Solid tumor treatment method and composition

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FIELD OF THE INVENTION

The present invention relates to a liposome composition and method, particularly for use in tumor diagnostics and/or therapeutics.

REFERENCES

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BACKGROUND OF THE INVENTION

It would be desirable, for extravascular tumor diagnosis and therapy, to target an imaging or therapeutic compound selectively to the tumor via the bloodstream. In diagnostics, such targeting could be used to provide a greater concentration of an imaging agent at the tumor site, as well as reduced background level of the agent in other parts of the body. Site-specific targeting would be useful in therapeutic treatment of tumors, to reduce toxic side effects and to increase the drug dose which can safely be delivered to a tumor site.

Liposomes have been proposed as a drug carrier for intravenously (IV) administered compounds, including both imaging and therapeutic compounds. However, the use of liposomes for site-specific targeting via the bloodstream has been severely restricted by the rapid clearance of liposomes by cells of the reticuloendothelial system (RES). Typically, the RES will remove 80-95% of a dose of IV injected liposomes within one hour, effectively out-competing the selected target site for uptake of the liposomes.

A variety of factors which influence the rate of RES uptake of liposomes have been reported (e.g., Gregoriadis, 1974; Jonah; Gregoriadis, 1972; Juliano; Allen, 1983; Kimelberg, 1976; Richardson; Lopez-Berestein; Allen, 1981; Scherphof; Gregoriadis, 1980; Hwang; Patel, 1983; Senior, 1985; Allen, 1983; Ellens; Senior, 1982; Hwang; Ashwell; Hakomori; Karlsson; Schauer; Durocher; Greenberg; Woodruff; Czop; and Okada). Briefly, liposome size, charge, degree of lipid saturation, and surface moieties have all been implicated in liposome clearance by the RES. However, no single factor identified to date has been effective to provide long blood halflife, and more particularly, a relatively high percentage of liposomes in the bloodstream 24 hours after injection.

In addition to a long blood halflife, effective drug delivery to a tumor site would also require that the liposomes be capable of penetrating the continuous endothelial cell layer and underlying basement membrane surrounding the vessels supplying blood to a tumor. Although tumors may present a damaged, leaky endothelium, it has generally been recognized that for liposomes to reach tumor cells in effective amounts, the liposomes would have to possess mechanisms which facilitate their passage through the endothelial cell barriers and adjacent basement membranes, particularly in view of the irregular and often low blood flow to tumors and hence limited exposure to circulating liposomes (Weinstein). Higher than normal interstitial pressures found within most tumors would also tend to reduce the opportunity for extravasation of liposomes by creating an outward transvascular movement of fluid from the tumor (Jain). As has been pointed out, it would be unlikely to design a liposome which would overcome these barriers to extravasation in tumors and, at the same time, evade RES recognition and uptake (Poznansky).

In fact, studies reported to date indicate that even where the permeability of blood vessels increases, extravasation of conventional liposomes through the vessels does not increase significantly (Poste). Based on these findings, it was concluded that although extravasation of liposomes from capillaries compromised by disease may be occurring on a limited scale below detection levels, its therapeutic potential would be minimal (Poste).

SUMMARY OF THE INVENTION

One general object of the invention is to provide a liposome composition and method which is effective for tumor targeting, for localizing an imaging or anti-tumor agent selectively at therapeutic dose levels in systemic, extravascular tumors.

The invention includes, in one aspect, a liposome composition for use in localizing a compound in a solid tumor, as defined in Section IV below, via the bloodstream comprising: The liposomes forming the composition (i) are composed of vesicle-forming lipids, and between 1-20 mole percent of an vesicle-forming lipid derivatized with a hydrophilic polymer, and (ii) have an average size in a selected size range between about 0.07-0.12 microns. The compound is contained in the liposomes in entrapped form (i.e., associated with the liposome membrane or encapsulated within the internal aqueous compartment of the liposome).

In a preferred embodiment, the hydrophilic polymer is polyethyleneglycol, polylactic, polyglycolic acid or a polylactic-polyglycolic acid copolymer having a molecular weight between about 1,000-5,000 daltons, and is derivatized to a phospholipid.

For use in tumor treatment, the compound in one embodiment is an anthracycline antibiotic or plant alkaloid, at least about 80% of the compound is in liposome-entrapped form, and the drug is present in the liposomes at a concentration of at least about 20 .mu.g and preferably above 50 .mu.g compound/.mu.mole liposome lipid in the case of the anthracycline antibiotics and 1 .mu.g/.mu.mole lipid in the case of the plant alkaloids.

In a related aspect, the invention includes a composition of liposomes characterized by:

(a) liposomes composed of vesicle-forming lipids and between 1-20 mole percent of a vesicle-forming lipid derivatized with a hydrophilic polymer,

(b) a blood lifetime, as measured by the percent of a liposomal marker present in the blood 24 hours after IV administration which is several times greater than that of liposomes in the absence of the derivatized lipids;

(c) an average liposome size in a selected size range between about 0.07-0.12 microns, and

(d) the compound in liposome-entrapped form.

Also disclosed is a method of preparing an agent for localization in a solid tumor, when the agent is administered by IV injection. In this case, following IV administration, the agent is carried through the bloodstream in liposome-entrapped form with little leakage of the drug during the first 48 hours post injection. By virtue of the low rate of RES uptake during this period, the liposomes have the opportunity to distribute to and enter the tumor. Once within the interstitial spaces of the tumor, it is not necessary that the tumor cells actually internalize the liposomes. The entrapped agent is released from the liposome in close proximity to the tumor cells over a period of days to weeks and is free to further penetrate into the tumor mass (by a process of diffusion) and enter tumor cells directly--exerting its anti-proliferative activity. The method includes entrapping the agent in liposomes of the type characterized above. One liposome composition preferred for transporting anthracycline antibiotic or plant alkaloid antitumor agents to systemic solid tumors would contain high phase transition phospholipids and cholesterol as this type of liposome does not tend to release these drugs while circulating through the bloodstream during the first 24-48 hours following administration.

In another aspect, the invention includes a method for localizing a compound in a solid tumor in a subject. The method includes preparing a composition of liposomes (i) composed of vesicle-forming lipids and between 1-20 mole percent of an vesicle-forming lipid derivatized with a hydrophilic polymer, (ii) having an average size in a selected size range between about 0.07-0.12 microns, and (iii) containing the compound in liposome-entrapped form. The composition is injected IV in the subject in an amount sufficient to localize a therapeutically effective dose of the agent in the solid tumor.

Also disclosed is a system for providing effective anti-tumor therapy for agents which possess intrinsic anti-tumor activity in vitro but, due to unfavorable biodistribution, toxicity and metabolism in vivo, do reach tumors in effective amounts by prior art methods of drug administration.

These and other objects and features of the present invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a general reaction scheme for derivatizing a vesicle-forming lipid amine with a polyalkylether;

FIG. 2 is a reaction scheme for preparing phosphatidylethanolamine (PE) derivatized with polyethyleneglycol via a cyanuric chloride linking agent;

FIG. 3 illustrates a reaction scheme for preparing phosphatidylethanolamine (PE) derivatized with polyethyleneglycol by means of a diimidazole activating reagent;

FIG. 4 illustrates a reaction scheme for preparing phosphatidylethanolamine (PE) derivatized with polyethyleneglycol by means of a trifluoromethane sulfonate reagent;

FIG. 5 illustrates a vesicle-forming lipid derivatized with polyethyleneglycol through a peptide (A), ester (B), and disulfide (C) linkage;

FIG. 6 illustrates a reaction scheme for preparing phosphatidylethanolamine (PE) derivatized with polylactic acid, polyglycolic acids and copolymers of the two;

FIG. 7 is a plot of liposome residence times in the blood, expressed in terms of percent injected dose as a function of hours after IV injection, for PEG-PE liposomes containing different amounts of phosphatidylglycerol;

FIG. 8 is a plot similar to that of FIG. 7, showing blood residence times of liposomes composed of predominantly unsaturated phospholipid components;

FIG. 9 is a plot similar to that of FIG. 7, showing the blood residence times of PEG-coated liposomes (solid triangles) and conventional, uncoated liposomes (solid circles);

FIG. 10 is a plot similar to that of FIG. 7, showing the blood residence time of polylactic or polyglycolic acid-coated liposomes (upper lines) and conventional uncoated liposomes (lower lines);

FIG. 11 is a plot showing the kinetics of doxorubicin clearance from the blood of beagle dogs, for drug administered IV in free form (open circles), in liposomes formulated with saturated phospholipids and hydrogenated phosphatidylinositol (HPI) (open squares), and in liposomes coated with PEG (open triangles);

FIGS. 12A and 12B are plots of the time course of doxorubicin uptake from the bloodstream by heart (solid diamonds), muscle (solid circles), and tumor (solid triangles) for drug administered IV in free (12A) and PEG-liposomal (12B) form;

FIG. 13 is a plot of the time course of uptake of doxorubicin from the bloodstream by J-6456 tumor cells implanted interperitoneally (IP) in mice, as measured as total drug (filled diamonds) as drug associated with tumor cells (solid circles) and liposome-associated form (solid triangles);

FIGS. 14A-14D are light micrographs showing localization of liposomes (small dark stained particles) in Kupfer cells in normal liver (14A), in the interstitial fluid of a C-26 colon carcinoma implanted in liver in the region of a capillary supplying the tumor cells (14B) and in the region of actively dividing C-26 tumor cells implanted in liver (14C) or subcutaneously (14D);

FIG. 15A-15C are plots showing tumor size growth in days following subcutaneous implantation of a C-26 colon carcinoma, for mice treated with a saline control (open circles), doxorubicin at 6 mg/kg (filled circles), epirubicin at 6 mg/kg (open triangles), or PEG-liposome-entrapped epirubicin at two doses, 6 mg/kg (filled triangles) or 12 mg/kg (open squares) on days 1, 8 and 15 (15A); for mice treated with saline (solid line), 6 mg/kg epirubicin (closed circles), 6 mg/kg epirubicin plus empty liposomes, (open circles), or PEG liposome entrapped at two doses, 6 mg/kg (filled triangles) and 9 mg/kg (open squares) on days 3, 10 and 17 (15B) or days 10, 17 and 24 (15C);

FIG. 16 is a plot showing percent survivors, in days following interperitoneal implantation of a J-6456 lymphoma, for animals treated with doxorubicin in free form (closed circles) or PEG-liposomal form (solid triangles), or untreated animals (open triangles); and

FIG. 17 is a plot similar to that in FIG. 15, showing tumor size growth, in days following subcutaneous implantation of a C-26 colon carcinoma, for animals treated with a saline control (filled circles), or animals treated with 10 mg/kg doxorubicin in free form (filled squares), or in conventional liposomes (open circles);

FIG. 18 shows plots of tumor size as a function of time following tumor implantation in animals, each treated with (A) (a) saline control, (B) 6 mg/kg free epirubicin, (C) PEG liposomes at 6 mg/kg, (D) PEG liposomes at 9 mg/kg, or (E) empty liposomes mixed with free epirubicin at 6 mg/kg in individual animals (10 animals per group), where (F) shows mean values for all five treatment groups for saline (open diamonds), free epirubicin, 6 mg/kg (filled circles), mixture of free drug and empty liposomes (open circles), and PEG liposomes with entrapped epirubicin at 6/mg/kg (filled triangles) and 9 mg/kg (open squares);

FIG. 19 is a plot showing the weight of animals expressed as percent change from pretreated levels for groups of seven mice which received on day 0, subcutaneous implantation of 10.sup.6 c-26 colon carcinoma cells, and which were injected intravenously on days 3, 10 and 17 with saline (closed circles), 6 mg/kg epirubicin (open circles), empty liposomes plug 6 mg/ky epirubicin (closed triangles), and PEG liposomes with entrapped epirubicin at 6 mg/kg (open triangles) or 9 mg/kg. (open squares);

FIG. 20 is a plot of weight changes in normal Sabra male mice untreated (open circles) or treated with four weekly intravenous injections on days 1, 8, 5 and 22 with a 10 mg/kg dose of either free doxorubicin (open triangles) or PEG liposomes with entrapped doxorubicin (open squares); and

FIG. 21 is a plot showing growth kinetics of syngeneic mammary carcinoma (MC2) for three groups of 20 animals implanted bilaterally with 10.sup.5 -10.sup.6 tumor cells subcutaneously on day 0 and treated on days 1, 8 and 15 with saline control, or 6 mg/kg free epirubicin or PEG liposomes containing entrapped epirubicin, as indicated.

DETAILED DESCRIPTION OF THE INVENTION

I. Preparation of Derivatized Lipids

FIG. 1 shows a general reaction scheme for preparing a vesicle-forming lipid derivatized with a biocompatible, hydrophilic polymer, as exemplified by polyethylene glycol (PEG), polylactic acid, and polyglycolic acid, all of which are readily water soluble, can be coupled to vesicle-forming lipids, and are tolerated in vivo without toxic effects. The hydrophilic polymer which is employed, e.g., PEG, is preferably capped by a methoxy, ethoxy or other unreactive group at one end or, alternatively, has a chemical group that is more highly reactive at one end than the other. The polymer is activated at one of its ends by reaction with a suitable activating agent, such as cyanuric acid, diimadozle, anhydride reagent, or the like, as described below. The activated compound is then reacted with a vesicle-forming lipid, such as a diacyl glycerol, including diacyl phosphoglycerols, where the two hydrocarbon chains are typically between 14-22 carbon atoms in length and have varying degrees of saturation, to produce the derivatized lipid. Phosphatidylethanol-amine (PE) is an example of a phospholipid which is preferred for this purpose since it contains a reactive amino group which is convenient for coupling to the activated polymers. Alternatively, the lipid group may be activated for reaction with the polymer, or the two groups may be joined in a concerted coupling reaction, according to known coupling methods. PEG capped at one end with a methoxy or ethoxy group can be obtained commercially in a variety of polymer sizes, e.g., 500-20,000 dalton molecular weights.

The vesicle-forming lipid is preferably one having two hydrocarbon chains, typically acyl chains, and a polar head group. Included in this class are the phospholids, such as phosphatidylcholine (PC), PE, phosphatidic acid (PA), phosphatidylinositol (PI), and sphingomyelin (SM), where the two hydrocarbon chains are typically between about 14-22 carbon atoms in length, and have varying degrees of unsaturation. Also included in this class are the glycolipids, such as cerebrosides and gangliosides.

Another vesicle-forming lipid which may be employed is cholesterol and related sterols. In general, cholesterol may be less tightly anchored to a lipid bilayer membrane, particularly when derivatized with a high molecular weight polymers, such as polyalkylether, and therefore be less effective in promoting liposome evasion of the RES in the bloodstream.

More generally, and as defined herein, "vesicle-forming lipid" is intended to include any amphipathic lipid having hydrophobic and polar head group moieties, and which (a) by itself can form spontaneously into bilayer vesicles in water, as exemplified by phospholipids, or (b) is stably incorporated into lipid bilayers in combination with phospholipids, with its hydrophobic moiety in contact with the interior, hydrophobic region of the bilayer membrane, and its polar head group moiety oriented toward the exterior, polar surface of the membrane. An example of a latter type of vesicle-forming lipid is cholesterol and cholesterol derivatives, such as cholesterol sulfate and cholesterol hemisuccinate.

According to one important feature of the invention, the vesicle-forming lipid may be a relatively fluid lipid, typically meaning that the lipid phase has a relatively low liquid to liquid-crystalline melting temperature, e.g., at or below room temperature, or relatively rigid lipid, meaning that the lipid has a relatively high melting temperature, e.g., up to 60.degree. C. As a rule, the more rigid, i.e., saturated lipids, contribute to greater membrane rigidity in a lipid bilayer structure and also contribute to greater bilayer stability in serum. Other lipid components, such as cholesterol, are also known to contribute to membrane rigidity and stability in lipid bilayer structures. As mentioned above, a long chain (e.g. C-18) saturated lipid plus cholesterol is one preferred composition for delivering anthracycline antibiotic and plant alkaloids anti-tumor agents to solid tumors since these liposomes do not tend to release the drugs into the plasma as they circulate through the bloodstream and enter the tumor during the first 48 hours following injection. Phospholipids whose acyl chains have a variety of degrees of saturation can be obtained commercially, or prepared according to published methods.

FIG. 2 shows a reaction scheme for producing a PEPEG lipid in which the PEG is derivatized to PE through a cyanuric chloride group. Details of the reaction are provided in Example 1. Briefly, methoxy-capped PEG is activated with cyanuric chloride in the presence in sodium carbonate under conditions which produced the activated PEG compound shown in the figure. This material is purified to remove unreacted cyanuric acid. The activated PEG compound is reacted with PE in the presence of triethyl amine to produce the desired PE-PEG compound shown in the figure. The yield is about 8-10% with respect to initial quantities of PEG.

The method just described may be applied to a variety of lipid amines, including PE, cholesteryl amine, and glycolipids with sugar-amine groups.

A second method of coupling a polyalkylether, such as capped PEG to a lipid amine is illustrated in FIG. 3. Here the capped PEG is activated with a carbonyl diimidazole coupling reagent, to form the activated imidazole compound shown in FIG. 3. Reaction with a lipid amine, such as PE leads to PEG coupling to the lipid through an amide linkage, as illustrated in the PEG-PE compound shown in the figure. Details of the reaction are given in Example 2.

A third reaction method for coupling a capped polyalkylether to a lipid amine is shown in FIG. 4. Here PEG is first protected at its OH end by a trimethylsilane group. The end-protection reaction is shown in the figure, and involves the reaction of trimethylsilylchloride with PEG in the presence of triethylamine. The protected PEG is then reacted with the anhydride of trifluoromethyl sulfonate to form the PEG compound activated with trifluoromethyl sulfonate. Reaction of the activated compound with a lipid amine, such as PE, in the presence of triethylamine, gives the desired derivatized lipid product, such as the PEG-PE compound, in which the lipid amine group is coupled to the polyether through the terminal methylene carbon in the polyether polymer. The trimethylsilyl protective group can be released by acid treatment, as indicated in the figure, or, alternatively, by reaction with a quaternary amine fluoride salt, such as the fluoride salt of tetrabutylamine.

It will be appreciated that a variety of known coupling reactions, in addition to those just described, are suitable for preparing vesicle-forming lipids derivatized with hydrophilic polymers such as PEG, polylactic acid, polyglycolic acid or polylactic-polyglycolic copolymers. For example, the sulfonate anhydride coupling reagent illustrated in FIG. 4 can be used to join an activated polyalkylether to the hydroxyl group of an amphipathic lipid, such as the 5'-OH of cholesterol. Other reactive lipid groups, such as an acid or ester lipid group may also be used for coupling, according to known coupling methods. For example, the acid group of phosphatidic acid can be activated to form an active lipid anhydride, by reaction with a suitable anhydride, such as acetic anhydride, and the reactive lipid can then be joined to a protected polyalkylamine by reaction in the presence of an isothiocyanate reagent.

In another embodiment, the derivatized lipid components are prepared to include a labile lipid-polymer linkage, such as a peptide, ester, or disulfide linkage, which can be cleaved under selective physiological conditions, such as in the presence of peptidase or esterase enzymes or reducing agents such as glutathione present in the bloodstream. FIG. 5 shows exemplary lipids which are linked through (A) peptide, (B), ester, and (C), disulfide containing linkages. The peptide-linked compound can be prepared, for example, by first coupling a polyalkylether with the N-terminal amine of the tripeptide shown, e.g., via the reaction shown in FIG. 3. The peptide carboxyl group can then be coupled to a lipid amine group through a carbodiimide coupling reagent conventionally. The ester linked compound can be prepared, for example, by coupling a lipid acid, such as phosphatidic acid, to the terminal alcohol group of a polyalkylether, using alcohol via an anhydride coupling agent. Alternatively, a short linkage fragment containing an internal ester bond and suitable end groups, such as primary amine groups can be used to couple the polyalkylether to the amphipathic lipid through amide or carbamate linkages. Similarly, the linkage fragment may contain an internal disulfide linkage, for use in forming the compound shown at C in FIG. 5. Polymers coupled to phospholipids via such reversible linkages are useful to provide high blood levels of liposomes which contain them for the first few hours post injection. After this period, plasma components cleave the reversible bonds releasing the polymers and the "unprotected" liposomes are rapidly taken up by the RES by the same mechanism as conventional liposomes.

It will be appreciated that the polymers in the derivatized lipids must be (a) safe for parenteral administration, both in terms of toxicity, biodegradability, and tissue compatibility, (b) compatible with stable lipid structure, and (c) amenable to liposome preparation and processing steps. These requirements are met by PEG polymers, and also by the thermoplastic polyester polymers polylactic acid and polyglycolic acid (also referred to as polylactide and polyglycolide) and copolymers of lactide and glycolide such as poly(lactide-co-glycolide). In particular, the polyester polymers are safe to administer because they biodegrade by undergoing random, nonenzymatic, hydrolytic cleavage of their ester linkages to form lactic acid and glycolic acid, which are normal metabolic compounds (Tice and Cowsar, and Wise et al.).

FIG. 6 illustrates a method for derivatizing polylactic acid, polyglycolic acid and polylactic-polyglycolic copolymers with PE. The polylactic acid is reacted, in the presence of PE,