Patent 6,828,306

The invention provides a pharmaceutical composition comprising a synthetic or naturally occurring charged phospholipid, which is formulated into a dosage form for administration to a subject or which is administered as a food additive. Negatively charged phospholipid composition increase the net negative charge on intravascular lipoproteins, enhance the clearance of cholesterol and regulate the function of lipolytic enzymes, retard prothrombin formation and aid in the clearance of virus and bacterial particles. Negatively charged lipid compositions can therefore be administered to humans and animals for the treatment of hyperlipidemia and blood coagulation disorders and to reduce the levels of virus, bacteria, and endotoxins in the blood stream. Positively charged lipid compositions can be administered to delay lipoprotein clearance from the plasma compartment and give longer duration of activity for drugs which are associated with lipoproteins.

Description of the Invention

This invention relates to charged lipid compositions. In one aspect, this invention relates to negatively charged (anionic) lipid compositions, and the use of these lipid compositions for changing lipoprotein charge in vivo to clear cholesterol and other substances from the blood stream. In another aspect, this invention relates to positively charged (cationic) lipid compositions, and the use of these compositions to prolong drug activity.

BACKGROUND OF THE INVENTION

Atherosclerosis leading to coronary vascular disease is a primary cause of mortality in the developed world. Atherosclerotic risk has been shown to be directly related to elevated plasma cholesterol levels. In plasma about 70% of cholesterol is esterified to long-chain fatty acids to form cholesteryl esters and these cholesteryl esters are bound to plasma lipoproteins. The lipoproteins involved in the transport of cholesterol and cholesteryl esters include low density lipoprotein (LDL), high density lipoprotein (HDL), and very-low density lipoprotein (VLDL).

While high levels of cholesterol associated with LDL have been linked to atherosclerotic risk (Schaefer et al., 1995), high HDL cholesterol levels may be protective against the development of heart disease (Miller et al., 1977). As a result there has been significant effort to develop therapies which effectively reduce the level of LDL cholesterol and raise the level of HDL cholesterol within an animal. HDL may play an anti-atherogenic role by promoting the clearance of cholesterol from the body (Eisenberg, 1984). Furthermore, Schwartz et al. (1978) disclose that cholesterol in HDL is specifically targeted for excretion from the body by the liver in the form of bile. However, current therapies directed to reduce the level of LDL cholesterol and raise the level of HDL cholesterol have not met with success.

The factors that regulate cholesterol flux to the liver are poorly understood but may involve two distinct systems; a cellular sterol regulatory system and an intravascular transport system. Excess extrahepatic cholesterol may be transported in HDL particles to the liver for excretion (Glomset, 1968). HDL has also been shown to be able to adsorb cholesterol and cholesteryl esters (CE) from cell membranes (Phillips et al., 1998). In addition, a second sterol transport pathway may include transfer of cholesterol from HDL to the rapidly turning over VLDL lipoprotein pool, followed by clearance of cholesterol by the liver (Tall, 1998).

The mechanism of intravascular sterol transport is also poorly understood, but may involve the concerted action of multiple proteins and enzymes. Two enzymes thought be involved in intravascular sterol transport is are lecithin:cholesterol acyltransferase (LCAT) and cholesterol ester transfer protein (CETP). At present it is thought that LCAT may form a concentration gradient to move sterol into and through the blood plasma compartment by promoting the conversion of free cholesterol (FC) to cholesterol esters (CE) on HDL particles (Jonas, 1987). CETP may then promote this lipid flux by moving the newly formed CE from HDL to an apoB containing lipoprotein pool (Lagrost, 1997).

All lipoprotein classes exhibit a net negative charge, due to both the apolipoprotein composition and its content of bound charged lipids (Davidson et al., 1994). However, individual bound lipids forming part of a lipoprotein can contribute either a net positive or a negative charge, or no charge at all, to the lipoprotein. Some phospholipids, when unbound, are negatively charged, some are positively charged, and some are electrically neutral. Examples of negatively charged phospholipids are phosphatidylinositol, phosphatidylserine, phosphatidylglycerol and phosphatidic acid. An example of an electrically neutral phospholipid is phosphatidylcholine. An example of a positively charged (cationic) phospholipid is dioleoyl trimethylammonium propane.

Williams U.S. Pat. No. 6,079,416 teaches administration of large liposomes containing phospholipids substantially free of sterols to treat hypercholesterolemia. Parker et al U.S. Pat. No. 5,614,507 teaches the injection of a bolus of phospholipid, with or without another electrically neutral lipid, to treat endotoxemia. However, the phospholipid compositions disclosed by Williams and Parker et al. are not electrically charged, so they would not act to change the electric charge of lipoproteins within the bloodstream. Instead, they act as a simple adsorbant to pick up and clear cholesterol or endotoxin from the blood.

U.S. Pat. No. 5,652,339 (Learch et al) and U.S. Pat. No. 5,128,318 (Levine et al) disclose the preparation of reconstituted high density lipoprotein (rHDL) particles, and suggest that rHDL particles may be used for drug administration and for treating diseases connected to lipids and lipodal substances. These rHDL particles said to be useable both in vivo or in vitro for removing lipid soluble materials (e.g. cholesterol, endotoxins) from cells or body fluids and aid in the treatment of hyperlipidemia and coronary atherosclerosis. Although, all lipoprotein classes exhibit a net negative charge as discussed above, these two patents teach nothing to increase the charge from that present in normal HDL.

Phosphatidylinositol (PI) is a negatively charged phospholipid found in all classes of lipoproteins and accounts for approximately 4% of the total phospholipid (PL) in HDL (Davidson et al., 1994). Incubation of PI with plasma or with isolated HDL, LDL or VLDL in vitro has shown that all of these lipoproteins can spontaneously absorb PI. However, little is known of what affects, or regulates, the amount of PI in different lipoprotein classes.

There is a need for novel compositions capable of enhancing hepatic clearance of lipoprotein particles thereby lowering cholesterol and tissue cholesterol, endotoxins and other lipid-soluble compounds such as some viruses and bacteria that associate with lipoprotein particles in vivo. There is also a need for methods to make use of such compositions. In particular, there is a need for such compositions, and methods for their use, which will preferentially lower the cholesterol associated with LDL. There is also a need for compositions, and methods for their use, which will retain drugs which associate with lipoproteins in the bloodstream, to increase the duration of the efficacy of such drugs.

SUMMARY OF THE INVENTION

The present invention provides negatively charged (anionic) lipid compositions that, when administered to an animal or subject, result in an increase in the in vivo lipoprotein negative electric charge. Associated with the increase in in vivo lipoprotein negative charge is an enhanced hepatic clearance of lipoprotein particles. The clearance of lipoprotein particles can be used for the clearance of cholesterol and has significant anti-atherogenic consequences. It can also be used to remove bacteria, endotoxin and viruses which associate with lipoproteins from the bloodstream, and for the treatment of lipid-associated diseases.

The invention also provides positively charged (cationic) lipid compositions which, when administered to an animal or subject, result in a decrease of the in vivo lipoprotein negative electric charge. Associated with the decrease in in vivo lipoprotein negative charge is a slowing of hepatic clearance of lipoprotein particles. This effect can be used to delay clearance of drugs which are associated with lipoproteins, thereby prolonging the efficacy of such drugs.

The present invention also comprises a pharmaceutical composition comprising a synthetic or naturally occurring negatively charged (anionic) phospholipid formulated into a dosage form for administration to a subject. If desired, the composition may comprise an admixture of two or more negatively charged phospholipids. The pharmaceutical composition is capable of mediating the level of lipid-associated compounds within an animal or subject. The invention also comprises the use of a negatively charged phospholipid composition as defined above for the production of a medicament useable to enhance clearance of cholesterol from the blood stream and cause the reduction in blood LDL and VLDL cholesterol levels.

The pharmaceutical compositions as discussed above can be provided in dosage forms comprising unilamellar vesicles multilamellar vesicles, multilamellar sheets, dispersions, micellar solutions, emulsions, microemulsions, pure lipid mixtures or any combination of these structures.

The present invention also relates to compositions as discussed above, wherein the dosage form is administered orally or as an injection intranasally or transdermally. In addition the composition may further comprise at least one pharmaceutically acceptable carrier.

The present invention also comprises an orally-administered food additive which comprises a charged phospholipid. When the charged phospholipid is a negatively charged phospholipid, the food additive is capable, when injested by a subject, of reducing the amount of cholesterol in the subject's bloodstream. A particularly preferred food additive comprises phosphatidylinosotol.

The present invention also relates to preferred compositions of those discussed above, wherein the charged phospholipid is a negatively charged phospholipid selected from phosphatidylinositol, phosphatidylserine, phosphatidylglycerol and/or phosphatidic acid or mixtures thereof, and particularly preferred compositions wherein the negatively charged phospholipid is phosphatidylinositol.

The invention also relates to a process for enhancing clearance of lipoprotein particles in vivo by administration of at least one negatively charged phospholipid. By increasing lipoprotein clearance, lipid-soluble compounds that associate with lipoprotein particles are also effectively removed. For example, as described herein, administration of charged phospholipid results in sterol mobilization into bile and excretion in faeces. Removal can therefore be accomplished of cholesterol, endotoxins, or lipoprotein-associated bacteria or virus particles. Without wishing to be bound by theory, it is thought that the administration of a pharmaceutical charged lipid composition to an animal or subject lowers serum levels of total cholesterol in the animal or subject, and inhibits the conversion of cholesterol to cholesteryl esters. Lipoprotein charge thus plays a role in regulating serum levels of lipoprotein-associated compounds, for example but not limited to free cholesterol and the like.

Also according to the invention, there is provided a method of lowering the level of cholesterol associated with LDL within a subject, the method comprising administering to the subject an effective amount of a composition comprising an anionic phospholipid composition.

Another aspect of the present invention pertains to the enhanced mobilization of cellular sterol, and the promotion of rapid clearance of both FC and CE from the plasma compartment, following the administration of a negatively charged phospholipid, (in a particularly preferred embodiment, PI) to a subject. Without wishing to be bound by theory, it is proposed that lipoprotein charge can affect cholesterol transport and that this process can be selectively manipulated by manipulating lipoprotein charge.

Other aspects of the invention provide methods of removing endotoxins, bacteria and virus particles, and treating lipid-associated diseases within an animal, including hyperlipidemia and atherosclerosis, by administering a negatively charged phospholipid to the animal or subject.

Another aspect of the invention provides a method for slowing the removal of lipoproteins, and drugs which associate with lipoproteins, from the bloodstream of an animal or subject by administering a positively charged phospholipid to such animal or subject. The invention also comprises compositions of positively charged phospholipids useful for such methods.

DESCRIPTION OF PREFERRED EMBODIMENTS

Lipoprotein charge is affected by its content of charged molecules, predominantly charged lipids such as PI and non-esterified free fatty acids (NEFA). PI is an anionic lipid found in all classes of lipoproteins and accounts for approximately 4% of the total phospholipid in HDL (Davidson et al. 1994)

The present invention provides for a pharmaceutical composition comprising a charged phospholipid, either natural, analog, or a combination thereof, and in some cases, a suitable carrier. A suitable carrier may include, but is not limited to, phosphate buffered saline, sodium cholate, uncharged synthetic or natural ampliphatic lipid, HDL particles, or any protein capable of binding one or more charged phospholipids. The carrier can be one which permits delayed or timed release of the phospholipid, as is known in the carrier art.

The negatively charged (anionic) phospholipids of the invention include any negatively charged phospholipid that increases the negative charge of lipoprotein in vivo. Preferred negatively charged phospholipids of the present invention, are phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA), or a mixture of any of these phospholipids. A particularly preferred phospholipid is phosphatidylinositol (PI).

The positively charged (cationic) phospholipids of the invention include any positively charged phospholipid that decreases the negative charge of lipoprotein in vivo. A preferred positively charged phospholipids is dioleoyl trimethylammonium propane (DOTAP).

The negatively charged phospholipid composition can be administered over a range of dosages that enhance clearance of cholesterol or other substances to be removed. When used in a pharmaceutical composition, administration in an amount from about 5 micromole to about 100 micromole per kg body weight of an animal or human subject is suitable. Preferably, the level of charged phospholipid administered to an animal or human subject is from about 5 micromole to about 20 micromole per kg body weight administered either in a single dose, or over a short period (eg. one hour). For example, which is not to be considered limiting, administration of PI in an amount from about 5 micromole to about 100 micromole per kg body weight of an animal or human subject enhances cholesterol mobilization as described herein.

When administration occurs by injection, the amount administered should preferably be enough to cause an increase in the negative surface potential on the surface of the HDL fraction of the blood of at least about 20% but preferably not more than 80% (ie. from a normal value of about -11 to -12.2 mV to a value of about -18 to -22 mV) for a transient period. Generally, the period lasts for at least one hour, and commences within a time of one minute to two hours after delivery to the body of the animal or human subject, although individual reactions may vary. The negative surface potential can conveniently be measured by removal of blood samples from the animal or human subject, and observing the migration of the lipoproteins in an agarose gel, in the manner known in the art. It is found that oral administration causes a less marked change in charge of the HDL fraction than administration by injection, (for example, not to be considered limiting, from about -10.9 mV to about 12 mV), but that the transient period lasts longer with oral administration.

Susequent doses, or continuous administration at a low level for a suitable period of time, can be given to maintain the increased negative surface potential for a longer period than single dose administration would give.

In one embodiment, the charged phospholipid composition can be used for the production of a medicament. The medicament containing a negatively charged phospholipid can be used to enhance clearance of cholesterol, or to remove endotoxin or virus particles. The medicament containing the positively charged phospholipid can be used to slow lipoprotein clearance from the blood stream, and thereby slow removal from the bloodstream of a drug that associates with lipoproteins. The medicament can optionally also contain a drug that associates with lipoproteins, for example a drug associated with HDL or rHDL particles.

The medicament can be delivered by injection, or transdermally (as by a patch or a subcutaneous injection) or orally. The carrier is chosen, as known to one skilled in the art, to be compatible with the type of administration. For some types of administration, no carrier is necessary.

In another embodiment, the charged phospolipid composition can be included in a food. The food can then be administered to the subject, enhancing that subject's clearance of cholesterol. The phospholipid compositions can for example be dried and admixed into solid foods, or they can be mixed as an emulsion into liquid foods which will not neutralize them (for example, milk), or they can be sprayed onto solid foods (such as for example cereals) and allowed to dry before the foods are administered.

To demonstrate the role lipoprotein charge plays in cholesterol metabolism in vivo, intravenous injection of an uncharged phospholipid (phosphatidylcholine, PC) or a charged, anionic phospholipid {phosphatidylinositol, (PI) or phosphatidylserine (PS)}--into a fasted rabbit was examined. Similar effects occur with other test animals. PC injection has a negligible effect on lipoprotein charge and composition, similar to that observed in a saline injected animal. However, PI injection causes a significant increase in the net negative surface charge of all lipoproteins (see FIG. 1, discussed further below). This change in net negative surface charge is observed rapidly, for example, but not limited to after about 10 min, and is followed by a gradual return to regular levels by about 24 h, however, these time periods may vary depending upon the animal being treated. Alterations in the net charge of lipoproteins has also been observed when plasma is incubated in the presence of PI or PS. PI and PS show similar results.

In the studies conducted as described herein, no major changes in the levels of, or the composition of, HDL or LDL are evident over a 24 h turnover period. Co-injection of [³ H]--FC revealed an increase in the rate of clearance of labelled cholesterol from the PI injected rabbit plasma (see FIG. 5, discussed further below). In addition, the rate of cholesterol esterification by lecithin:cholesterol acyltransferase was almost completely inhibited in the PI animals (See FIG. 3, discussed further below.).

By the term "charged phospholipid" is meant a natural or synthetic glycerophospholipid which is electrically charged at neutral pH. A "negatively charged phospholipid" (also known as an "anionic phospholipid") has a negative charge at neutral pH. A "positively charged phospholipid" (also known as a "cationic phospholipid") has a positive charge at neutral pH.

Persons skilled in the art will understand that glycerophospholipids comprise a glycerol backbone wherein one of the hydroxyls is linked to a polar phosphate-containing group and the other two hydroxyls are linked to hydrophobic groups. Also evident to someone skilled in the art is that glyceride nomenclature is often defined in terms of a stereospecific numbering (sn) system, but that other stereochemical conventions such as D/L and R/S may be used, with most natural glycerophospholipids having the R or D configuration. The present invention contemplates charged phospholipids having either R or S (or equivalently D or L) configurations.

Most natural glycerophospholipids have hydrophobic groups attached to the sn-1 and sn-2 positions of the glycerol backbone, while a phosphate is usually attached at the sn-3 position. The hydrophobic groups usually comprise long chain hydrocarbons that are linked through ester or ether linkages. Thus, the present invention fully contemplates charged phospholipids comprising hydrophobic groups linked through ester or ether linkages. The hydrophobic groups may be any known in the art, for example but not wishing to be limiting, the hydrophobic groups may comprise saturated fatty acids such as lauric, myristic, palmitic, stearic, arachidic acid, or unsaturated fatty acids such as, but not limited to palmitoleic, oleic, linoleic, arachidonic acid, or any combination thereof.

The phosphate group of a glycerophospholipid is usually attached to the glycerol moiety at the sn-3 position and this phosphate group is linked to a head-group moiety. In phosphatidylinositol, the headgroup is inositol, while in phosphatidylserine, the headgroup is serine. The charged phospholipids of the present invention can have a phosphate group esterified to any hydroxyl of glycerol, with the remaining glyceryl hydroxyls esterified or attached via an ether bond to a hydrophobic group as defined above. In addition, the headgroup may optionally be substituted.

By the term "pharmaceutical composition" or "charged phospholipid composition" it is meant a composition comprising a charged phospholipid, as discussed above, that is formulated into an appropriate dosage form in the presence of a suitable carrier for administering to a subject. The carrier may consist of a buffer, for example but not limited to phosphate buffered saline, or a protein capable of associating with an charged phospholipid as described herein. For example, without limitation, the charged phospholipid can be formulated into structures such as unilamellar vesicles, multilamellar vesicles, multilamellar sheets, dispersions, micellar solutions, emulsions, microemulsions, or any combination thereof in the presence of phosphate buffered saline and the like as would be known to one of skill in the art, HDL particles, or any protein capable of associating with or binding charged phospholipid.

The formulations described above represent aqueous formulations. However, the charged phospholipid can be formulated as a solid, such as, without limitation, a powder that may form structures such as one or more of those discussed above when added to an aqueous solution. Also, the pharmaceutical composition may optionally include the addition of one or more pharmaceutically acceptable excipients as would be known to someone of skill in the art. For example, but not meaning to be limiting, the pharmaceutical composition may comprise other lipids to facilitate the formation of vesicular structures in solution. Alternatively pharmaceutical acceptable excipients such as but not limited to, hydrophilic phase components, lipophilic phase components and surfactants may be optionally included if the charged phospholipid is to be delivered in the form of an emulsion or microemulsion. Pharmaceutically acceptable excipients may also include, but are not limited to, solvents, buffers, antioxidants, and stabilizers as is known in the art.

The pharmaceutical composition of the present invention may be administered to a subject by any method known in the art. For example, and without limitation, the pharmaceutical composition can be administered orally or the pharmaceutical composition may be injected such as but not limited to intravenous injection, intramuscular injection, or intraperitoneal injection. However someone of skill in the art will understand that certain dosage forms may be more suitable to specific modes of administration. For example, if the pharmaceutical composition is to be administered intravenously as a vesicular solution, it is preferable for the charged phospholipid composition to be administered as unilamellar vesicles of cross-section sufficiently small so that they will not get caught in the microvasculature of a subject.

When the charged phospholipids of the present invention are administered as a food additive, they can be administered as part of any solid or liquid food with which they do not react or complex in such a way as to lose their charge. It is particularly preferred to administer the charged phospholipid or a mixture of charged phospholipids having the same charge in a dry state on or comminged in conventional cereal products, although other foods can also be used. In case of either a pharmaceutical or a food product, sufficient is administered to result in a change, for a period of at least 10 minutes, of the in vivo charge on the blood lipoproteins by at least 20%. The negatively charged phospholipids increase the negative charge of the lipoproteins, while the positively charged phospholipids decrease it.

Claim 1 of 12 Claims

What is claimed is:

1. A method for treating a lipid-associated condition selected from dyslipidemia and atherosclerosis in a mammal, the method comprising administering to the mammal a therapeutically effective amount of phosphatidylinositol for achieving one or more of the following changes in serum lipid levels in said mammal: (1) an increase in the level of serum HDL-cholesterol; (2) a reduction in the level of serum VLDL-cholesterol and LDL-cholesterol; and (3) a reduction in the level of serum triglycerides.

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