Fisher, 1999, Circulation 100: 1762-63; Tangirala et al, 1999, Circulation100 (17): 1816-22; Fan et al, 1999, Atherosclerosis 147 (1): 139-45; Decert et al, 1999, Circulation100 (11): 1230-35; Boisvert et al, 1999, arioscler.Thromb. Vaval.19 (3): 1994-30; Benoit et al, 1999, Circulation 99 (1): 105-10; Hoeos et al, 1998, Clostrich.J.12.19 (3): 18812; 1994-32; Valsilicone et al, 1995-11; Valsilicone et al, 94; 1995-11: 2, 1995-32; Ostre et al; Ostre. 11: 11; 1995-35; 1995, Ostre. J.11; 1995, 1995-11; 1995, Ostre et al; 1995, Ostre. 11; 1995, 2. J.11; 1995, 2. J. 12; 1995, 2. J.),32; 2. J., Ostremul. J.),32, 1994, proc.nat.acad.sci.usa 91 (20): 9607-11; paszty et al, 1994, j.clin.invest.94 (2): 899-903; she et al, 1992, chi. med.j. (Engl) 105 (5): 369-73; rubin et al, 1991, Nature 353 (6341): 265 to 67; she et al, 1990, ann.ny acad.sci.598: 339-51; ran, 1989, Chung Hua Ping Li Hsueh Tsa Chih (also translated as: Zhonghua Bing Lixue Za Zhi)18 (4): 257-61; quezado et al, 1995, J.Pharmacol.exp.Ther.272 (2): 604-11; duverger et al, 1996, ariioscler.thromb.vase.biol.16 (12): 1424-29; kopfter et al, 1994, Circulation; 90(3): 1319-27; miller et al, 1985, Nature 314 (6006): 109-11; ha et al, 1992, biochim. biophysis. acta1125 (2): 223-29; beitz et al, 1992, Prostagladins Leukot. essence. Fatty Acids47 (2): 149-52) HDL has been generally considered to be "favorable" cholesterol as a conclusion (see, e.g., Zhang, et al, 2003 Circulation 108: 661-663).

The "protective" effect of HDL has been demonstrated in a number of studies (e.g., Miller et al, 1977, Lancet 1 (8019): 965-68; Whayne et al, 1981, Atherosclerosis 39: 411-19). In these studies, elevated concentrations of LDL appear to be associated with increased cardiovascular risk, while higher HDL concentrations appear to have cardiovascular protective effects. In vivo studies have further demonstrated the protective effects of HDL, which suggests that HDL injected into rabbits may block the formation of cholesterol-induced arterial lesions (Badimon et al, 1989, Lab. invest.60: 455-61) and/or may induce regression of the arterial lesions (Badimon et al, 1990, J. Clin. invest.85: 1234-41).

3.1 reverse cholesterol transport, HDL and Apolipoprotein A-I

The Reverse Cholesterol Transport (RCT) pathway functions to eliminate cholesterol from most tissues outside the liver and is critical to maintaining the structure and function of most cells in the body. RCT consists essentially of three steps: (a) cholesterol efflux, i.e., the initial removal of cholesterol by a distinct peripheral cell pool; (b) cholesterol is esterified via lecithin: cholesterol Acyltransferase (LCAT) to prevent the efflux of cholesterol from re-entering the cell; and (c) uptake of HDL cholesterol and cholesterol esters into hepatocytes for hydrolysis, followed by recirculation, storage, secretion or metabolism into bile acids.

LCAT (a key enzyme in RCT) is produced by the liver and circulates in plasma associated with HDL components. LCAT converts cell-derived cholesterol into cholesterol esters which are sequestered in the HDL for which removal is indicated (see Jonas 2000, biochim. biophysis. acta1529 (1-3): 245-56). Cholesteryl Ester Transfer Protein (CETP) and phospholipid transfer protein (PLTP) are devoted to the further engineering of the circulating HDL population. CETP transports cholesteryl esters produced by LCAT to other lipoproteins, particularly ApoB-containing lipoproteins such as VLDL and LDL. PLTP supplies lecithin to HDL. HDL triglycerides are metabolized by extracellular hepatic triglyceride lipase, and lipoprotein cholesterol is removed in the liver via several mechanisms.

The functional characteristics of HDL particles are determined primarily by their major apolipoprotein components, such as ApoA-I and ApoA-II. Also observed are traces of ApoC-I, ApoC-II, ApoC-III, ApoD, ApoA-IV, ApoE, ApoJ associated with HDL. HDL exists in a variety of different sizes and mixtures of the above components depending on the remodeling state of the RCT metabolic cascade or pathway processes.

Each HDL particle typically comprises at least 1 molecule, and typically 2 to 4 molecules of ApoA-I. HDL particles may also include only ApoE (γ -LpE particles), which is also thought to be responsible for cholesterol efflux, as described by Prof. Gerd Assmann (see, e.g., von Eckardstein et al, 1994, Curr Opin Lipidol.5 (6): 404-16). ApoA-I is synthesized as preproapoproteins A-I by the liver and small intestine and is secreted as propoapoproteins A-I (proApoA-I) and rapidly cleaved to yield ApoA-I in the form of plasma, a polypeptide chain of 243 amino acids (Brewer et al, 1978, biochem. Biophys. Res. Commun.80: 623-30). preproApoA-I injected directly into the bloodstream in an experiment may also be cleaved to form ApoA-I in plasma form (Klon et al 2000 Biophys. J.79 (3): 1679-85; Segrest et al 2000, curr. Opin. Lipidol.11 (2): 105-15; Segrest et al 1999, J.biol. chem.274 (45): 31755-58).

ApoA-I comprises 6 to 8 different 22-amino acid alpha-monocycles or functional repeats, often spaced apart by a proline linker moiety. The repeat units exist in an amphiphilic helical configuration (Segrest et al, 1974, FEBS Lett.38: 247-53) and have the major biological activities of ApoA-I, namely lipid binding and Lecithin Cholesterol Acyltransferase (LCAT) activity.

ApoA-I forms the following three types of stable complexes with lipids: small lipid-free complexes known as pre- β -1 HDL; flat disk-like particles comprising polar lipids (phospholipids and cholesterol), called pre- β -2 HDL; and as globular or mature HDL (HDL)₃And HDL₂) Comprising polar and non-polar lipids. Most of the HDL in the circulating population includes ApoA-I and ApoA-II ("AI/AII-HDL components"). However, HDL components that include only ApoA-I ("AI-HDL components") appear more effective in RCT. Some areEpidemiological studies have demonstrated the hypothesis that the Apo-AI-HDL fraction is resistant to atherosclerosis (Parra et al, 1992, Arterioscler. Thromb.12: 701-07; Decossin et al, 1997, Eur. J. Clin. invest.27: 299-307).

HDL is made up of several populations of particles with different sizes, different lipid compositions, and apolipoprotein compositions. They may be separated according to their properties including their hydration density, apolipoprotein composition and charge properties. For example, pre- β -HDL is characterized as having a surface charge less than that of mature α -HDL. Due to this charge difference, pre- β -HDL and mature α -HDL have different electrophoretic mobilities in agarose gels (David et al, 1994, J.biol.chem.269 (12): 8959-8965).

The metabolism of pre- β -HDL is also different from that of mature α -HDL. Pre- β -HDL has two metabolic fates: removed from plasma and metabolized by the kidneys or reconstituted into intermediate size HDL that is preferentially degraded by the liver (Lee et al, 2004, J. lipid Res.45 (4): 716-.

Although the mechanism of cholesterol transfer from the cell surface (i.e., cholesterol efflux) is unknown, the lipid-free complex, pre- β -1 HDL, is believed to be the preferred receptor for cholesterol transferred from peripheral tissues involved in RCT (see Davidson et al, 1994, J.biol. chem.269: 22975-82; Bielicki et al, 1992, J.Lipid Res.33: 1699; Rothblat et al, 1992, J.Lipid Res.33: 1091-97; and Kawano et al, 1993, Biochemistry 32: 5025-28; Kawano et al, 1997, Biochemistry 36: 9816-25). During this process of cholesterol recruitment from the cell surface, pre- β -1 HDL is rapidly converted to pre- β -2 HDL. PLTP can increase the rate of pre- β -2 HDL disc (disc) formation, but lacks data indicating the role of PLTP in RCT. LCAT preferentially reacts with discotic small (pre- β) HDL and globular (i.e., mature) HDL, transferring the 2-acyl group of lecithin or other phospholipids to the free hydroxyl residues of cholesterol to form cholesteryl esters (retained in HDL) and lysolecithin. The LCAT reaction requires ApoA-I as an activator; that is, ApoA-I is a natural cofactor for LCAT. The conversion of sequestered cholesterol in HDL to its ester prevents the reentry of cholesterol into the cell, with the net result that cholesterol is removed from the cell.

The cholesterol esters in the mature HDL particles in the ApoAI-HDL fraction (i.e., comprising ApoA-I without ApoA-II) are removed via the liver and processed into bile more efficiently than those derived from HDL comprising ApoA-I and ApoA-II (a1/AII-HDL fraction). This may be due in part to more efficient binding of ApoAI-HDL to the liver cell membrane. The HDL receptor has been postulated to exist, and the class B type I scavenger receptor (SR-BI) has been identified as the HDL receptor (Acton et al, 1996, Science 271: 518-20; Xu et al, 1997, Lipid Res.38: 1289-98). SR-BI is abundantly expressed in steroid-producing tissues (e.g., adrenal glands) and liver (Landshulz et al, 1996, J.Clin.invest.98: 984-95; Rigotti et al, 1996, J.biol.chem.271: 33545-49). For a review of HDL receptors, see Broutin et al, 1988, anal.biol.chem.46: 16-23.

The initial lipidation via the ATP-binding cassette transporter AI is crucial for the formation of plasma HDL and for the cholesterol efflux capacity of pre- β -HDL particles (Lee and Parks, 2005, Curr. Opin. Lipidol.16 (1): 19-25). According to these authors, this initial lipidation allows the pre- β -HDL to act more efficiently as a cholesterol receptor and prevents the ApoA-I from rapidly associating with pre-existing plasma HDL particles, resulting in a stronger cholesterol efflux capacity of the pre- β -HDL particles.

CETP may also play an important role in RCT. Changes in CETP activity or its receptors VLDL and LDL play an important role in the "remodeling" of the HDL population. For example, in the absence of CETP, HDL becomes an unremovable large particle. (for reviews of RCT and HDL, see Fielding and Fielding, 1995, J.lipid Res.36: 211-28; Barrans et al, 1996, biochem.Biophys.acta 1300: 73-85; Hirano et al, 1997, Arterioscler.Thromb.Vase.biol.17 (6): 1053-59).

HDL also plays an important role in the reverse transport of other lipids and nonpolar molecules, as well as in detoxification, i.e., the transport of lipids from cells, organs, and tissues to the liver for metabolism and excretion. Such lipids include Sphingomyelin (SM), oxidized lipids and lysophosphatidylcholine. For example, Robins and Fasulo (1997, J.Clin.invest.99: 380-84) have demonstrated that HDL stimulates the transport of phytosterols via the liver into bile secretions.

ApoA-I, the major component of HDL, can associate with SM in vitro. When ApoA-I is reconstituted in vitro using Bovine Brain Sm (BBSM), the maximal reconstitution rate is produced at a temperature of 28 ℃ near the transition temperature of BBSM (Swaney, 1983, j.biol.chem.258(2), 1254-59). At a BBSM: ApoA-I ratio of 7.5: 1 or less (wt/wt), reconstituted homogeneous HDL particles were formed, each particle comprising three molecules of ApoA-I and having a BBSM: ApoA-I molar ratio of 360: 1. The HDL particles are disc-like complexes under an electron microscope, similar to complexes obtained by recombining ApoA-I with phosphatidylcholine at elevated phospholipid/protein ratios. However, at a BBSM: ApoA-I ratio of 15: 1(wt/wt), larger diameter discotic complexes with higher molar ratios of phospholipid to protein (535: 1) are formed. These complexes are significantly larger, more stable and more resistant to denaturation than ApoA-I complexes formed using phosphatidylcholine.

The increase in Sphingomyelin (SM) in the early cholesterol receptors (pre- β -HDL and gamma-migrating lipoproteins including ApoE) indicates that SM can enhance the ability of these particles to promote cholesterol efflux (Dass and lessup, 2000, J.Pharm.Pharmacol.52: 731-61; Huang et al, 1994, Proc.Natl.Acad.Sci.USA 91: 1834-38; Fielding and Fielding 1995, J.lipid Res.36: 211-28).

3.2 protection mechanisms for HDL and ApoA-I

Recent research on the mechanism of HDL protection has focused on apolipoprotein A-I (ApoA-I), the major component of HDL. Higher plasma concentrations of ApoA-I have been associated with loss or reduction of coronary lesions (Maciejko et al, 1983, N.Engl. J.Med.309: 385-89; Sedlis et al, 1986, issue 73: 978-84).

Injection of ApoA-I or HDL in experimental animals can produce significant biochemical changes, such that the extent and severity of atherosclerotic lesions is reduced. Following initial reports by Maciejko and Mao (1982, Arteriosclerosis 2: 407a), Badimon et al (1989, Lab. invest.60: 455-61; 1989, J. Clin. invest.85: 1234-41), it was found that the extent of atherosclerotic lesions (45% reduction) and cholesterol ester content (58.5% reduction) in cholesterol fed rabbits could be significantly reduced by injection of HDL (d ═ 1.063-1.325 g/ml). They also found that injection of HDL could produce a regression of approximately 50% of established lesions. Esper et al (1987, Arteriosclerosis 7: 523a) have demonstrated that injection of HDL can significantly alter the plasma lipoprotein composition of Watanabe rabbits with hereditary hypercholesterolemia that can progress to early arterial lesions. In these rabbits, the injection of HDL can result in more than a two-fold ratio between protective HDL and atherogenic LDL.

The potential of HDL to prevent arterial disease in animal models is further understood by the observation that ApoA-I exerts fibrinolytic activity in vitro (Saku et al, 1985, Thromb. Res.39: 1-8). Ronneberger (1987, Xth int. Congr. Pharmacol, Sydney, 990) demonstrated that ApoA-I can increase fibrinolysis in Beagle dogs and Cynomologous monkeys. Similar activity was found in human plasma in vitro. Ronneberger was able to demonstrate a reduction in lipid deposition and arterial plaque formation in ApoA-I treated animals.

In vitro studies have shown that complexes of ApoA-I with lecithin promote the efflux of free cholesterol from cultured arterial smooth muscle cells (Stein et al, 1975, biochem. Biophys. acta, 380: 106-18). HDL also reduces the proliferation of these cells by this mechanism (Yoshida et al, 1984, exp. MoI Pathol.41: 258-66).

It has also been demonstrated that the modulation of plasma HDL concentrations through the ABC1 transporter using HDL perfusion therapy including ApoA-I or ApoA-II mimetic peptides results in efficacy in the treatment of cardiovascular disease (see, e.g., Brewer et al, 2004, Arterioscler, Thromb, Vase, biol. 24: 1755-.

Has already been used forTwo naturally occurring human mutants of ApoA-I were isolated in which the arginine residue was mutated to cysteine. In apolipoprotein A-I_Milan(ApoA-I_M) In (b), the substitution occurs at residue 173, but in apolipoprotein A-I_Paris(ApoA-I_p) Such a substitution occurs at residue 151 (francischini et al, 1980, j. clin. invest.66: 892-900; weisgraber et al, 1983, j.biol.chem.258: 2508-13; bruckert et al, 1997, Atheroscleosis 128: 121-28; daum et al, 1999, j.moi.med.77: 614-22; klon et al, 2000, biophys.j.79 (3): 1679-85). Comprising ApoA-I_MOr ApoA-I_pReconstituted HDL particles of disulfide-linked homodimers were similar in their ability to clear Dimyristoylphosphatidylcholine (DMPC) emulsion and their ability to promote cholesterol efflux as compared to reconstituted HDL particles comprising wild-type ApoA-I (Calabresi et al, 1997b, Biochemistry 36: 12428-33; France schini et al, 1999, Arterioscler. Thromb. Vase. biol.19: 1257-62; Daum et al, 1999, J.MoI.Med.77: 614-22). In both mutants, heterozygous individuals reduced HDL concentrations, but paradoxically, they also reduced the risk of Atherosclerosis (France schini et al, 1980, J.Clin.invest.66: 892-; Weisgaber et al, 1983, J.biol.chem.258: 2508-13; Bruckert et al, 1997, Atheroschesis 128: 121-28). Although reconstituted HDL particles including either variant have reduced potency when compared to reconstituted HDL particles including wild-type ApoA-I, they are capable of activating LCAT (Calabrei et al, 1997a, biochem. Biophys. Res. Commun.232: 345-49; Daum et al, 1999, J.MoI.Med.77: 614-22).

ApoA-I_MThe mutant is inherited as an autosomal dominant trait; and eight generations of vectors within this family have been identified (Gualandri et al, 1984, am. J. hum. Genet. 37: 1083-97). Single ApoA-I_MThe carrier state is characterized by a significantly reduced concentration of HDL cholesterol. Although a single vector significantly reduced HDL cholesterol concentration, it did not significantly represent any increased risk of arterial disease. In fact, these subjects were found to be "protected" by studying pedigree profilesIt is protected from atherosclerosis (Sirtori et al, 2001, Circulation, 103: 1949-.

ApoA-I_MThe possible mechanism of protection in the vector mutants seems to be linked to ApoA-I_MStructural modifications of the mutant have been implicated in which an alpha-helix is deleted and exposed hydrophobic residues are added (France schini et al, 1985, J.biol.chem.260: 1632-35). The absence of multiple alpha-helical compact structures increases the flexibility of the molecule, which is more easily associated with lipids than conventional ApoA-I. Furthermore, apolipoprotein-lipid complexes are more susceptible to denaturation, suggesting that in mutants, lipid delivery may also be improved.

Bielicki et al (1997, Arterioscler.Thromb.Vase.biol.17 (9): 1637-43) have demonstrated ApoA-I_MCompared to wild-type ApoA-I, has a limited ability to recruit membrane cholesterol. Furthermore, from ApoA-I_MPrimary HDL formation associated with membrane lipids is predominantly 7.4nm particles rather than the larger 9-and 11-nm complexes formed by wild-type ApoA-I. These observations suggest Arg in the ApoA-I base sequence₁₇₃→Cys₁₇₃The replacement interferes with the normal process of cellular cholesterol recruitment and nascent HDL buildup. This mutation is clearly associated with a reduced efficacy of cholesterol removal from the cells. Thus, its atherogenic properties may be independent of RCT.

Due to Arg₁₇₃→Cys₁₇₃The most significant structural change that is substituted is ApoA-I_MDimerization (Bielicki et al, 1997, Arterioscler.Thromb.Vase.biol.17 (9): 1637-43). ApoA-I_MCan form homodimers with itself and heterodimers with ApoA-II. Studies on blood components including mixtures of apolipoproteins have shown that the presence of dimers and complexes in the circulation is responsible for the extended half-life of apolipoprotein elimination. Such an increase in elimination half-life has been observed in clinical studies of vector mutants (Gregg et al, 1988, NATO ARWon Human ApolipoproteinsMutants: from Gene Structure to PhototypiciExpression, Limone S G). Other studies have shown ApoA-I_MDimer (ApoA-I)_M/ApoA-I_M) Can act as an inhibitor of HDL particle interconversion in vitro (francischini et al, 1990, j.biol.chem.265: 12224-31).

3.3 treatment of dyslipidemia and related disorders

Disorders of lipid metabolism are diseases associated with elevated serum cholesterol and triglyceride concentrations and lower serum HDL: LDL ratios, and include hyperlipidemia (particularly hypercholesterolemia), coronary heart disease, coronary artery disease, vascular and perivascular diseases, and cardiovascular diseases such as atherosclerosis. Syndromes associated with atherosclerosis, such as intermittent claudication caused by arterial insufficiency, may also be included. There are a number of treatments currently available for reducing high serum cholesterol and triglycerides associated with disorders of lipid metabolism. However, each treatment has its own drawbacks and deficiencies in view of efficacy, side effects, and the population suffering from the disease.

Bile acid binding resins are a class of drugs that interrupt the recirculation of bile acids from the intestine to the liver, such as cholestyramine (Questran Light)Bristol-Myers Squibb) and colestipol hydrochloride (Colestid)Upjohn Corp.). When taken orally, these positively charged resins bind negatively charged bile acids in the intestine. Since the resins are not absorbed by the intestine, they are excreted with bile acids. However, the use of such resins can only reduce serum cholesterol concentrations by about 20% at best and is associated with gastrointestinal side effects including constipation and some vitamin deficiencies. In addition, since the resin may also be combined with other drugs, it must be taken at least one hour before or four to six hours after the resin is ingestedOther oral medications are taken, thereby complicating the regimen for patients with heart disease.

Statins are cholesterol-lowering drugs that block cholesterol synthesis by inhibiting HMGCoA reductase, a key involved in the cholesterol biosynthesis pathway. Statins, such as lovastatin (Mevacor), are sometimes used in combination with bile acid binding resins) Simvastatin (Zocor)) Pravastatin (pravastatin)) Fluvastatin (Lescol)) And atorvastatin (Lipitor)). Statins significantly lower serum cholesterol and serum LDL concentrations and slow the progression of coronary atherosclerosis. However, serum HDL cholesterol concentrations only moderately increased. The mechanism of attenuation of LDL action may involve a decrease in VLDL concentration and induction of LDL receptor cellular expression, which results in decreased production and/or increased LDL metabolism. Side effects, including liver and kidney dysfunction, are associated with the use of these drugs (Physicians Desk Reference, 56 th edition, 2002) Medical Economics).

Nicotinic acid (niacin) is a water-soluble vitamin B-complex which is used as a dietary supplement and an antihyperlipidemic agent. Niacin reduces VLDL formation and is effective in lowering LDL. In some cases, niacin is used in conjunction with the bile acid binding resin. Niacin can increase HDL when used in sufficient doses, but its effectiveness is limited by serious side effects when used in such high doses. NiaspanIs an extended release niacin form that produces fewer side effects than pure niacin. Niacin/lovastatin) Is a formulation containing niacin and lovastatin, which combines the benefits of each drug.

Fibrates (Fibrates) are a class of lipid lowering drugs used to treat various forms of hyperlipidemia (i.e., elevated serum triglycerides) that may also be associated with hypercholesterolemia. Fibrates appear to decrease the VLDL fraction and increase HDL moderately, however, the effect of these drugs on serum cholesterol is variable. In the United states, fibrates such as clofibrate (Atromid-S)) Fenofibrate (Tricor)) And bezafibrate (Bezalip)) Has been approved for use as a hypolipidemic agent, but has not yet been approved for use as a hypercholesterolemic agent. For example, clofibrate is a hypolipidemic agent that acts (via an unknown mechanism) to lower serum triglycerides by reducing the VLDL fraction. Although serum cholesterol can be reduced in certain patient subpopulations, its biochemical response to drugs is variable and it is not always possible to predict which patients will achieve favorable results. Atromid-SIt does not show the curative effect of preventing coronary heart disease. The chemically and pharmacologically relevant drug gemfibrozil (Lopid)) Is a lipid regulatorWhich moderately lowers serum triglycerides and VLDL cholesterol, and moderately increases HDL cholesterol-HDL₂And HDL₃Subcomponents and ApoA-I and A-II (i.e., AI/AMT-HDL components). However, the lipid response is heterogeneous (heterogeneous), especially in different patient populations. Furthermore, although a prophylactic effect of coronary heart disease was observed in male patients between the ages of 40-55 who had no history or symptoms of coronary heart disease, these results could not be unambiguously extrapolated to other patient populations (e.g., women, elderly and younger men). In fact, no therapeutic effect was observed in patients with established coronary heart disease. Serious side effects are associated with the use of fibrates, and include toxic effects, such as malignancy (particularly gastrointestinal cancer), gallbladder disease, and an increased incidence of non-coronary death.

Oral estrogen replacement therapy is believed to be useful in reducing hypercholesterolemia in postmenopausal women. However, an increase in HDL may be accompanied by an increase in triglycerides. Of course, estrogen therapy is limited to a specific patient population (postmenopausal women) and is associated with serious side effects including the induction of malignancy, gallbladder disease, thromboembolic disease, liver adenoma, elevated blood pressure, glucose intolerance, and hypercalcemia.

Other drugs that may be used to treat hyperlipidemia include ezetimibe (Zetia)(ii) a Merck) which blocks or inhibits the absorption of cholesterol. However, inhibitors of ezetimibe have shown some toxicity.

Accordingly, there is a need for safe drugs that are more effective in lowering serum cholesterol, increasing HDL serum concentrations, and preventing and/or treating dyslipidemia and/or diseases, disorders, and/or disorders associated with dyslipidemia.

For example, HDL and recombinant forms of ApoA-I complexed with phospholipids can act as absorbents/scavengers for nonpolar or amphiphilic molecules such as cholesterol and derivatives (oxysterol, oxidized sterols, phytosterols, etc.), cholesterol esters, phospholipids and derivatives (oxidized phospholipids), triglycerides, oxidized products, and Lipopolysaccharides (LPS) (see, e.g., Casas et al, 1995, j.surg.res.nov 59 (5): 544-52). HDL may also function as a scavenger of TNF- α and other lymphokines. HDL may also function as a carrier for human serum paraoxonase, such as PON-1, -2, -3. Paraoxonase is an HDL associated esterase that is important for protecting cellular components against oxidation. Oxidation of LDL occurs during oxidative stress, which appears to be directly linked to the formation of atherosclerosis (Aviram, 2000, Free Radic. Res.33 Suppl: S85-97). Paraoxonase appears to play an important role in the susceptibility to atherosclerosis and cardiovascular disease (Aviram, 1999, MoI. Med. today 5 (9): 381-86). Human serum paraoxonase (PON-1) binds to High Density Lipoprotein (HDL). Its activity is inversely related to atherosclerosis. PON-1 hydrolyzes organophosphates and protects against atherosclerosis by inhibiting the oxidation of HDL and Low Density Lipoprotein (LDL) (Aviram, 1999, MoI. Med. today 5 (9): 381-86). Experimental studies have shown that this protective effect is related to the ability of PON-1 to hydrolyze specific lipid peroxides in oxidized lipoproteins. Interventions that maintain or promote PON-1 activity may help delay the onset of atherosclerosis and coronary heart disease.

HDL further has the effects of an antithrombotic agent and a fibrinogen reducing agent as well as the effect as a hemorrhagic shock drug (cockericll et al, WO 01/13939, published 3/1 in 2001). HDL, and in particular ApoA-I, has been shown to successfully exchange the lipopolysaccharide produced by sepsis for lipid particles comprising ApoA-I, thereby functionally neutralizing the lipopolysaccharide (Wright et al, WO 9534289, published 12/21 1995; Wright et al, U.S. Pat. No. 5,928,624 issued 7/27/1999; Wright et al, U.S. Pat. No. 5,932,536 issued 8/3/1999).

However, large amounts of vehicle are required due to the therapeutic administrationLipoprotein and due to the expensive cost of protein production, the current consideration of lower overall yields of production is that ApoA-I, ApoA-I_M、ApoA-I_pAnd other variants, and reconstituted HDL, have limited therapeutic use. Early clinical trials have shown that doses ranging between 1.5-4g of protein per infusion are useful for treating cardiovascular disease. The number of infusions required for complete treatment is unknown. (see, e.g., Eriksson et al, 1999, Circulation100 (6): 594-98; Carlson, 1995, Nutr.Metab.Cardiovasc.Dis.5: 85-91; Nanjee et al, 2000, Arterioscler.Thromb.Val.biol.20 (9): 2148-55; Nanjee et al, 1999, Arterioscler.Thromb.Val.19 (4): 979-89; Nanjee et al, 1996, Arterioscler.Thromb.Val.16 (9): 1203-14). Accordingly, there is a need to develop new methods for the treatment and/or prevention of diseases, conditions and/or disorders of lipid metabolism.

Citation or identification of any reference in section 2 or any other section of this application shall not be construed as an admission that such reference is available as prior art to the present invention.

4. Summary of the invention

The present disclosure provides charged lipoprotein complexes, compositions comprising the complexes, and methods of using the complexes to treat and/or prevent a variety of disorders and conditions, including dyslipidemia and/or various diseases, disorders, and/or conditions associated therewith. The complex is typically a lipoprotein that includes two components (an apolipoprotein component and a lipid component) and includes, as a key component, a specific amount of a charged phospholipid (or a mixture of two or more different, usually similarly charged phospholipids). The charged phospholipid may have a positive or negative charge at physiological pH, but in various embodiments is a negative charge, and in some embodiments the charged phospholipid comprises one or more phosphatidylinositols, phosphatidylserines, phosphatidylglycerols, and/or phosphatidic acids.

The apolipoprotein component includes one or more proteins, peptides or peptide analogs that are capable of mobilizing cholesterol included in the complex (referred to as "apolipoproteins"). A specific example of such an apolipoprotein is ApoA-I. Other specific examples are described further below.

The lipid component typically includes one or more neutral and charged phospholipids, and may optionally include other lipids, such as triglycerides, cholesterol esters, lysophospholipids, and various analogs and/or derivatives thereof. In some embodiments, the charged lipoprotein complex does not include such optional lipids.

A neutral phospholipid may be any phospholipid having a net charge of about zero at physiological pH. In some embodiments, the neutral phospholipid is a zwitterion having a net charge of about zero at physiological pH. In some embodiments, the neutral phospholipid comprises lecithin (also known as phosphatidylcholine or "PC"). In some embodiments, the neutral phospholipid comprises sphingomyelin ("SM"). In some embodiments, the neutral phospholipid comprises a mixture of lecithin and SM. In various embodiments of the charged lipoprotein complexes, the lipid component comprises lecithin or SM, at least one charged phospholipid, and other optional lipids, said lipoprotein complexes being referred to as "ternary" complexes in that they comprise the following three "major" components: apolipoproteins, lecithin or sphingomyelin, and charged phospholipids. In various embodiments of the charged lipoprotein complexes, the lipid component includes lecithin and SM, at least one charged phospholipid, and other optional lipids, which are referred to as "quaternary" complexes.

The total amount of charged phospholipids, including the lipid component, of the charged lipoprotein complexes may vary, but typically ranges from about 0.2 to 10 wt%. In some embodiments, the lipid component comprises about 0.2 to 2 wt%, 0.2 to 3 wt%, 0.2 to 4 wt%, 0.2 to 5 wt%, 0.2 to 6 wt%, 0.2 to 7 wt%, 0.2 to 8 wt%, or 0.2 to 9 wt% of the charged phospholipid in total. In some embodiments, the lipid component comprises about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 wt% charged phospholipid, and/or any range including these values as endpoints. In some embodiments, the lipid component comprises about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 wt% charged phospholipid up to about 4, 5, 6, 7, 8, 9, or 10 wt% charged phospholipid.

The total amount of neutral phospholipids including the lipid component can also vary and is dependent on the amount of charged phospholipids and any optional lipids included. In embodiments that do not include optional lipids, the lipid component will typically include about 90 to 99.8 wt% total neutral phospholipids.

As mentioned above, the neutral phospholipid may comprise lecithin, SM or a mixture of lecithin and SM. The lecithin and/or SM may comprise a bulk (bulk) of the neutral phospholipid, or alternatively, the neutral phospholipid may comprise a neutral phospholipid in addition to the lecithin and/or SM. In embodiments where the neutral phospholipid comprises lecithin, but not SM, the neutral phospholipid typically comprises about 5 to 100 wt% lecithin. In some embodiments, the neutral phospholipid comprises 100 wt% lecithin.

In embodiments where the neutral phospholipid includes SM and does not include lecithin, the neutral phospholipid typically includes about 5 to 100 wt% SM. In some embodiments, the neutral phospholipid comprises 100 wt% SM.

In embodiments where the neutral phospholipid comprises a mixture of lecithin and SM, the amount of the mixture including all of the neutral phospholipids, as well as the relative amount of lecithin and SM included in the mixture (i.e., the molar ratio of lecithin: SM) can vary. Typically, the neutral phospholipid will comprise about 5 to 100 wt% of the lecithin/SM mixture. In some embodiments, all of the neutral phospholipids included are lecithin and SM (i.e., a 100 wt% mixture of lecithin and SM).

The molar ratio of lecithin to SM (lecithin: SM) can vary, but is typically in the range of about 20: 1 to 1: 20. In some embodiments, the molar ratio of lecithin to SM ranges from about 10: 3 to 10: 6. In other embodiments, the molar ratio of lecithin to SM ranges from about 1: 20 to 3: 10.

If included, the optional lipid will typically include about 50 wt% or less of the lipid component. In some embodiments, the lipid component includes less than about 30 wt% of the total amount of optional lipids. In particular embodiments, the lipid component includes less than about 5 wt%, 10 wt%, or 20 wt% of the total amount of optional lipids.

The molar ratio of lipid to apolipoprotein in the charged lipoprotein complex may also vary. In some embodiments, the charged lipoprotein complex comprises a molar ratio of lipid to apolipoprotein ranging from about 2: 1 to about 200: 1. In some embodiments, the molar ratio of lipid to apolipoprotein is about 50: 1.

The charged lipoprotein complexes described herein can take a variety of shapes, sizes and forms, ranging from micellar structures to small discoidal particles similar to naturally-occurring pre- β HDL particles, to larger discoidal particles similar to naturally-occurring α -HDL particles, to larger discoidal particles similar to naturally-occurring HDL particles₂Or HDL₃Large spherical particles of (2). The desired size and shape of the charged lipoprotein complexes described herein can be controlled by adjusting the composition to weight (or molar) ratio of the lipids comprising the lipid component, as well as adjusting the molar ratio of lipid to apolipoprotein, as is known in the art (see, e.g., Barter et al, 1996, J.biol.chem.271: 4243-4250).

In some embodiments, the charged lipoprotein complex is in the form of a disk-shaped particle, wherein the lipid component consists essentially of about 90 to 99.8 wt% total of all neutral phospholipids and about 0.2 to 10 wt% total of all negative charged phospholipids. The disk-shaped particles can be large (e.g., having a oblate diameter of about 10 to 14 nm) or small (e.g., having a oblate diameter of about 5 to 10 nm). The size of the discotic particles can be controlled by adjusting the molar ratio of lipid to apolipoprotein, as is known in the art (see, e.g., Barter et al, 1996, supra). For example, size exclusion column chromatography (size exclusion column chromatography) can be used to determine the size of the particles.

The pharmaceutical compositions generally comprise a charged lipoprotein complex as described herein, and may optionally comprise one or more pharmaceutically acceptable carriers, excipients and/or diluents. In some embodiments, the pharmaceutical composition is packaged in unit doses suitable for administration. For example, in some embodiments, a composition comprising a unit dose of a dried (e.g., lyophilized) charged lipoprotein complex is packaged in a sealed vial. Such compositions are suitable for reconstitution with water, physiological solution (such as saline), or buffer, and administration via injection. Such compositions may optionally include one or more anti-caking agents and/or anti-coagulants that facilitate reconstitution of the charged complex, or one or more buffers, sugars or salts (such as sodium chloride) designed to adjust the pH, osmotic pressure and/or salinity of the reconstituted suspension.

The charged lipoprotein complexes and compositions described herein are expected to affect and/or promote cholesterol efflux and/or elimination of cholesterol, and thus the complexes and compositions are expected to be useful in the treatment and/or prevention of a variety of conditions and disorders, examples including dyslipidemia and/or diseases, conditions and/or disorders associated with dyslipidemia or associated with lipid depletion, accumulation or exclusion (e.g., lipo-deposition, cellular degradation)/or non-polar molecules (such as toxins, extravasations, etc.). Non-limiting examples of such diseases, disorders and/or associated conditions that may be treated or prevented using the charged lipoprotein complexes and compositions described herein include peripheral vascular disease, hypertension, inflammation, alzheimer's disease, restenosis, atherosclerosis, and various clinical manifestations of atherosclerosis, such as shock, hemorrhagic shock, transient ischemic attacks, myocardial infarction, acute coronary syndrome, angina, renovascular hypertension, renovascular insufficiency, intermittent claudication, critical limb ischemia, resting pain, and gangrene, to name a few.

The methods generally comprise administering to a subject an effective amount of a charged lipoprotein complex or pharmaceutical complex (composition) described herein sufficient to treat or prevent a particular indication. The complexes and/or compositions can be administered alone (as monotherapy) or, alternatively, they can be administered in combination with other therapeutic agents that can be used to treat and/or prevent dyslipidemia and/or a condition, disease, and/or disorder associated with dyslipidemia. Non-limiting examples of therapeutic agents that can be administered in conjunction with the charged lipoprotein complexes and compositions described herein include bile acid binding resins, HMG CoA reductase inhibitors (statins), nicotinic acid, resins, cholesterol absorption inhibitors, and fibrates.

While not intending to be bound by any theory of operation, it is believed that the charged phospholipids including lipid components provide the charged lipoprotein complexes and compositions described herein with improved therapeutic properties over conventional lipoprotein complexes. One key difference between small discoidal pre- β HDL, which is degraded in the kidney, and large discoidal and/or spherical HDL, which is recognized by the liver where storage, recirculation, metabolism (as bile acids) or elimination (in the bile) of cholesterol is a function of the charge of the particles. The small discoidal pre-beta HDL has less negative surface charge than the large discoidal and/or spherical HDL having a negative charge. While not intending to be bound by any theory of operation, it is believed that the more negative charge is one of the factors that trigger the liver to recognize particles, and thus avoid metabolizing the particles via the kidneys. The belief that the charged lipoprotein complexes and compositions described herein remain in the circulation for longer than conventional lipoprotein complexes can be attributed in part to the presence of charged phospholipids, or the charge can affect the half-life of the lipoprotein in a charge-dependent manner. Longer circulation (residence) times for lipoproteins are expected to favor cholesterol flow (giving the complex more time to accumulate cholesterol) and esterification (giving LCAT more time to catalyze the esterification reaction). The charge may also increase the probability of capturing and/or removing cholesterol, thereby facilitating substantial removal of cholesterol. Thus, it is desirable that the charged lipoprotein complexes and compositions described herein provide therapeutic benefits over conventional lipoprotein therapies, yet require less complex and/or composition to be administered, and less times to be administered.

5. Brief description of the drawings

FIG. 1 provides a chromatogram of an uncharged lipoprotein complex consisting of pro-Apo-AI (33 wt%) and sphingomyelin (67 wt%);

FIG. 2 provides a chromatogram of an embodiment of a charged lipoprotein complex consisting of pro-Apo-AI (33 wt%), sphingomyelin (65 wt%) and phosphatidylglycerol (2 wt%);

FIG. 3 provides a graph of total free cholesterol in HDL of rabbits as a function of time following administration of a control, uncharged lipoprotein complex (curve labeled IIA), or charged lipoprotein complex embodiment as described herein (curve labeled IIB); and

figure 4 provides a graph of the average amount of free cholesterol measured in the HDL of rabbits as a function of time following administration of a control, uncharged lipoprotein complex (group IIA, two animals) or a charged lipoprotein complex embodiment as described herein (group IIB, two animals).

6. Detailed description of the preferred embodiments

The present disclosure provides, among other things, charged lipoprotein complexes and compositions useful for treating and/or preventing dyslipidemia and/or diseases, disorders, and/or conditions associated with dyslipidemia. As discussed in the summary of the invention section, the charged lipoprotein complex includes two major components, an apolipoprotein component and a lipid component, and includes, as key components, a specific amount of one or more charged phospholipids.

The charged lipoprotein complex can be isolated from natural sources, such as from human serum (referred to herein as an "isolated charged lipoprotein complex"), or the charged lipoprotein complex can be prepared or reconstituted from its individual components (referred to herein as a "reconstituted charged lipoprotein complex"). As will be appreciated by those skilled in the art, the reconstituted charged lipoprotein complexes are advantageous in a variety of applications because the identity and amount of the various components of the reconstituted charged lipoprotein complex can be selectively controlled.

6.1 Apolipoprotein and Apolipoprotein peptide

The nature of the apolipoproteins in the charged lipoprotein complexes, including the apolipoprotein component, is not critical to success. In fact, any apolipoprotein and/or derivative or analogue thereof that provides a therapeutic and/or prophylactic benefit as described herein can be included in the charged complex. Furthermore, any alpha helical peptide or peptide analogue or any other type of molecule that "mimics" the activity of an apolipoprotein (e.g. ApoA-I) may activate LCAT or form a disc-like particle when associated with a lipid, and so it may include charged complexes and is therefore included within the definition of "apolipoprotein". Examples of suitable apolipoproteins include, but are not limited to, ApoA-I, ApoA-II, ApoA-IV, ApoA-V, and ApoE in the form of preproapoproteins; human ApoA-I, ApoA-II, ApoA-IV and ApoE in both its native and mature forms; and activated polymorphs, isoforms, variants and mutants and truncated forms, the most common of which is ApoA-I_M(ApoA-I_M) And ApoA-I_p(ApoA-I_p). Apolipoprotein mutants are also known to contain cysteine residues and may also be used (see, e.g., U.S. 2003/0181372). The apolipoprotein may be in monomeric or dimeric form, while the dimer may be a homodimer or a heterodimer. For example, the following homodimers and heterodimers (feasible) may be used: pro-ApoA-I and mature ApoA-I (Duverger et al, 1996,Arterioscler.Thromb.Vase.Biol.16(12)：1424-29)、ApoA-I_M(France schini et al, 1985, J.biol.chem.260: 1632-35), ApoA-I_P(Daum et al, 1999, J.MoI.Med.77: 614-22), ApoA-II (Shelness et al, 1985, J.biol.chem.260 (14): 8637-46; Shelness et al, 1984, J.biol.chem.259 (15): 9929-35), ApoA-IV (Duverger et al, 1991, Euro.J.biochem.201 (2): 373-83), ApoE (McLean et al, 1983, J.biol.chem.258 (14): 8993-9000), ApoJ and ApoH. The apolipoprotein may include residues corresponding to elements that facilitate its isolation (such as His-tag) or other elements designed for other purposes, so long as the apolipoprotein retains some biological activity when included in a complex.

As is well known in the art, such apolipoproteins may be purified from animal sources (particularly from human sources) or produced recombinantly, see, e.g., Chung et al, 1980, j.lipid res.21 (3): 284-91; cheung et al, 1987, j. lipid res.28 (8): 913-29 see also U.S. patent nos. 5,059,528, 5,128,318, 6,617,134 and U.S. publication nos. 20002/0156007, 2004/0067873, 2004/0077541 and 2004/0266660.

Peptides and peptide analogs corresponding to apolipoproteins and mimetics ApoA-I, ApoA-I suitable as apolipoproteins for use in the charged complexes and compositions described herein_MNon-limiting examples of agonists of ApoA-II, ApoA-IV and Apo-E activity are disclosed in the following references: U.S. patent nos. 6,004,925, 6,037,323, and 6,046,166 (to Dasseux et al); U.S. patent No. 5,840,688 (to Tso); U.S. publications 2004/0266671, 2004/0254120, 2003/0171277, and 2003/0045460 (Fogelman); and U.S. publication 2003/0087819 (of Bielicki), the entire disclosure of which is incorporated herein by reference. These peptides and peptide analogs may include L-amino acids or D-amino acids or mixtures of L-amino acids and D-amino acids. The peptide and peptide analogs may also include one or more non-peptide or amide linkages, such as one or more of the well-known peptide/amide isosteres. Any peptide synthesis technique known in the art may be used to carry outSuch "peptide and/or peptidomimetic" apolipoproteins are synthesized or prepared by peptide synthesis techniques including, for example, techniques described in U.S. patent nos. 6,004,925, 6,037,323, and 6,046,166.

The charged complex may comprise a single type of apolipoprotein, or a mixture of two or more different apolipoproteins, which may be derived from the same or different species. Although not required, the charged lipoprotein complex preferably includes an apolipoprotein derived from the amino acid sequence of the animal species being treated or an apolipoprotein corresponding thereto, to avoid inducing an immune response to treatment. The use of apolipoprotein peptide mimetics may also induce or avoid an immune response.

6.2 Phospholipids

The lipid component of the charged complexes and compositions include the following two types of phospholipids: neutral phospholipids and charged phospholipids. As used herein, a "neutral phospholipid" is a phospholipid having a net charge of about zero at physiological pH. In various embodiments, the neutral phospholipid is a zwitterion, although other types of net charge neutral phospholipids are known and can be used. The neutral phospholipids include one or both of lecithin and/or SM, and may optionally include other neutral phospholipids. In some embodiments, the neutral phospholipid comprises lecithin but does not comprise SM. In other embodiments, the neutral phospholipid includes SM but does not include lecithin. In yet another embodiment, the neutral phospholipids include lecithin and SM. All of these specific exemplary embodiments may also include neutral phospholipids in addition to lecithin and/or SM, but in various embodiments such additional neutral phospholipids are not included.

The nature of the SM used is not critical to success. Thus, as used herein, the expression "SM" includes not only sphingomyelin derived from natural sources, but also analogues and derivatives of naturally occurring SM that are not affected by LCAT hydrolysis, as are naturally occurring SM. SM is a phospholipid with a structure very similar to lecithin, but it is not consistent with lecithin in that it does not have a glycerol backbone and thus does not have ester linkages to acyl chains. In contrast, SM has a ceramide backbone in which an amide bond is linked to an acyl chain. SM is not a substrate for LCAT and it is generally not hydrolyzed by LCAT. However, SM may act as an LCAT inhibitor or may reduce LCAT activity by diluting the concentration of substrate phospholipid. Since SM is not hydrolyzed, it can be held up for a longer time in the cycle. This feature is expected to provide charged lipoprotein complexes including SM with longer duration of pharmacological action (cholesterol flux) and which are able to acquire more lipids, especially cholesterol, than apolipoprotein complexes not including SM (see, e.g., the apolipoprotein complex described in U.S. publication No. 2004/0067873, the entire disclosure of which is incorporated herein by reference). This effect may result in a less frequent or lower dose of treatment of the lipoprotein complexes including SM compared to lipoprotein complexes not including SM.

In fact, the SM may originate from any source. For example, SM can be obtained from milk, eggs or brain. SM analogs or derivatives can also be used. Non-limiting examples of useful SM analogs and derivatives include, but are not limited to, palmitoyl sphingomyelin, stearoyl sphingomyelin, D-erythrose-N-16: 0-sphingomyelin and its dihydroisomers, D-erythrose-N-16: 0-dihydrosphingomyelin.

Sphingomyelin isolated from natural sources can be artificially enriched with a particular saturated or unsaturated acyl chain. For example, milk sphingomyelin (Avanti sphingomyelin, Alabaster, Ala.) is characterized by having saturated long acyl chains (i.e., acyl chains with 20 or more carbon atoms). Egg sphingomyelin, in contrast, is characterized by having short acyl chains that are saturated (i.e., acyl chains having less than 20 carbon atoms). For example, while only about 20% of milk sphingomyelin includes C16:0(16 carbon, saturated) acyl chains, about 80% of egg sphingomyelin includes C16:0 acyl chains. The composition of milk sphingomyelin can be enriched with acyl chain components comparable to egg sphingomyelin and vice versa by using solvent extraction.

To make SM have a specific acyl chain, it can be semi-synthetic. For example, milk sphingomyelin can be first purified from milk, and then one particular acyl chain, such as the C16:0 acyl chain, can be cleaved and replaced with another. SM can also be completely synthesized, e.g. by large scale synthesis. See, e.g., Dong et al, U.S. patent No. 5,220,043 entitled "synthetic sof D-ervthro-sphingomotilins," issued 6/15/1993; weis, 1999, chem.phys.lipids 102 (1-2): 3-12.

The length and saturation of the acyl chain including semi-synthetic or synthetic SM can be selectively varied. The acyl chain may be saturated or unsaturated, and may contain from about 6 to about 24 carbon atoms. Each chain may contain the same number of carbon atoms, or alternatively, each chain may contain a different number of carbon atoms. In some embodiments, a semi-synthetic or synthetic SM comprises mixed acyl chains such that one chain is saturated and the other chain is unsaturated. In SM with such mixed acyl chains, the length of the chains may be the same or different. In other embodiments, the acyl chains of the semi-synthetic or synthetic SM may both be saturated or unsaturated. Furthermore, the chains may contain the same or different numbers of carbon atoms. In some embodiments, the two acyl chains comprising semi-synthetic or synthetic SM are the same. In particular embodiments, the chain is identical to the acyl chain of a naturally occurring fatty acid, such as oleic acid, palmitic acid, or stearic acid. In another specific embodiment, both acyl chains are saturated and both contain 6 to 24 carbon atoms. Table 1 below provides non-limiting examples of acyl chains present in commonly formed fatty acids that may be included in semi-synthetic and synthetic SM:

as with SM, the nature of the lecithin used is not critical to success. Further, also like SM, lecithin may be derived or isolated from natural sources, or it may be obtained synthetically. Examples of suitable lecithins isolated from natural sources include, but are not limited to, egg phosphatidylcholine and soy phosphatidylcholine. Other non-limiting examples of suitable lecithins include dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, 1-myristoyl-2-palmitoylphosphatidylcholine, 1-palmitoyl-2-myristoylphosphatidylcholine, 1-palmitoyl-2-stearoylphosphatidylcholine, 1-stearoyl-2-palmitoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine, 1-oleoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholine, ether derivatives thereof, or the like.

As with SM, lecithin derived or isolated from natural sources can be enriched in specific acyl chains. As discussed above in connection with SM, in embodiments where semi-synthetic or synthetic lecithin is employed, the properties of the acyl chains may be selectively altered. In some embodiments of the charged complexes described herein, the two acyl chains on the lecithin are the same. In some embodiments of the charged lipoprotein complex comprising SM and lecithin, the acyl chains of SM and lecithin are all the same. In particular embodiments, the acyl chain is identical to that of myristic, palmitic, oleic or stearic acid.

The lipid component also includes a charged phospholipid. As used herein, a "charged phospholipid" is a phospholipid having a net charge at physiological pH. The charged phospholipid may comprise a single type of charged phospholipid or a mixture of two or more different, typically similarly charged phospholipids. In some embodiments, the charged phospholipid is a negatively charged glycerophospholipid. The nature of the charged phospholipids is not critical to success. Specific examples of suitable negatively charged phospholipids include, but are not limited to, phosphatidylglycerol (phosphatidylglycerol), phosphatidylinositol (phosphatidyllinosotol), phosphatidylserine, phosphatidylglycerol, and phosphatidic acid. In some embodiments, the negatively charged phospholipid comprises one or more phosphatidylinositols, phosphatidylserines, phosphatidylglycerols, and/or phosphatidic acids.

Like SM and lecithin, negatively charged phospholipids can be obtained from natural sources or prepared by chemical synthesis. As discussed above in connection with SM, in embodiments employing synthetic negatively charged phospholipids, the properties of the acyl chains can be selectively altered. In some embodiments of the charged lipoprotein complexes described herein, the two acyl chains on the negatively charged phospholipid are the same. In some embodiments of the ternary and quaternary charged lipoprotein complexes described herein, the acyl chains on the SM, lecithin and negatively charged phospholipid are all the same. In particular embodiments, the charged phospholipid and/or SM each has a C16:0 or C16:1 acyl chain. In another specific embodiment, the acyl chain of the charged phospholipid, lecithin and/or SM is identical to the acyl chain of palmitic acid. In yet another specific embodiment, the acyl chain of the charged phospholipid, lecithin and/or SM is identical to the acyl chain of oleic acid.

The total amount of negatively charged phospholipids including the charged complexes can vary. Typically, the lipid component will comprise about 0.2 to 10 wt% negatively charged phospholipids. In some embodiments, the lipid component comprises about 0.2 to 1 wt%, 02 to 2 wt%, 02 to 3 wt%, 0.2 to 4 wt%, 0.2 to 5 wt%, 0.2 to 6 wt%, 0.2 to 7 wt%, 0.2 to 8 wt%, or 0.2 to 9 wt% of the negatively charged phospholipid in total. In some embodiments, the lipid component comprises about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 wt% negatively charged phospholipid and/or any range including these values as endpoints. In some embodiments, the lipid component comprises about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 wt% negatively charged phospholipid up to about 4, 5, 6, 7, 8, 9, or 10 wt% negatively charged phospholipid.

It is expected that the negatively charged phospholipids included in the charged lipoprotein complexes described herein will provide complexes with higher stability (in solution) and products with longer shelf life than conventional complexes. Furthermore, it is expected that the use of negatively charged phospholipids can minimize particle aggregation (e.g., by charge repulsion), thereby effectively increasing the number of available complexes present in a given dosage regimen and facilitating targeted recognition of the complexes by the liver rather than the kidney.

In vivo, some apolipoproteins are exchanged from one lipoprotein complex to another (this is true for apolipoprotein ApoA-I). During such exchanges, the apolipoproteins typically carry with them one or more phospholipid molecules. Due to this property, the charged lipoprotein complexes described herein can be expected to "seed" negatively charged phospholipids into endogenous HDL, thereby converting the negatively charged phospholipids into alpha particles that are more resistant to renal elimination. Thus, administration of the charged lipoprotein complexes and compositions described herein is expected to increase the concentration of serum HDL, and/or alter the half-life of endogenous HDL as well as the metabolism of endogenous HDL. It is expected to alter cholesterol metabolism and reverse lipid transport.

The lipid component may optionally include additional lipids in addition to the neutral and charged phospholipids. Virtually any type of lipid can be used, including, but not limited to, lysophospholipids, galactocerebrosides, gangliosides, cerebrosides, glycerides, triglycerides, and cholesterol and derivatives thereof.

Even if such optional lipids are included, they typically include only less than about 50 wt% lipid component, although more optional lipids may be included in some examples. In some embodiments, the lipid component of the charged lipoprotein complex does not include an optional lipid.

As shown in the summary section, the total amount of neutral phospholipids of the lipid component comprising the charged lipoprotein complex can vary depending on the total amount of charged phospholipids included and whether any optional lipids are included, and typically ranges from about 50 to 99.8 wt%. In particular embodiments that do not include optional lipids, neutral phospholipids are typically included in a total amount of about 90 to 99.8 wt%. Suitable lecithin to SM molar ratios in the lipid component including lecithin and SM are described in the summary section.

In particular embodiments, the charged lipoprotein complex is a ternary complex in which the lipid component consists essentially of about 90 to 99.8 wt% SM and about 0.2 to 10 wt% negatively charged phospholipids, e.g., about 0.2-1 wt%, 0.2-2 wt%, 0.2-3 wt%, 0.2-4 wt%, 0.2-5 wt%, 0.2-6 wt%, 0.2-7 wt%, 0.2-8 wt%, 0.2-9 wt%, or 0.2-0 wt% negatively charged phospholipids, in total. In another specific embodiment, the charged lipoprotein complex is a ternary complex in which the lipid component consists essentially of about 90 to 99.8 wt% lecithin and about 0.2 to 10 wt% negatively charged phospholipid, e.g., about 0.2-1 wt%, 0.2-2 wt%, 0.2-3 wt%, 0.2-4 wt%, 0.2-5 wt%, 0.2-6 wt%, 0.2-7 wt%, 0.2-8 wt%, 0.2-9 wt%, or 0.2-10 wt% negatively charged phospholipid, in total.

In yet another specific embodiment, the charged lipoprotein complex is a quaternary complex in which the lipid component consists essentially of about 9.8 to 90 wt% SM, about 9.8 to 90 wt% lecithin, and about 0.2 to 10 wt% negatively charged phospholipids, e.g., about 0.2 to 1 wt%, 0.2 to 2 wt%, 0.2 to 3 wt%, 0.2 to 4 wt%, 0.2 to 5 wt%, 0.2 to 6 wt%, 0.2 to 7 wt%, 0.2 to 8 wt%, 0.2 to 9 wt% to 0.2 to 10 wt% negatively charged phospholipids, in total.

The complex may also optionally include other proteins, such as Paraoxonase (PON) or LCAT, antioxidants, cyclodextrins, and/or other materials that help to trap cholesterol in the core or on the surface of the complex. The complex may optionally be pegylated (e.g., covered with polyethylene glycol or other polymer) to extend the circulation half-life.

As understood by those skilled in the art, the molar ratio of the lipid component to the apolipoprotein component in the charged lipoprotein complexes described herein may vary and depends on, among other factors, the identity of the apolipoprotein comprising the apolipoprotein component, the identity and amount of the charged phospholipid comprising the lipid component, and the desired size of the charged lipoprotein complex. Since the biological activity of an apolipoprotein such as ApoA-I is believed to be mediated by an amphipathic helix comprising the apolipoprotein, ApoA-I protein equivalents may conveniently be used to express the molar ratio of lipid to apolipoprotein in the apolipoprotein. It is generally accepted that ApoA-I contains 6-10 amphipathic helices and that it depends on the method used to calculate the helix. Other apolipoproteins may be expressed in terms of ApoA-I equivalents, depending on the number of amphipathic helices that the apolipoprotein contains. For example, ApoA-I, which is normally present as a disulfide bridged dimer_MCan be expressed as 2 equivalents of ApoA-I, since ApoA-I is present per molecule_MContains twice as many amphipathic helices as ApoA-I per molecule. In contrast, a peptide apolipoprotein containing one amphipathic helix may be represented as 1/10-1/6 equivalents of ApoA-I, since it contains amphipathic helices per molecule that are 1/10-1/6 per molecule of ApoA-I. Generally, charged lipoprotein complexes (defined herein as "R_i") the molar ratio of lipid to ApoA-I equivalents ranges from about 2: 1 to 100: 1. In some embodiments, R_iAbout 50: 1. The weight ratio can be obtained using phospholipids with MW of about 650-800.

Can be modified by changing R_iTo control the size of the charged lipoprotein complex. In other words, R_iThe smaller the disc. For example, large disc-shaped discs typically have an R_iIn the range of about 200: 1 to 100: 1, while small disk-shaped disks generally have an R_iIn the range of about 100: 1 to 30: 1.

In some particular embodiments, the charged lipoprotein complex is a large disk-shaped disc containing 2-4 equivalents of ApoA-I (e.g., 2-4 molecules of ApoA-I, 1-2 molecules of ApoA-I)_MDimer or 6-10 single helical peptide molecules) and,1 molecule of charged phospholipid and a total of 400 molecules of neutral phospholipid. In other particular embodiments, the charged lipoprotein complex is a small disk-shaped disc containing 2-4 equivalents of ApoA-I, 1 molecule of charged phospholipid and a total of 200 molecules of neutral phospholipid.

The various apolipoproteins and/or phospholipid molecules including the charged lipoprotein complexes may be labeled using any art-known, detectable label, including stable isotopes (e.g.,¹³C、¹⁵N、²h, etc.), a radioisotope (e.g.,¹⁴C、³H、¹²⁵i, etc.), fluorophores, chemiluminescent, or enzyme labels.

6.3 method for preparing charged lipoprotein complexes

The charged lipoprotein complexes described herein can be prepared in a variety of forms including, but not limited to, vesicles, liposomes, proteoliposomes, micelles, and discotic particles. The charged lipoprotein complexes can be prepared using a variety of methods well known to those skilled in the art. A number of techniques that can be used to prepare liposomes or proteoliposomes can be used. For example, the apolipoprotein can be sonicated (using a sonication bath or probe sonicator) with a suitable phospholipid to form a complex. Alternatively, the apolipoprotein may be combined with preformed lipid vesicles to spontaneously form charged lipoprotein complexes. The charged lipoprotein complex can also be formed by detergent dialysis or by using an extrusion device or by homogenization, for example, a mixture of apolipoprotein, charged phospholipid SM and/or lecithin and a detergent such as cholate is dialyzed to remove the detergent and reconstitute the charged lipoprotein complex (see, e.g., Jonas et al, 1986, Methods in enzymol.128: 553-82).

In certain embodiments, the charged lipoprotein complexes are prepared by the cholate dispersion method described in example 1 of U.S. publication No. 2004/0067873, the disclosure of which is incorporated herein by reference. Briefly, dried lipids are in NaHCO₃Hydration was performed in buffer, followed by vortexing and sonication until all lipids were dispersed. The cholate solution was added and the mixture was incubated for 30 minutes with regular vortexing and sonication until the mixture became clear, at which point it was shown that lipid cholate micelles formed. NaHCO added with original ApoA-I₃Buffered and the solution incubated at about 37 ℃ to 50 ℃ for 1 hour. The ratio of lipid to pro-ApoA-I in solution may range from 1: 1 to 200: 1 (moles/mole), but in some embodiments the ratio is a lipid weight to protein weight ratio (wt/wt) of about 2: 1.

Bile salts may be removed by methods well known in the art. For example, cholate may be removed by dialysis, ultrafiltration, or the cholate molecules may be removed by adsorption onto affinity beads (affinity beads) or resins. In one embodiment, affinity BEADS, such as BIO-BEADS(Bio-Rad laboratories) was added to the charged lipoprotein complex and cholate preparation to adsorb cholate. In another embodiment, the preparation, such as a micellar preparation of a charged lipoprotein complex with cholate, is passed through a column packed with affinity beads.

In a particular embodiment, the BIO-BEADS is prepared by loading the formulation into a syringeIn the above, cholate was removed from the charged lipoprotein complex preparation. The syringe was then sealed with a septum and incubated overnight at 4 ℃ with shaking. Before use, the solution is injected through BIO-BEADSRemoving cholate in BIO-BEADSIs adsorbed by the beads.

The charged lipoprotein complexes are expected to have extended circulating half-lives when the complexes have a size and density similar to HDL, particularly similar to HDL in the pre- β -1 or pre- β -2 HDL populations. A stable formulation with a longer shelf life can be prepared by freeze-drying. For example, the co-freeze drying process described below provides a stable formulation and makes the formulation/particle preparation process easier to perform. U.S. Pat. No. 6,287,590 also describes co-lyophilization (Dasseux et al, entitled "Peptide/lipid complex formation by co-lyophilization", published 2001, 9, 11), which is incorporated herein by reference in its entirety. Frozen charged lipoprotein complexes can be used to prepare bulk supplies (bulk supplies) for pharmaceutical reconstitution or as individual aliquots or dosage units that can be reconstituted by rehydration with sterile water or a suitable buffer prior to administration to a subject.

U.S. patent nos. 6,004,925, 6,037,323, 6,046,166 and 6,287,590, which are incorporated herein by reference in their entirety, disclose a simple method for preparing charged lipoprotein complexes having HDL-like properties. This method involves co-lyophilization of apolipoprotein with a lipid solution in an organic solvent (or solvent mixture) and formation of a charged lipoprotein complex from the lyophilized powder during hydration, and has the following advantages: (1) the method requires few steps; (2) the method uses a low-cost solvent; (3) most or all of the included ingredients are used to form the designed complex, thus avoiding waste of starting materials common to other processes; (4) lyophilized complexes are formed which are stable during storage, thus allowing reconstitution of the resulting complex immediately prior to use; (5) the resulting complex usually does not need further purification after formation and before use; (6) avoiding toxic compounds, including detergents such as cholates; and (7) the manufacturing process is easy to industrialize and suitable for GMP production (i.e., in an endotoxin-free environment).

In some embodiments, the charged lipoprotein complexes are prepared using a co-freeze drying process, which is generally known in the art. Briefly, the co-lyophilization step involves dissolving the apolipoprotein ("Apo") in an organic solvent or solvent mixture with the phospholipid, or dissolving Apo and phospholipid separately and mixing them together. The desired characteristics of the solvent or solvent mixture are: (i) the relative polarity of the medium capable of dissolving the hydrophobic lipids and the amphiphilic proteins, (ii) the solvent should be of class 2 or 3 according to FDA solvent guidelines (federal gazette, 247, volume 62) to avoid potential toxicity associated with residual organic solvents, (iii) have a lower boiling point to ensure easier solvent removal during lyophilization, (iv) have a higher melting point to provide faster freezing, higher condensation temperatures, and thus less lyophilization vessels. In a preferred embodiment, glacial acetic acid is used. Combinations such as methanol, glacial acetic acid, xylene or cyclohexane may also be used.

The Apo/lipid solution is then freeze-dried to obtain a homogeneous Apo/lipid powder. The conditions of freeze-drying may be optimised to obtain rapid evaporation of the solvent and to minimise the amount of residual solvent in the freeze-dried Apo/lipid powder. The choice of freeze-drying conditions can be determined by one skilled in the art and depends on the nature of the solvent, the type and size of container (e.g. bottle), the solution contained, the fill volume and the performance of the freeze-dryer used. To remove organic solvents and successfully form the complex, the concentration of the lipid/Apo solution before lyophilization may range from 10 to 50mg/ml concentration equivalent of Apo a-I, and from 20 to 100mg/ml concentration of lipid.

The Apo-lipid complex is formed spontaneously upon hydration of the Apo-lipid lyophilized powder with an aqueous medium having a suitable pH and osmotic pressure. In some embodiments, the medium may also contain stabilizers such as sucrose, trehalose, glycerol and others. In some embodiments, to form the lipid complex, the solution must be heated several times above the transition temperature. For successful formation of charged lipoprotein complexes, the molar ratio of lipid to protein can be from 2: 1 to 200: 1 (expressed as ApoA-I equivalent) and is preferably a lipid to protein weight ratio (wt/wt) of about 2: 1. The powder was hydrated to obtain a final complex concentration of about 5-30mg/ml expressed as ApoA-I protein equivalent.

In some embodiments, by NH of Apo solution₄CO₃The solution was freeze-dried to obtain Apo powder. A homogeneous solution of Apo and lipid is formed by dissolving lipid powder with Apo in glacial acetic acid. The solution is then lyophilized and a charged lipoprotein complex similar to HDL is formed by hydrating the lyophilized powder with an aqueous medium.

In some embodiments, homogenization is used to prepare Apo-lipid complexes. The method is also useful for the preparation of Apo soy-PC complexes and it is generally used for the formulation of ApoA-I_M-a POPC complex. Homogenization may be more suitable for the formation of charged lipoprotein complexes. Briefly, the method comprises the steps of: by Ultraturex^TMA suspension of lipids in an aqueous Apo solution is formed and the resulting lipid-protein suspension is homogenized using a high pressure homogenizer until the suspension becomes a clear milky white solution and a complex is formed. Temperatures above the lipid transition are used during homogenization. The solution is homogenized for a further period of 1-14 hours and pressurized.

In some embodiments, charged lipoprotein complexes can be formed by co-lyophilization of phospholipids and peptide or protein solutions or suspensions. Homogeneous solutions of peptides/proteins, charged phospholipids, SM and/or lecithin (plus any other selected phospholipids) in organic solvents or organic solvent mixtures can be lyophilized, and charged lipoprotein complexes can be spontaneously formed by hydrating the lyophilized powder with an aqueous buffer. Examples of organic solvents or mixtures thereof include, but are not limited to, acetic acid and xylene, acetic acid and cyclohexane, and methanol and xylene.

Suitable ratios of protein (peptide) to lipid can be determined empirically so that the resulting complex has suitable physical and chemical properties, i.e., generally (but not necessarily) similar size to HDL. The resulting mixture of Apo and lipid in solvent is frozen and lyophilized to dryness. Sometimes additional solvent must be added to the mixture to facilitate lyophilization. It is desirable that the lyophilized product be capable of being stored for an extended period of time and remain stable.

The lyophilized product can be reconstituted to obtain a solution or suspension of the charged lipoprotein complex. To this end, the lyophilized powder may be rehydrated with an aqueous solution to a suitable volume (typically 5-20mg charged lipoprotein complex/ml) suitable for use, for example, for intravenous injection. In a preferred embodiment, the lyophilized powder is rehydrated with a phosphate buffer, bicarbonate, or physiological saline solution. The mixture may be agitated or vortexed to smooth rehydration. Generally, this reconstitution step should be performed at or above the phase transition temperature of the lipid component of the complex. Within a few minutes of reconstitution, a clear preparation of reconstituted charged lipoprotein complex should be obtained.

Other methods include spray drying, in which the solution is sprayed out and the solvent is evaporated (at elevated temperature or reduced pressure). The lipid and apolipoprotein may be dissolved in the same solvent or different solvents. The powder charge can then be filled into bottles.

Mechanical methods can also be used to mix the apolipoprotein with a lyophilized powder of the lipid. The homogeneous powder containing the apolipoprotein and lipid may then be hydrated to spontaneously form a complex of the appropriate size and appropriate lipid to apolipoprotein molar ratio.

Aliquots of the resulting reconstituted formulation may be characterized to confirm that the complex in the formulation has a desired particle size distribution, such as that of HDL. Identification of the reconstituted formulation may be performed using any method known in the art including, but not limited to, size exclusion filtration (size exclusion filtration), gel filtration, column filtration, gel permeation chromatography, and native gel electrophoresis.

For example, after hydration of the lyophilized charged lipoprotein powder or at the end of homogenization, or at the end of cholate dialysis to form Apo-lipid particles similar to HDL, its size, concentration, final pH of the resulting solution, and osmotic pressure, and in some instances, the integrity of the lipids and/or apolipoproteins. The particle size of the resulting charged lipoprotein particles is a determining factor in their integrity, and therefore this measurement typically includes the characteristics of the particles.

In some embodiments, Gel Permeation Chromatography (GPC) can be used, such as with a 1X 30cm Superdex^TMColumn (Pharmacia Biotech) and UV detector. The complex was eluted using a bicarbonate buffer comprising 140mM NaCl and 20mM sodium bicarbonate and delivered at a flow rate of 0.5 ml/min. Typical amounts of injected complex are 0.1 to 1mg based on protein weight. The complex can be detected by absorbance at 280 nm.

Protein and lipid concentrations in the charged lipoprotein particle solution can be measured by any method known in the art, including but not limited to protein and phospholipid analysis and chromatography, such as HPLC, gel filtration chromatography, GC coupled to various detectors including mass spectrometry, UV or diode arrays, fluorescence, elastic light scattering, and others. Lipid and protein integrity can also be determined by the same chromatographic techniques as well as peptide spectroscopy, SDS-page gels, N-and C-terminal protein sequences and standard assays for detecting lipid oxidation.

The homogeneity and/or stability of the charged lipoprotein complexes or compositions described herein can be measured by any method known in the art, including, but not limited to, chromatography, such as gel filtration chromatography. For example, in some embodiments, a single peak or a limited number of peaks are associated with a stable complex. The stability of the complex can be determined by monitoring the appearance of new peaks over time. The appearance of a new peak becomes a marker for recombination in the complex due to particle instability.

The optimal ratio of phospholipids to apolipoproteins in the charged complex can be determined using any number of functional assays known in the art including, but not limited to, gel electrophoresis mobility assays, size exclusion chromatography, interaction with HDL receptors, recognition via ATP-binding cassette transporter (ABCA1), hepatic uptake, and pharmacokinetics/pharmacodynamics. For example, gel electrophoresis mobility detection can be used to determine the optimal ratio of phospholipid to apolipoprotein in a charged complex. The charged complexes described herein should exhibit electrophoretic mobilities similar to native pre- β -HDL or α -HDL particles. Thus, in some embodiments, native pre- β -HDL or α -HDL particles can be used as standards for determining the mobility of charged complexes.

As another example, size exclusion chromatography can be used to determine the particle size of the charged complexes described herein as compared to native pre- β -HDL particles. Native pre- β -HDL particles typically do not exceed 10-12nm, and the periphery of the discoidal particles typically ranges from 7-10 nm.

As another example, HDL receptors can be used in functional assays to identify which complex is closest to the native pre- β -HDL particle, or which complex is most effective in removing and/or mobilizing cholesterol or lipids from cells. In one assay, the complex can be tested for its ability to bind to the ABCA-1 receptor. Such detection relies on the independent removal of cholesterol to distinguish ABCA-1. While ApoA-I is believed to be the best ligand for such assays, complexes such as small micelles or small discotic particles are also potential ABCA-I ligands. ABCA-1 binding assays that can be used are described in Brewer et al, 2004, Arterioscler, Thromb, Vase, biol.24: 1755-1760).

As another example, it is known that cells expressing ABCA1 recognize free ApoA-1 and reduce native pre- β -HDL particles to a lesser extent (Brewer et al, 2004, Arteriosclar. Thromb. Vase. biol. 24: 1755) 1760 in these embodiments, ABCA1 cells recognizing native pre- β -HDL particles can be compared to any one of the charged complexes described herein in order to identify complexes that are extremely similar to native pre- β -HDL particles.

One relatively simple method for identifying charged complexes that are extremely similar to native pre- β -HDL particles is to perfuse the liver with a solution containing reconstituted charged complexes and measure the amount taken up by the liver.

In some embodiments, the pharmacokinetics/pharmacodynamics (PK/PD) of the charged complex can be measured using a single injection in rabbits. In these embodiments, the concentration of ApoA-1 is used as an indicator of kinetics. Pharmacodynamics, i.e., the amount of cholesterol flowing on the baseline and the amount of cholesterol in the HDL fraction, can be measured after one injection. PK and PD depend on the nature of the phospholipid, the composition of the phospholipid, the molar ratio of lipid to apolipoprotein and the concentration of phospholipid in the complex. For example, Dipalmitoylphosphatidylcholine (DPPC)/ApoA-1 complex has a longer half-life than Egg Phosphatidylcholine (EPC)/ApoA-I complex. The sphingomyelin/ApoA-1 complex has a longer half-life than the EPC/ApoA-1 complex. Human ApoA-1 has a half-life in humans of about 5 to 6 days.

In another embodiment, the pharmacodynamics of the charged complex can be measured by tracking the rate of esterification of cholesterol in the HDL fraction over time. LCAT is the only enzyme responsible for the esterification of cholesterol in the blood. The rate of cholesterol esterification is a good parameter for assessing the quality of the particles. LCAT acts as a molecular probe, and if the quaternary complex is recognized by LCAT, the esterification rate is higher. This means that the surface is ideal, the charge is ideal, the morphology is ideal and both substrates are reactive (LCAT first hydrolyses the acyl chain of the phospholipid (esterase activity) and subsequently esterifies the free OH of cholesterol (esterase activity) to form cholesterol esters) and at the correct concentration. Furthermore, it means that the particles have good size and composition to dissolve and capture the products of the following reactions: lysophospholipids and cholesterol esters, otherwise the reaction will stop.

6.4 pharmaceutical compositions

The pharmaceutical compositions contemplated by the summary of the invention comprise as an active ingredient a charged lipoprotein complex contained in a pharmaceutically acceptable carrier suitable for in vivo administration and delivery. Since the peptide may contain acidic and/or basic termini and/or side chains, the peptidomimetic apolipoproteins may be included in the composition in the form of a free acid or base, or in the form of a pharmaceutically acceptable salt. Modified proteins, such as amidated, acylated, acetylated or pegylated proteins, may also be used.

Injectable compositions include sterile suspensions, solutions or emulsions of the active ingredient in aqueous or oily vehicles. The composition may also include various formulation ingredients such as suspending, stabilizing and/or dispersing agents. Compositions for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, and may include an added preservative. For infusion, the composition may be supplied in an infusion bag made of a material compatible with the charged lipoprotein complex, such as ethylene vinyl acetate or any other compatible material known in the art.

Alternatively, the injectable compositions may be provided in powder form for constitution with a suitable vehicle, including, but not limited to, sterile, pyrogen-free water, buffer, dextrose solution, and the like, before use. For this purpose, Apo can be lyophilized or a co-lyophilized charged lipoprotein complex can be prepared. The stored compositions may be supplied in unit dosage form and reconstituted prior to use in vivo.

For prolonged delivery, the active ingredient may be formulated as a depot composition for implant administration, e.g., subcutaneous, intradermal, or intramuscular injection. Thus, for example, Apo-lipid complexes or apolipoproteins may be formulated alone with suitable polymeric or hydrophobic materials (e.g. as an emulsion in an acceptable oil) or in phospholipid foams or ion exchange resins.

Alternatively, transdermal delivery systems prepared as suction cups or patches can be used, which slowly release the active ingredient absorbed transdermally. For this purpose, a penetration enhancer may be used to facilitate transdermal penetration of the active ingredient. Particular benefits are obtained by introducing the charged complexes described herein into nitroglycerin patches for patients with ischemic heart disease and hypercholesterolemia.

Alternatively, local or intramural (intravascular wall) delivery may be performed using a catheter or injector (see, e.g., U.S. publication 2003/0109442).

If desired, the composition may be presented in a pack or dispenser device which may include one or more unit dosage forms containing the active ingredient. For example, the package may comprise a metal or plastic foil, such as a blister pack. The packaging or dispenser device may be accompanied by instructions for administration.

6.5 methods of treatment

Indeed, the charged lipoprotein complexes and compositions described herein can be used for a variety of purposes for which lipoprotein complexes have been shown to be useful. In particular embodiments, the complexes and compositions may be used to treat or prevent dyslipidemia and/or virtually any disease, disorder and/or condition associated with dyslipidemia. As used herein, the term "dyslipidemia" or "dyslipidemic" refers to an abnormal increase or decrease in plasma lipid concentration, including but not limited to changes in lipid concentration associated with: coronary heart disease, coronary artery disease, cardiovascular disease, hypertension, restenosis, vascular or peripheral vascular disease, disorders of lipid metabolism, dyslipoproteinemia, high concentrations of low density lipoprotein cholesterol, high concentrations of very low density lipoprotein cholesterol, low concentrations of high density lipoprotein, high concentrations of lipoprotein lp (a) cholesterol, high concentrations of apolipoprotein B, atherosclerosis (including the treatment and prevention of atherosclerosis), hyperlipidemia, hypercholesterolemia, Familial Hypercholesterolemia (FH), Familial Complex Hyperlipidemia (FCH), lipoprotein lipase deficiencies, such as hypertriglyceridemia, alpha-hypolipoproteinemia, and hypercholesterolemia (hypercholesterolemia).

Dyslipidemia-associated diseases include, but are not limited to, coronary heart disease, coronary artery disease, acute coronary syndrome, cardiovascular disease, hypertension, restenosis, vascular or peripheral vascular disease, disorders of lipid metabolism, dyslipoproteinemia, high concentrations of low-density lipoprotein cholesterol, high concentrations of very low-density lipoprotein cholesterol, low concentrations of high-density lipoprotein, high concentrations of lipoprotein lp (a) cholesterol, high concentrations of apolipoprotein B, atherosclerosis (including the treatment and prevention of atherosclerosis), hyperlipidemia, hypercholesterolemia, Familial Hypercholesterolemia (FH), Familial Complex Hyperlipidemia (FCH), lipoprotein lipase deficiencies, such as hypertriglyceridemia, alphaproteolipidemia, and hypercholesterolemia.

It is expected that phospholipids in the dosage range of about 2 to 25 times lower than the currently known effective dosages in the art (expressed as ApoA-I equivalents) can be used to effectively treat or prevent diseases or can bring about improved therapeutic effects by using the charged lipoprotein complexes and compositions described herein.

In one embodiment, the method comprises a method of treating or preventing a disease associated with dyslipidemia, comprising the steps of: administering to the subject an effective amount of a charged lipoprotein complex or composition described herein such that, at least one day after administration, a serum concentration of free or complexed apolipoprotein in the range of about 10mg/dL to 300mg/dL greater than the baseline concentration (initial) prior to administration is achieved.

In another embodiment, the method comprises a method of treating or preventing a disease associated with dyslipidemia, comprising the steps of: administering to the subject an effective amount of a charged lipoprotein complex or composition described herein such that, at least one day after administration, a circulating plasma concentration of the HDL-cholesterol component that is at least about 10% higher than the initial HDL-cholesterol component prior to administration is achieved.

In another embodiment, the method comprises a method of treating or preventing a disease associated with dyslipidemia, comprising the steps of: administering to the subject an effective amount of a charged lipoprotein complex or composition described herein such that circulating plasma concentrations of the HDL-cholesterol component of 30 and 300mg/dL are achieved between 5 minutes and 1 day after administration.

In another embodiment, the method comprises a method of treating or preventing a disease associated with dyslipidemia, comprising the steps of: administering to the subject an effective amount of a charged lipoprotein complex or composition described herein such that circulating plasma concentrations of cholesterol esters of 30 and 300mg/dL are achieved between 5 minutes and 1 day after administration.

In yet another embodiment, the method comprises a method of treating or preventing a disease associated with dyslipidemia, comprising the steps of: administering to the subject an effective amount of a charged lipoprotein complex or composition described herein such that at least one day after administration, an increased fecal cholesterol secretion of at least about 10% greater than the baseline (initial) concentration prior to administration is obtained.

The charged lipoprotein complexes or compositions described herein can be used alone or in combination therapy with other agents for treating or preventing the aforementioned conditions. Such treatment includes, but is not limited to, simultaneous or sequential administration of the included drugs. For example, in the treatment of hypercholesterolemia or atherosclerosis, the charged lipoprotein formulation can be administered with any one or more of the cholesterol-lowering therapies currently in use, such as bile acid resins, nicotinic acid, statins, cholesterol absorption inhibitors, and/or fibrates. Since each drug acts on a different target in the synthesis and transport of cholesterol, such a combination regimen may lead to specific beneficial effects, i.e., bile acid resins affect cholesterol recirculation, chylomicrons and LDL populations; nicotinic acid mainly affects VLDL and LDL populations; statins inhibit cholesterol synthesis, reduce the LDL population (or increase the expression of LDL receptors); the charged lipoprotein complexes described herein, in turn, affect RCT, increase HDL and promote cholesterol efflux.

In another embodiment, the charged lipoprotein complexes or compositions described herein can be used in combination with a fibrate to treat or prevent coronary heart disease, coronary artery disease, cardiovascular disease, hypertension, restenosis, vascular or peripheral vascular disease, disorders of lipid metabolism, dyslipoproteinemia, high concentrations of low density lipoprotein cholesterol, high concentrations of very low density lipoprotein cholesterol, low concentrations of high density lipoprotein, high concentrations of lipoprotein lp (a) cholesterol, high concentrations of apolipoprotein B, atherosclerosis (including the treatment and prevention of atherosclerosis), hyperlipidemia, hypercholesterolemia, Familial Hypercholesterolemia (FH), Familial Complex Hyperlipidemia (FCH), lipoprotein lipase deficiencies such as hypertriglyceridemia, hypoalphalipoproteinemia, and hypercholesterolemia. Exemplary formulations and treatment regimens are described below.

The charged lipoprotein complexes or compositions described herein can be administered by any suitable route that ensures circulatory bioavailability. An important feature of an embodiment comprising SM is that the charged lipoprotein complex can be administered at a dose 1-10% lower than the expected effective dose for administration of apolipoprotein (Apo) or Apo peptide alone; and at a dose 2-25 times lower than the effective dose required for Apo-soy PC (or Apo-egg PC or Apo-POPC) administration. It is desirable to administer as low a dose of apolipoprotein (for intravenous injection) as about 40mg to 2g apolipoprotein per person every 2 to 10 days, rather than the large amount of apolipoprotein required for currently available treatment regimens (20 mg/kg to 100mg/kg per 2 to 5 days per 1.4g to 8g average adult body weight).

The charged lipoprotein complexes or compositions described herein may be administered in doses that increase the small HDL components, such as pre-beta, pre-gamma and pre-beta-like HDL components, alpha HDL components, HDL3 and/or HDL2 components. In some embodiments, the dose is effective to achieve a reduction in atherosclerotic plaques as measured via an imaging technique, such as Magnetic Resonance Imaging (MRI) or intravascular ultrasound (IVUS). Parameters tracked by IVUS include, but are not limited to, the percentage of change in atheroma volume from baseline and the change in total atheroma volume; parameters that are MRI tracked include, but are not limited to, those of IVUS, as well as lipid composition and calcification of plaque.

Regression of plaques (time 0 versus time t) can be measured at the end of the last infusion, or within weeks after the last infusion, or within 3 months, 6 months, or 1 year after treatment initiation using the patient as a self-control.

Optimal administration can be achieved by parenteral routes of administration, which include Intravenous (IV), Intramuscular (IM), intradermal, Subcutaneous (SC), and Intraperitoneal (IP). In certain embodiments, administration is via a syringe, inhaler, or catheter. In some embodiments, the charged lipoprotein complex is administered by injection, subcutaneous implantation pump or depot preparation in an amount to achieve a circulating serum concentration equivalent to that obtained by parenteral administration. For example, the complex may also be absorbed into a stent or other device.

Administration can be achieved by a variety of different treatment regimens. For example, several intravenous injections may be administered periodically throughout the day, where the cumulative total volume of injections does not reach the daily toxic dose. Alternatively, the intravenous injection may be administered about every 3 to 15 days, preferably about every 5 to 10 days, and most preferably about every 10 days. In yet another alternative, increasing doses may be administered, beginning at a dose between each administration (50-200mg) about 1 to 5 times, followed by a repeat of the dose between 200mg and 1g at each administration. Depending on the patient's needs, it may be administered by a slow infusion lasting more than 1 hour, by a fast infusion for 1 hour or less, or by a single fast bolus injection.

In some embodiments, regular administration of injections may be performed, followed by 6 months to 1 year of cessation, followed by another series of administrations. The injection maintenance series may then be administered annually or every 3 to 5 years. The series of injections may be administered within one day (perfused to maintain a particular plasma concentration of the complex), within several days (e.g., four injections over an eight day period), or within several weeks (e.g., four injections over a four week period), and then restarted six months to one year later.

Other routes of administration may be used. For example, absorption through the gastrointestinal tract may be achieved by oral routes of administration (including, but not limited to, swallowing, buccal and sublingual routes) provided by suitable formulations (e.g., enteric coatings) which may be used to avoid or minimize degradation of the active ingredient in harsh environments such as the oral mucosa, stomach and/or small intestine. Alternatively, administration via mucosal tissue, such as vaginal and rectal modes of administration, may be employed to avoid or minimize degradation in the gastrointestinal tract. In other embodiments, the formulations of the present invention may be administered transdermally (e.g., transdermally) or by inhalation. It will be appreciated that the preferred route may vary according to the condition, age and compliance of the recipient.

The actual dosage of the charged lipoprotein complexes or compositions described herein may vary depending on the route of administration.

Data obtained from the animal model systems described in U.S. patent nos. 6,004,925, 6,037,323, and 6,046,166 (issued to dasseeux et al, incorporated herein by reference in its entirety) indicate that ApoA-I peptides are associated with HDL components and are expected to have a half-life of about 5 days in humans. Thus, in some embodiments, the charged lipoprotein complexes can be administered by intravenous injection at the following doses: every 2 to 10 days, between about 0.1g-1g of charged lipoprotein complex per adult human per average body weight is administered.

Toxicity and therapeutic efficacy of various charged lipoprotein complexes can be determined using standard pharmaceutical procedures in cell culture or using experimental animals for determining LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is a therapeutic index and it can be expressed as the ratio LD50/ED 50. Charged lipoprotein complexes exhibiting higher therapeutic indices are preferred. Non-limiting examples of trackable parameters include liver function transaminase (not exceeding 2 × normal baseline levels). It indicates that too much cholesterol is transported to the liver and cannot be mobilized in such large amounts. The effect on the red blood cells can also be monitored, as cholesterol efflux from the red blood cells can embrittle the red blood cells or affect their shape.

Patients may receive treatment for days to weeks before, during, or after taking medical measures (e.g., prophylactic treatment). The administration can be performed sequentially or concurrently with another interventional therapy, such as angioplasty, carotid ablation, rotoblader, or organ transplantation (e.g., heart, kidney, liver, etc.).

In certain embodiments, the charged lipoprotein complex is administered to a patient whose cholesterol synthesis is controlled by a statin or cholesterol synthesis inhibitor. In other embodiments, the charged lipoprotein complexes are administered to a patient receiving treatment with a binding resin (e.g., cholestyramine-binding semi-synthetic resin) or fiber (e.g., plant fiber) to capture bile salts and cholesterol to increase bile acid secretion and reduce blood cholesterol concentrations.

6.6 other uses

For example, the charged lipoprotein complexes and compositions described herein can be used in vitro assays to measure serum HDL for diagnostic purposes. Since ApoA-I, ApoA-II and Apo peptides are associated with HDL components in serum, charged lipoprotein complexes can be used as "markers" for HDL populations and pre-beta 2 HDL populations. In addition, the charged lipoprotein complexes can be used as markers for HDL subpopulations that are active in RCT. To this end, the charged lipoprotein complexes can be added to or mixed with a serum sample of the patient and, after a suitable incubation time, the HDL fraction can be analyzed by detecting the introduced charged lipoprotein complexes. This can be achieved by using a labeled charged lipoprotein complex (e.g., radiolabel, fluorescent label, enzyme label, dye, etc.) or by using an immunoassay with antibody (or antibody fragment) specificity for the charged lipoprotein complex.

Alternatively, imaging methods may use labeled charged lipoprotein complexes (e.g., CAT scans, MRI scans) to image the circulatory system or to monitor RCT, or to image HDL that accumulates in the place of fatty streaks, atherosclerotic lesions, etc., where HDL is active in cholesterol efflux.

Examples and data relating to the preparation and properties of certain pro-ApoA-1 lipid complexes are described in U.S. patent publication No. 2004/0067873, which is incorporated herein by reference in its entirety.

Data obtained from animal model systems using certain pro-ApoA-1 lipid complexes are described in U.S. patent publication No. 2004/0067873, which is incorporated herein by reference in its entirety.

7. Examples of the embodiments

Example 1: preparation of ProApoA-I, sphingomyelin and phosphatidylglycerol

A lyophilisate of the original ApoA-I protein containing about 90mg of protein per 100mL vial is supplied by Unitrede Biotechnology, Institut Meurice, Hte Ecole Luciade Brouckere, 1Avenue Emile Gryzon, B-1070 Anderecht, Belgium. Lot number 20060202. The protein was stored at about 4 ℃ prior to use. Prior to lyophilization, the concentration of pro-ApoA-I was 3.225mg/mL, with urea at about 0.011 mg/mL. A solution of pro-ApoA-I was prepared by dissolving about 630mg of pro-ApoA-I in 25.6mL of acetic acid/5% water. The final concentration of the solution was 25 mg/mL.

Sphingomyelin (Coatsome) from eggsNM-10) was supplied by NOF corporation, l-56, Oohama-Cho, Amagasaki-Shi, 660-. Lot number 0502ES 1. Sphingomyelin is stored at about-20 ℃ before use. The purity of sphingomyelin was 99.1%. By dissolving 799.4mg of purified sphingomyelinThe sphingomyelin solution was prepared dissolved in 16mL of acetic acid/5% water to give a final concentration of 50 mg/mL.

Sodium salt of 1, 2-dipalmitoyl-SN-glyceryl-3-phosphatidylglycerol (DPPG-Na, Coatsome)MG-6060LS) was supplied from NOF corporation, 1-56, Oohama-Cho, Amagasaki-Shi, 660-. Lot number 0309651L. DPPG-Na was stored at about 4 ℃ prior to use. The purity of DPPG-Na is 99.2%. A DPPG-Na solution was prepared by dissolving 49.1mg of DPPG-Na in 1mL of acetic acid/5% water to give a final concentration of 50 mg/mL.

Example 2: preparation of uncharged control lipoprotein complexes

An uncharged control lipoprotein complex consisting of pro-Apo-AI (33 wt%) and sphingomyelin (67 wt%) was prepared as described below.

The preparation of uncharged control lipoprotein complex was prepared by mixing 5.6mL of pro-Apo-AI at a concentration of 25mg/mL with approximately 5.6mL of sphingomyelin at a concentration of 50mg/mL in a 100mL glass vial. The resulting mixture was filtered through a 0.22 μm nylon filter. The mixture was heated at about 50 ℃ and then frozen in liquid nitrogen with manual stirring. Immediately after freezing, the bottle was placed in a lyophilizer for 15 hours. After lyophilization, the vial was placed under vacuum at about 40 ℃ for 4 hours. The resulting formulation was stored at about 4 ℃ prior to use.

14mL of a solution containing 140mM NaCl and 20mM NaHCO₃The solution of (a) was added to a glass vial containing the uncharged control lipoprotein complex lyophilized formulation. The resulting solution was adjusted to a basic pH by adding 0.75mL of 1M NaOH to 20mL of solution. The solution was stirred manually, heated at about 50 ℃ and then placed in an ultrasonic bath for at least 1 hour. The concentration of pro-ApoA-I in the resulting formulation was 10 mg/mL. Injecting the formulation into an HPLC system to detect the presence of uncharged lipoprotein complexesA compound (I) is provided. FIG. 1 provides an example of an HPLC chromatogram of an uncharged lipoprotein complex prepared as described herein.

Example 3: preparation of the tested charged lipoprotein complexes

Charged lipoprotein complexes consisting of pro-Apo-AI (33 wt%), sphingomyelin (65 wt%) and phosphatidylglycerol (2 wt%) were prepared as described below.

The preparation of charged lipoprotein complex was prepared by mixing 5.6mL of pro-ApoA-I at a concentration of 25mg/mL with about 5.6mL of sphingomyelin at a concentration of 50mg/mL and about 0.15mL of DPPG-NA at a concentration of 50mg/mL in a 100mL glass vial, and the resulting mixture was subsequently filtered through a 0.22 μm nylon filter. The mixture was heated at about 50 ℃ and frozen in liquid nitrogen with manual stirring. Immediately after freezing, the bottle was placed in a lyophilizer for 15 hours. After lyophilization, the vial was placed under vacuum at about 40 ℃ for 4 hours. The resulting formulation was stored at about 4 ℃ prior to use.

14mL of 140mM NaCl and 20mM NaHCO₃Adding into glass bottle containing the above lyophilized preparation. The resulting solution was adjusted to a basic pH by adding 0.75mL of 1M NaOH to 20mL of solution. The solution was stirred manually, heated at about 50 ℃ and then placed in an ultrasonic bath for at least 1 hour. The concentration of pro-ApoA-I in the resulting formulation was 10 mg/mL. The formulation was injected into an HPLC system to detect the presence of uncharged lipoprotein complexes. FIG. 2 provides an example of an HPLC chromatogram of a charged lipoprotein complex prepared as described herein.

Example 4: animal model system

Cholesterol mobilized by the uncharged complexes described above as well as by charged complexes was determined using new zealand male rabbits weighing between 3 and 4 kg. The animals were supplied by CEGAV in france and identified individually with unique ear tattoos. Rabbits were housed individually in individual cages at the Avogadro (France) animal facility. The housing and care of the animals meet the criteria of directive 86/609/EEC. The Avogadro animal facility has protocol number B3118801, obtained by the french veterinary agency. All animals were similarly managed and given proper care for their health according to common practice and current Standard Operating Protocols (SOPs) by avogado. Regularly cleaning the equipment and the animal house in due time.

The conditions of the animal chamber were as follows: temperature: 22 ± 2 ℃, relative humidity: 55. + -. 15% and a cycle of 12 hours light/12 hours dark was performed. Temperature and relative humidity were recorded daily and the raw data of the study was retained. Each rabbit was observed once a day, and any abnormal results observed were recorded and reported to the experimental responsible.

Animals were acclimated for at least 7 days before study initiation. Animals were randomly received a granular control diet on a daily basis. Water was ad libitum throughout the study.

Animals were fasted overnight prior to administration of the complex. Animals were weighed just prior to administration of the complex. The complex was administered intravenously at a dose ratio of 15mg/kg, which is equivalent to 1.5 mL/kg. The volume administered is based on weight. Feeding was resumed approximately 6 hours after complex administration. The details of treatment recorded included dose calculation, dose administered, date and time of administration.

Animals were fasted overnight before blood samples were collected. Blood samples were taken from the jugular vein or from the auricular vein. Blood was drawn from the jugular vein using a syringe fitted with a needle and EDTA (approximately 1mL of blood was drawn at the time of each sampling). Blood samples were stored at about 4 ℃ immediately after collection to avoid blood sample changes. Blood samples were centrifuged (3500 g for 10 minutes at about 5 ℃). Plasma samples were separated and aliquoted (at least 3 aliquots of 200. mu.L (aliquot A, B, C)), and then stored at about-80 ℃. The remaining blood clot is discarded.

Example 5: cholesterol mobilization by charged lipoprotein complexes

Control lipoprotein complex (formulation IIA) or charged lipoprotein complex (formulation IIB) was prepared as described above and administered to rabbits (15mg complex/kg body weight); two rabbits each.

Blood samples (1ml) were taken before dose administration, 5min, 15min, 30min, 1h, 2h, 3h and 6h after dose administration. Plasma samples were analyzed for total cholesterol, free cholesterol, and triglycerides according to the disclosed methods (see, e.g., Usui, s. et al, 2002, j. lipid res., 43: 805-14). The esterified cholesterol concentration was calculated by subtracting the free cholesterol content from the total cholesterol content. Figure 3 shows the free cholesterol in HDL results for each animal. Figure 4 shows the mean values of two animals including the control group (group IIA) and the test group (group IIB).

As expected, both the control lipoprotein complex and the test lipoprotein complex mobilized cholesterol, with a comparison of the test group mean to the control group mean indicating increased mobilization of the test group.

All references are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual text, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

The citation of any publication 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.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope thereof. The particular embodiments described are offered by way of example only, and the invention is not limited solely to the terms of the appended claims, but also covers the full scope of equivalents to which such claims are entitled.

Claims (25)

1. A lipoprotein complex comprising ApoA-I apolipoprotein and a lipid component, wherein the lipid component consists essentially of sphingomyelin and 3 wt% negatively charged phospholipid, and the molar ratio of the lipid component to the ApoA-I apolipoprotein ranges from about 2: 1 to 200: 1.

2. The lipoprotein complex of claim 1 in which the negatively charged phospholipid is selected from the group consisting of phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, phosphatidic acid and mixtures thereof.

3. The lipoprotein complex of claim 1 in which the negatively charged phospholipid is a phosphatidylglycerol.

4. The lipoprotein complex of claim 1 in which the sphingomyelin comprises D-erythrose-sphingomyelin and/or D-erythrose-dihydrosphingomyelin.

5. The lipoprotein complex of any one of claims 1 to 4 in which the acyl chains of the sphingomyelin or the negatively charged phospholipid are independently of each other selected from saturated, monounsaturated and polyunsaturated hydrocarbons containing 6 to 24 carbon atoms.

6. The lipoprotein complex of claim 5 in which each acyl chain of the sphingomyelin and/or the negatively charged phospholipid is the same.

7. The lipoprotein complex of claim 5 in which the acyl chains of the sphingomyelin and the negatively charged phospholipid contain the same number of carbon atoms.

8. The lipoprotein complex of claim 5 in which the acyl chains of the sphingomyelin and the negatively charged phospholipid have different degrees of saturation.

9. The lipoprotein complex of claim 1 in which the ApoA-I apolipoprotein is selected from human mature ApoA-I, mature ApoA-I_MilanMature ApoA-I_ParisAnd mixtures thereof.

10. The lipoprotein complex of claim 1 in which the ApoA-I apolipoprotein is in monomeric form.

11. The lipoprotein complex of claim 1 in which the ApoA-I apolipoprotein is human mature ApoA-I apolipoprotein and the negatively charged phospholipid is phosphatidylglycerol.

12. The lipoprotein complex of claim 1 in which the molar ratio of the lipid component to the ApoA-I apolipoprotein ranges from about 200: 1 to 100: 1.

13. The lipoprotein complex of claim 1 in which the molar ratio of the lipid component to the ApoA-I apolipoprotein ranges from about 100: 1 to 30: 1.

14. The lipoprotein complex of claim 1 in which the ApoA-I apolipoprotein is human mature ApoA-I apolipoprotein, the sphingomyelin is egg sphingomyelin, the negatively charged phospholipid is phosphatidylglycerol, and the molar ratio of the lipid component to the ApoA-I apolipoprotein ranges from about 200: 1 to 100: 1.

15. The lipoprotein complex of claim 1 in which the ApoA-I apolipoprotein is human mature ApoA-I apolipoprotein, the sphingomyelin is egg sphingomyelin, the negatively charged phospholipid is phosphatidylglycerol, and the molar ratio of the lipid component to the ApoA-I apolipoprotein ranges from about 100: 1 to 30: 1.

16. A pharmaceutical composition comprising a lipoprotein complex according to any one of claims 1-15 and a pharmaceutically acceptable carrier, diluent and/or excipient.

17. Use of the lipoprotein complex of any one of claims 1-15 in the manufacture of a medicament for treating dyslipidemia or a disease associated with dyslipidemia in a subject, wherein the dyslipidemia or the disease associated with dyslipidemia is peripheral vascular disease, hypertension, inflammation, alzheimer's disease, restenosis, atherosclerosis and multiple clinical manifestations of atherosclerosis; or coronary heart disease, coronary artery disease, acute coronary syndrome, cardiovascular disease, hypertension, restenosis, vascular or peripheral vascular disease, lipid metabolism disorders, dyslipoproteinemia, high concentrations of low density lipoprotein cholesterol, high concentrations of very low density lipoprotein cholesterol, low concentrations of high density lipoprotein, high concentrations of lipoprotein lp (a) cholesterol, high concentrations of apolipoprotein B, atherosclerosis, hyperlipidemia, hypercholesterolemia, familial complex hyperlipidemia, lipoprotein lipase deficiency, alpha-hypolipoproteinemia, or hypercholesterolemia.

18. The use according to claim 17, wherein the multiple clinical manifestations of atherosclerosis are shock, hemorrhagic shock, transient ischemic attacks, myocardial infarction, acute coronary syndrome, angina pectoris, renovascular hypertension, renovascular insufficiency, intermittent claudication, critical limb ischemia, resting pain and gangrene.

19. The use of claim 17 or 18, wherein the lipoprotein complex is used in an amount that increases the serum level of free or complexed ApoA-I protein in the subject by about 10-300mg/dL compared to a baseline level.

20. The use of claim 17 or 18, wherein the amount of lipoprotein complex administered is sufficient to deliver about 1 to 100mg/kg of ApoA-I apolipoprotein.

21. The use of claim 17 or 18, wherein the lipoprotein complex is formulated for intravenous administration.

22. The use according to claim 17 or 18, wherein the medicament is for the treatment of dyslipidemia.

23. The use according to claim 17 or 18, wherein the medicament is for the treatment of a disease associated with dyslipidemia.

24. The use according to claim 17 or 18, wherein the lipoprotein complex is formulated for adjunctive administration with bile acid resins, nicotinic acid, statins, fibrates and/or cholesterol absorption inhibitors.

25. The use according to claim 17 or 18, wherein the lipoprotein complex is formulated for administration in the form of a pharmaceutical composition comprising the lipoprotein complex and a pharmaceutically acceptable carrier, diluent and/or excipient.