Polyethylene glycol modified ceramide lipids and liposome uses thereof

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BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel polyethylene glycol (PEG) derivatized lipids, their method of preparation and their use in liposomes or other lipid-based carriers. More specifically, the present invention includes PEG-Ceramide lipids and their inclusion in liposomes for use in drug delivery.

2. The Relevant Art

Liposomes are vesicles comprised of concentrically ordered lipid bilayers which encapsulate an aqueous phase. Liposomes form when lipids, molecules which typically comprise a polar head group attached to one or more long chain aliphatic tails, such as phospholipids, are exposed to water. Upon encountering such media the lipids aggregate to form a structure in which only the polar head groups are exposed to the external media to form an external shell inside which the aliphatic tails are sequestered. See, e.g., Lehninger, PRINCIPLES OF BIOCHEMISTRY (Worth, 1982). Liposomes can entrap a variety of bioactive or pharmaceutical agents for delivery of these agents to cells and tissues in vivo. See, e.g., U.S. Pat. No. 5,185,154 to Lasic, et al.; European Patent Application No. 526,700 to Tagawa, et al.; and U.S. Pat. No. 5,013,556 to Woodle, et al.

Liposomes can alter the biodistribution and rate of delivery of an encapsulated bioactive agent in a number of ways. For example, drugs encapsulated in liposomes are protected from interactions with serum factors which may chemically degrade the drug. The size of the liposome compared to the free drug also affects its access to certain sites in the body; this property can be advantageous in limiting drug delivery to certain sites. Uptake by the reticuloendothelial system (RES) can be inhibited by including factors on the liposome surface that inhibit protein association with the liposome or liposome interactions with RES cells, for example, by using PEG-lipids with other lipids such as ganglioside GM.sub.1. See, Woodle, supra.

A variety of liposome structures can be formed using one or more lipids. Typical classes of liposome structures include small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), or multilamellar vesicles (MLVs). The construction of liposomes and their application as delivery systems is described in the art. See, e.g., LIPOSOMES, Marc J. Ostro, ed. (Marcel Dekker 1983).

Liposomes have been prepared by derivatizing existing lipid systems to form new liposome structures. For example, polyethyleneglycol (PEG) derivatized lipids have been developed. See Woodle, supra.

Typically, PEG-lipids are prepared by derivatization of the polar head group of a diacylglycerophospholipid, such as distearoylphosphatidylethanolamine (DSPE), with PEG. These phospholipids usually contain two fatty acyl chains bonded to the 1- and 2- position of glycerol by ester linkages. Unfortunately, these acyl groups are susceptible to cleavage under acidic or basic conditions. The resulting hydrolytic products, such as analogs of lysophospholipid and glycerophosphate, do not remain associated with the bilayer structure of the liposome. Such dissociation may weaken the integrity of the liposome structure, leading to significant leakage of the bioactive agent or drug from the liposome and contributing to instability during storage, and thus shortened shelf-life of the liposome product. In addition, the loss of these hydrolysis products, such as PEG-lysophospholipid, from the liposome would negate the benefits otherwise resulting from the presence of the PEG-phospholipid.

Lipid stability is important in the development of liposomal drug delivery systems. This is especially relevant when a transmembrane pH gradient is used to entrap or encapsulate the bioactive agent in the liposome, as very acidic (pH 2-4) or basic (pH 10-12) conditions may be used to achieve efficient drug uptake and retention. Therefore, it is desirable to develop PEG-lipids that are less susceptible to hydrolysis, thereby, increasing the liposome circulation longevity.

SUMMARY OF THE INVENTION

In one aspect, the present invention includes novel PEG-lipids such as the PEG-modified ceramide lipids of Formula I: ##STR1## wherein:

R.sup.1, R.sup.2, and R.sup.3 are independently hydrogen, C.sub.1 -C.sub.6 alkyl, acyl, or aryl;

R.sup.4 is hydrogen, C.sub.1 -C.sub.30 alkyl, C.sub.2 -C.sub.30 alkenyl, C.sub.2 -C.sub.30 alkynyl, or aryl;

R.sup.5 is hydrogen, alkyl, acyl, aryl, or PEG;

X.sup.1 is --O--, --S--, or --NR.sup.6 --, where R.sup.6 is hydrogen, C.sub.1 -C.sub.6 alkyl, acyl or aryl; or when R.sup.5 is PEG and b is 1, X.sup.1 is also --Y.sup.1 -alk-Y.sup.2 --;

Y is --NR.sup.7 --, where R.sup.7 is hydrogen, C.sub.1 -C.sub.6 alkyl, acyl or aryl, or Y is --O--, --S-- or --Y.sup.1 -alk-Y.sup.2 --, wherein Y.sup.1 and Y.sup.2 are independently amino, amido, carboxyl, carbamate, carbonyl, carbonate, urea, or phosphoro; and alk is C.sub.1 -C.sub.6 alkylene;

PEG is a polyethylene glycol with an average molecular weight from about 550 to about 8,500 daltons optionally substituted by C.sub.1 -C.sub.3 alkyl, alkoxy, acyl or aryl; wherein a is 0 or 1; and b is 1 unless R.sup.5 is PEG wherein b is 0 or 1.

More preferred are those compounds wherein R.sup.1, R.sup.2, R.sup.3, and R.sup.5 are hydrogen; R.sup.4 is alkyl; X.sup.1 is O, Y is succinate; and PEG has an average molecular weight of about 2,000 or about 5,000 daltons and is substituted with methyl at the terminal hydroxyl position.

Also preferred are those compounds wherein R.sup.1, R.sup.2, R.sup.3, and R.sup.5 are hydrogen, R.sup.4 is alkyl; X.sup.1 is O; Y is --NH--; and PEG has an average molecular weight of about 2,000 or about 5,000 daltons and is substituted with methyl at the terminal position.

Other preferred lipid compounds are those wherein R.sup.1, R.sup.2, R.sup.3, and R.sup.5 are hydrogen; R.sup.4 is C.sub.7 -C.sub.23 alkyl, X.sup.1 is O; Y is succinate; and PEG has an average molecular weight of about 2,000 daltons and is substituted with methoxy at the terminal hydroxyl position; more preferred are those lipid compounds wherein R.sup.4 is C.sub.13 -C.sub.19 alkyl.

In another aspect, the present invention includes liposomes or other lipid-based carriers including the above-described PEG-Ceramide lipids. Preferred liposome compositions include the preferred lipids described above. In construction of the liposomes, various mixtures of the described PEG-Ceramide lipids can be used in combination and in conjunction with other lipid types, such as DOPE and DODAC, as well as DSPC, SM, Chol and the like, with DOPE and DODAC preferred. Typically, the PEG-Ceramide will comprise about 5 to about 30 mol % of the final liposome construction, but can comprise about 0.0 to about 60 mol % or about 0.5 to about 5 mol %. More preferred lipid compositions are those wherein a drug or a biological agent is encapsulated within the liposome. The invention also includes lipid complexes whereby the PEG-Ceramide lipid comprises about 0.01 to about 90 mol % of the complex.

In still another aspect, the present invention includes methods for delivering therapeutic agents such as drugs and vaccines to a patient in need thereof comprising administering to the patient a therapeutically effective amount of such therapeutic agent in a liposome or a lipid-based carrier of the invention. Also provided are kits for preparing labeled liposomes, containing the PEG-Ceramide lipids, and pharmaceutical formulations containing liposomes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B illustrate graphically the circulation lifetimes of the PEG modified ceramide liposomes of the invention. FIG. 1A shows plasma clearance of 100 nm liposomes prepared of distearylphosphatidylcholine (DSPC)/Cholesterol (Chol) (55:45 mol %; open circles), DSPC/Chol/PEG.sub.2000 Ceramide (50:45:5 mol %; filled circles), and DSPC/Chol/PEG.sub.5000 Ceramide (50:45:5 mol %; filled squares). FIG. 1B shows plasma clearance of 100 nm liposomes prepared of DSPC/Chol (55:45 mol %; open circles), Sphingomyelin (SM)/Chol (55:45 mol %; filled circles), and Sphingomyelin/Chol/PEG.sub.2000 Ceramide (50:45:5 mol %; open squares).

FIG. 2 graphically shows that the incorporation of PEG modified ceramide into liposomal vincristine formulations does not adversely affect drug retention characteristics. Vincristine retention by Sphingomyelin/Chol (55:45 mol %; filled circles) liposomes within the circulation is not affected by incorporation of PEG.sub.2000 Ceramide (50:45:5 mol %; open squares). Vincristine retention is also shown for DSPC/Chol (55:45 mol %; open circles) and SM/Chol/PEG-PE (Phosphatidylethanolamine) (50:45:5 mol %; filled squares).

FIG. 3 shows the lipid circulation half-life (T.sub.1/2) values (in hours) of various SM/cholesterol liposomes, including those containing PEG.sub.2000 -ceramides of various fatty amide chain lengths and those containing PEG.sub.2000 -DSPE (distearolyphosphatidylethanolamine).

FIG. 4 illustrates the circulation half-life values (T.sub.1/2) (in hours) of the vincristine/lipid ratios (vincristine retention) for liposomes, containing vincristine with various PEG.sub.2000 -ceramides, as well as PEG.sub.2000 -DSPE.

FIG. 5 presents the circulation half-life values (T.sub.1/2) (in hours) of vincristine-containing liposomes.

FIG. 6 graphically shows the effect of increasing concentrations of PEG-Ceramide (C20) on biodistribution of liposomes in the blood and liver. .sup.3 H-labeled liposomes composed of DOPE (dioleoylphosphatidylethanolamine), 15 mol % DODAC (N,N-dioleoyl-N,N-dimethylammonium chloride) and the indicated concentrations of PEG-Ceramide (C20) were injected i.v. into mice. Biodistribution was examined at 1 hour after injection, and the data were expressed as a percentage of the injected dose in the blood (uppe