SOLID TUMOR TREATMENT METHOD AND COMPOSITION

Abstract: A liposome composition for delivering a compound to a solid tumor via the bloodstream. The liposomes, which contain the agent in entrapped form, are composed of vesicle-forming lipids and between 1-20 mole percent of a vesicle-forming lipid derivatized with hydrophilic polymer, and have sizes in a selected size range between 0.07 and 0.12 microns. After intravenous administration, the liposomes are taken up by the tumor within 24-48 hours, for site-specific release of entrapped compound into the tumor. In one composition for use in treating a solid tumor, the compound is an anthracycline antibiotic drug which is entrapped in the liposomes at a concentration of greater than about 50 mu g agent/ mu Mole liposome lipid.

Description: SOLID TUMOR TREATMENT METHOD AND COMPOSITION 1. Fielα of the Invention The present invention relates to a liposome composi¬ tion and method, particularly for use in tumor diagnos¬ tics and/or therapeutics. 2. References Allen, T.M., (1S81) Biochem. Biophys. Acta 640. 385397. Allen, T.M., and Everest, J. (1983) J. Phar¬ macol. Exp. Therap. 226. 539-544. Altura, B.M. (1980) Adv. Microcirc. _9_. 252-294. Alving, CR. (1984) Biochem. Soc. Trans. 12. 342344. Ashwell, G., and Morell, A.G. (1974) Adv. Enzymo¬ logy 41^ 99-128. Czop, J.K. (1978) Proc. Natl. Acad. Sci. US 75:3831. Durocher, J.P., et al. (1975) Blood 45:11. Ellens, H., et al. (1981) Biochim. Biophys. Act 674. 10-18. Gabizon, A., Shiota, R. and Papahadjopoulos, D. (1989) J. Natl. Cancer Inst. JJl, 1484-1488. Gabizon, A., Huberty, J., Straubinger, R.M., Price, D. C. and Papahadjopoulos, D. (1988-1989) J. Liposom Resh. 1 , 123-135. SUBSTITUTE SHEET Gregoriadis, G., and Ryman, B.E. (1972) Eur. J. Biochem. 24, 485-491. Gregoriadis, G., and Neerunjun, D. (1974) Eur. J. Biochem. __, 179-185. Gregoriadis, G., and Senior, J. (1980) FEBS Lett. 119, 43-46. Greenberg, J.P., et al (1979) Blood 53:916. Hakomori, S. (1981) Ann. Rev. Biochem. 50, 733-764. Hong, K., Friend, D., Glabe, C. and Papahadjopoulos (1984) Biochem. Biophys. Acta 732,320-323. Hwany.. K.J., et al. (1980) Proc. Natl. Acad. Sci. USA 77:4030- Jain, K.J. (1989) J. Natl. Can. Inst. 81, 570-576. Jonah, M.M., e+-. al. (1975) Biochem. Biophys. Acta 401, 336-348. Juliano, R.L., and Stamp, D. (1975) Biochem. bio¬ phys. Res. Commun. 63. 651-653. Karlsson, K.A. (1982) In: Biological Membranes, Vol. 4, D. Chapman (ed.) Academic Press, N.Y., pp. 1-74. Kimelberg, H.K., et al. (1976) Cancer Res. 36,2949- 2957. Kirby, CJ. and Gregoriadis (1984) Ir: Liposome Technology, Vol. 3, G. Gregoriadis (ed.) CRC Press, Boca Raton, FL., p. 19. Lee, K.C, et al., J. Immunology 125:86 (1980). Lopez-Berestein, G., et al. (1984) Cancer Res. 44, 375-378. Martin, F.J. (1990) In: Specialized Drug Delivery Systems - Manufacturing and Production Technology, P. Tyle (ed.) Marcel Dekker, New York, pp. 267-316. Okada, N. (1982) Nature 299:261. Poste, G., et al., in "Liposome Technology" Volume 3, page 1 (Gregoriadis, G., et al, eds.), CRC Press, Boca Raton (1984) ; SUBSTITUTE SHEET Poznansky, M.J., and Juliano, R.L. (1984) Pharmacol. Rev. 36. 277-336. Richardson, V.J., et al. (1979) Br. J. Cancer _4J3, 3543. Saba, T.M. (1970) Arch. Intern. Med. 126. 1031-1052. Schaver, R. (1982) Adv. Carbohydrate Chem. Biochem. £0:131. Scherphof, T., et al. (1978) Biochi .Biophys. Act 542, 296-307. Senior, J., and Gregoriadis, G. (1982) FEBS Lett. 145, 109-114. Senior, J., et al. (1985) Biochim. Biophys. Act 839, 1-8. Szoka, F., Jr., ct al. (1978) Proc. Natl. Acad. Sci. USA 75:4194. Szoka, F., Jr., et al. (1980) Ann. Rev. Biophys. Bioeng. _9:467. einstein, J.W., et al., Pharmar Ther, 24:20 (1984) . Woodruff, J.J., et al. (1969) J. Exp. Med. 129:551. 3. Background of the Invention It would be desirable, for extravascular tumo diagnosis and therapy, to target an imaging or therapeu tic compound selectively to the tumor via the blood stream. In diagnostics, such targeting could be used t provide a greater concentration of an imaging agent a the tumor site, as well as reduced background levels o the agent in other parts of the body. Site-specifi targeting would be useful in therapeutic treatment o tumors, to reduce toxic side effects and to increase th drug dose which can safely be delivered to a tumor site. SUBSTITUTE SHEET 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., Gregoria¬ dis, 1974; Jonah; Gregoriadis, 1972; Juliano; Allen, 1983; Kimelberg, 1976; Richardson; Lopez-Berestein; Allen, 1981; Scherpho±; Gregoriadis, 1980; Hwang; Patel, 1983; Senior, 1985; Allen, 1983; Ellens; Senior, 1982; Hwang; Ashwell; Hakomori; Karlsson; Schauer; Durocher; Greenberg; Woodruff; Czop; and Olrada) . 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 lipo¬ somes 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 endo- thelial 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 adja- SUBSTITUTE SHEET cent basement membranes, particularly in view of the low blood flow to tumors and hence limited exposure to circu¬ lating liposomes (Weinstein) . Higher than normal inter¬ stitial pressures found within most tumors would also tend to reduce the opportunity for extravasation of lipo¬ somes by creating a 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 over¬ come these barriers to extravasation in tumors and, at the sa ?. time, evade RES recognition and uptake (Poz- nanski) . In fact, studies reported to date indicate that even where the permeability of blood vessels increases, extra¬ vasation 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 bei w detection levels, its therapeutic potential would be minimal (Poste) . 4. 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 blood- stream 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. SUBSTITUTESHEET 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 this context, vesicle-forming lipid is defined as any lipid that by itself or in combination with other lipids forms bilayer structures. In a preferred embodiment, the hydrophilic polymer is polyethyleneglycol, poly lactic poly glycoloc acid 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 alka¬ loid, 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 μg compound/μmole liposome lipid in the case of the anthracycline antibio¬ tics and and 1 μg/μmoles lipid in the case of the plant alkaloids. In a related aspect, the invention includes a com- position of liposomes characterized by: (a) liposomes composed of vesicle-forming lipids and between 1-20 mole percent of an 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 adminis¬ tered by IV injection. In this case, following IV admi¬ nistration the agent is carried through the bloodstream SUBSTITUTE SHEET in liposome-entrapped form with little leakage of th drug during the first 48 hours post injection. By virtu of the low rate of RES uptake during this period, th liposomes have the opportunity to distribute to and ente the tumor. Once within the interstitial spaces of th tumor, it is not necessary that the tumor cells actuall internalize the liposomes. The entrapped agent is re leased from the liposome in close proximity to the tumo cells over a period of days to weeks and is free t further penetrate into the tumor mass (by a process o diffusion) and enter tumor cells directly - exerting its anti-proliferative activity. The method includes entrap¬ ping the agent in liposomes of the type characterize above. One liposome composition preferred for transpor - ing anthracycline antibiotic or plant alkaloid anti-tumor agents to systemic solid tumors would contain high phase transition phospholipids and cholesterol as this type o 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 metho for localizing a compound in a solid tumor in a subject. The method includes preparing a composition of liposo.nes (i) composed of vesicle-forming lipids and between 1-20 mole percent of an vesicle-forming lipid derivatized wit a hydrophilic polymer, (ii) having an average size in selected size range between about 0.07-0.12 microns, an (iii) containing the compound in liposome-entrapped form. The composition is injected IV in the subject in a amount sufficient to localize a therapeutically effectiv dose of the agent in the solid tumor. These and other objects and features of the presen invention will become more fully apparent when the fol lowing detailed description of the invention is read i SUBSTITUTE SHEET conjunction with the accompanying drawings. Brief Description of the Drawings Figure 1 illustrates a general reaction scheme for derivatizing a vesicle-forming lipid amine with a polyal- kylether; Figure 2 is a reaction scheme for preparing phospha- tidylethanolamine (PE) derivatized with polyethylene- glycol via a cyanuric chloride linking agent; Figure 3 illustrates a reaction scheme for preparing phosphatidylethanolamine (PE) derivatized with polyethy- leneglycol by -ϊieans of a diimidazole activating reagent; Figure 4 illustrates a reaction scheme for preparing phosphatidylethanolamine (PE) derivatized with polyethy- leneglycol by means of a trifluoromethane sulfonate reagent; Figure 5 illustrates a vesicle-forming lipid deriva¬ tized with polyethyleneglycol through a peptide (A) , ester (B) , and disulfide (C) linkage; Figure 6 illustrates a reaction scheme for preparing phosphatidylethanolamine (PE) derivatized with poly lactic acid; Figure 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 lipo¬ somes containing different amounts of phosphatidylglyce- rol; Figure 8 is a plot similar to that of Figure 7, showing blood residence times of liposomes composed of predominantly unsaturated phospholipid components; Figure 9 is a plot similar to that of Figure 7, showing the blood residence times of PEG-coated liposomes (solid triangles) and conventional, uncoated liposomes (solid circles) ; SUBSTITUTE SHEET Figure 10 is a plot similar to that of Figure 7, showing the blood residence time of polylactic acid- coated liposomes (solid squares) polyglycolic acid-coated liposomes (open triangles) ; Figure 11 is a plot showing the kinetics of doxoru¬ bicin 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 lipo- somes coated, with PEG (open triangles) ; Figures 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; Figure 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 drag associated with tumor cells (solid circles) and liposome-associated form (solid triangles) ; Figures 14A-14D are light micrographs showing loca¬ lization 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) ; Figure 15A-C 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), epiru- bicin at 6 mg/kg (open triangles) , or PEG-liposome-en- trapped epirubicin at two doses, 6mg/kg (filled trian- SUBSTITUTE SHEET gles) 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 entrap- ped at two doses, 6 mg/kg (filled triangles) and 9 mg/kg (open squares) on days 3 and 10 (15B) or days 10 and 17 (15C) ; Figure 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 tri¬ angles) , or untreated animals (open triangles) ; and Figure 17 is a plot similar to that in Figure 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 ani¬ mals treated with 10 mg/kg doxorubicin in free form (filled squares) , or in conventional liposomes (open circles) . Detailed Description of the Invention I. Preparation of Derivatized Lipids Figure 1 shows a general reaction scheme for prepa- ring a vesicle-forming lipid derivatized 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 em¬ ployed, e.g., PEG, is preferably capped by a methoxy, ethoxy or other unreactive group at one end or, alterna¬ tively, has a chemical group that is more highly reactive at one end than the other. The polymer is activated at SUBSTITUTE SHEET one of its ends by reaction with a suitable activatin agent, such as cyanuric acid, diimadozle, anhydrid reagent, or the like, as described below. The activate compound is then reacted with a vesicle-forming lipid, such as a diacyl glycerol, including diacyl phosphogly cerols, where the two hydrocarbon chains are typicall between 14-22 carbon atoms in length and have varyin degrees of saturation, to produce the derivatized lipid. Phosphatidylethanol-amine (PE) is an example of a phos- pholipid 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 poly¬ mer, or the two groups may be joined in a concerte 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 phos- pholipids, such as phosphatidylcholine (PC) , PE, phos phatidic acid (PA), phosphatidylinositol (PI), and sphin- gomyelin (SM) , where the two hydrocarbon chains ar typically between about 14-22 carbon atoms in length, an have varying degrees of unsaturation. Also included i this class are the glycolipids, such as cerebrosides an gangliosides. Another vesicle-forming lipid which may be employe is cholesterol and related sterols. In general, choles terol may be less tightly anchored to a lipid bilaye membrane, particularly when derivatized with a hig molecular weight polymers, such as polyalkylether, an therefore be less effective in promoting liposome evasio SUBSTITUTE SHEET of the RES in the bloodstream. More generally, and as defined herein, "vesicle forming lipid" is intended to include any amphipathi lipid having hydrophobic and polar head group moieties, and which (a) by itself can form spontaneously int bilayer vesicles in water, as exemplified by phospholi pids, or (b) is stably incorporated into lipid bilayer in combination with phospholipids, with its hydrophobi moiety in contact with the interior, hydrophobic regio of the bilayer membrane, and its polar head group moiet oriented toward the exterior, polar surface of the mem brane. An example of a latter type of vesicle-formin lipid is cholesterol and cholesterol derivatives, such a cholesterol sulfate and cholesterol hemisuccinate. According to one important feature of the invention, the vesicle-forming lipid may be a relatively flui lipid, typically meaning that the lipid phase has relatively low liquid to liquid-crystalline meltin temperature, e.g., at or below room temperature, o relatively rigid lipid, meaning that the lipid has relatively high melting temperature, e.g., up to 60°C. As a rule, the more rigid, i.e., saturated lipids, con tribute to greater membrane rigidity in a lipid bilaye structure and also contribute to greater bilayer stabi lity in serum. Other lipid components, such as choleste¬ rol, are also known to contribute to membrane rigidity and stability in lipid bilayer structures. A long chai (e.g. C-18) saturated lipid plus cholesterol is on preferred composition for delivering anthracycline anti biotic and plant alkaloids anti-tumor agents to soli tumors since these liposomes do not tend to release th drugs into the plasma as they circulate through th bloodstream and enter the tumor during the first 48 hour following injection. Phospholipids whose acyl chain have a variety of degrees of saturation can be obtained commercially, or prepared according to published methods. Figure 2 shows a reaction scheme for producing a PE- PEG 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 mate- rial 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 vari¬ ety 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 Figure 3. Here the capped PEG is activated with a carbonyl diimidazole coupling reagent, to form the activated imidazole compound shown in Figure 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 poly¬ alkylether to a lipid amine is shown in Figure 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 trimethylsilylchlo- ride with PEG in the presence of triethylamine. The protected PEG is then reacted with the anhydride of trifluoromethyl sulfonate to form the PEG compound acti- SUBSTITUTE SHEET vated 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 deriva¬ tized with hydrophilic polymers such as PEG, . For exam- pie, the sulfonate anhydride coupling reagent illustrated in Figure 4 can be used to join an activated polyalkyl¬ ether 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 phosphatidi-; 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 isothio- cyanate reagent. In another embodiment, the derivatized lipid com¬ ponents are prepared to include a labile lipid-polymer linkage, such as a peptide, ester, or disulfide linkage, which can be cleaved under selective physiological condi¬ tions, such as in the presence of peptidase or esterase enzymes or reducing agents such as glutathione present in the bloodstream. Figure 5 shows exemplary lipids which are linked through (A) peptide, (B) , ester, and (C) , SUBSTITUTE SHEET disulfide containing linkages. The peptide-linked com pound can be prepared, for example, by first coupling polyalkylether with the N-terminal amine of the tripep tide shown, e.g., via the reaction shown in Figure 3. The peptide carboxyl group can then be coupled to a lipi amine group through a carbodiimide coupling reagent con ventionally. The ester linked compound can be prepared, for example, by coupling, a lipid acid, such as phosphati dic acid, to the terminal alcohol group of a polyalkyl ether, using alcohol via an anhydride coupling agent. Alternatively, an short linkage fragment containing a internal ester bond and suitable end groups, such a primary amine groups can be used to couple the polyalkyl ether to the amphipathic lipid through amide or carbamat linkages. Similarly, the linkage fragment may contain a internal disulfide linkage, for use in forming the com pound shown at C in Figure 5. Polymers coupled to phos pholipids via such reversible linkages are useful t provide high blood levels of liposomes which contain the for the first few hours post injection. After thi period, plasma components cleave the reversible bond releasing the polymers and the "unprotected" liposome are rapidly taken up by the RES. Figure 6 illustrates a method for derivatizin polylactic acid with PE. The polylactic acid is reacted, in the presence of PE, with dicyclohexylcarboimid (DCCI) , as detailed in Example 4. Similarly, a vesicle forming lipid derivatized with polyglycolic acid may b formed by reaction of polyglycolic acid or glycolic aci with PE in the presence of a suitable coupling agent, such as DCCI, also as detailed in Example 4. The vesi cle-forming lipids derivatized with either polylacti acid or polyglycolic acid form part of the inventio herein. Also forming part of the invention are liposome containing these derivatized lipids, in a 1-20 mole percent. II. Preparation of Liposome Composition A. Lipid Components The lipid components used in forming the liposomes of the invention may be selected from a variety of vesi¬ cle-forming lipids, typically including phospholipids, sphingolipids and sterols. As will be seen, one require- ment of the liposomes of the present invention is long blood circulation lifetime. It is therefore useful to establish a standardized measure of blood lifetime which can be used for evaluating the effect of lipid components on blood halflife. One method used for evaluating liposome circulation time in vivo measures the distribution of IV injected liposomes in the bloodstream and the primary organs of the RES at selected times after injection. In the stan¬ dardized model which is used herein, RES uptake is mea- sured by the ratio of total liposomes in the bloodstream to total liposomes in the liver and spleen, the principal organs of the RES. In practice, age and sex matched mice are injected IV through the tail vein with a radiolabtled liposome composition, and each time point is determined by measuring total blood and combined liver and spleen radiolabel counts, as detailed in Example 5. Since the liver and spleen account for nearly 100% of the initial uptake of liposomes by the RES, the blood- /RES ratio just described provides a good approximation of the extent of uptake from the blood to the RE3 in vivo. For example, a ratio of about 1 or greater indi¬ cates a predominance of injected liposomes remaining in the bloodstream, and a ratio below about 1, a predomi¬ nance of liposomes in the RES. For most of the lipid SUBSTITUTE SHEE compositions of interest, blood/RES ratios were calcu lated at 1,2, 3, 4, and 24 hours post injection. The liposomes of the present invention include 1-2 mole percent of the vesicle-forming lipid derivatize with a hydrophilic polymer, described in Section I. According to one aspect of the invention, it has bee discovered that blood circulation halflives in thes liposomes is largely independent of the degree of satura tion of the phospholipid components making up the lipo somes. That is, the phospholipid components may b composed of predominantly of fluidic, relatively unsatu rated, acyl chains, or of more saturated, rigidifyin acyl chain components. This feature of the invention i seen in Example 6, which examines blood/RES ratios i liposomes formed with PEG-PE, cholesterol, and PC havin varying degrees of saturation (Table 4)-. As seen fro the data in Table 5 in the example, high blood/RES ratio were achieved in substantially ail of the liposome for mulations, independent of the extent: of lipid u satura tion in the bulk PC phospholipid, and no systemati trend, as a function of degree of lipid saturation, wa observed. Accordingly, the vesicle-forming lipids may b selected to achieve a selected degree of fluidity o rigidity, to control the stability of the liposomes i serum and the rate of release of entrapped drug from th liposomes in the bloodstream and/or tumor. The vesicle forming lipids may also be selected, in lipid saturatio characteristics, to achieve desired liposome preparatio properties. It is generally the case, for example, tha more fluidic lipids are easier to formulate and down-siz by extrusion and homogenization methods than more rigi lipid compositions. Similarly, it has been found that the percentage of cholesterol in the liposomes may be varied over a wide range without significant effect on observed blood/RES ratios. The studies presented in Example 7A, with refer- ence to Table 6 therein, show virtually no change in blood/RES ratios in the range of cholesterol between 0-30 mole percent. It has also been found, in studies conducted in support of the invention, that blood/RES ratios are also relatively unaffected by the presence of charged lipid components, such as phosphatidylglycerol (PG) . This can be seen from Figure 7, which plots percent loss of encap¬ sulated marker for PEG-PE liposomes containing either 4.7 mole percent PG (triangles) or 14 mole percent PG (cir- cles) . Virtually no difference in liposome retention in the bloodstream over a 24 hour period was observed. The option of including negative charge in the liposome without aggravating RES uptake provides a number of potential advantages. Liposomes suspensions which con- tain negative charge tend to be less sensitive to aggre¬ gation in high ionic strength buffers and h nce physical stability is enhanced. Also, negative charge present in the liposome membrane can be used as a formulation tool to effectively bind high amounts of cationic drugs. The vesicle-forming lipid derivatized with a hydro¬ philic polymer is.present in an amount preferably between about 1-20 mole percent, on the basis of moles of deriva¬ tized lipid as a percentage of total moles of vesicle- forming lipids. It will be appreciated that a lower mole ratio, such as 0.0 mole percent, may be appropriate for a lipid derivative with a large molecular weight polymer, such as one having a molecular weight of 100 kilodaltons. As noted in Section I, the hydrophilic polymer in the derivatized lipid preferably has a molecular weight between about 200-20,000 daltons, and more preferabl between about 500-5,000 daltons. Example 7B, whic examines the effect of very short ethoxy ether moietie on blood/RES ratios indicates that polyether moieties o greater than about 5 carbon ethers are required t achieve significant enhancement of blood/RES ratios. B. Preparing the Liposome Composition The liposomes may be prepared by a variety of tech niques, such as those detailed in Szoka et al, 1980. On method for preparing drug-containing liposomes is th reverse phase evaporation method described by Szoka et a and in U.S. Patent No. 4,235,871. The reverse phas evaporation vesicles (REVs) have typical average size between about 2-4 microns and are predominantly oligo lamellar, that is, contain one or a few lipid bilaye shells. The method is detailed in Example 4A. Multilamellar vesicles (MLVs) can be formed b simple lipid-film hydration techniques. In this proce dure, a mixture of liposome-forming lipids of the typ detailed above dissolved in a suitable organ:c solvent i evaporated in a vessel to form.,a thin film, which is the covered by an aqueous medium, as detailed in Example --IB The lipid film hydrates to form MLVs, typically wit sizes between about 0.1 to 10 microns. In accordance with one important aspect of th invention, the liposomes are prepared to have substan tially homogeneous sizes in a selected size range betwee about 0.07 and 0.12 microns. In particular, it has bee discovered that liposomes in this size range are readil able to extravasate into solid tumors, as discussed i Section III below, and at the same time, are capable o carrying a substantial drug load to a tumor (unlike smal unilamellar vesicles, which are severely restricted i drug-loading capacity) . One effective sizing method for REVs and MLVs in¬ volves extruding an aqueous suspension of the liposomes through a series of polycarbonate membranes having a selected uniform pore size in the range of 0.03 to 0.2 micron, typically 0.05, 0.08, 0.1, or 0.2 microns. The pore size of the membrane corresponds roughly to the largest sizes of liposomes produced by extrusion through that membrane, particularly where the preparation is extruded two or more times through the same membrane. This method of liposome sizing is used in preparing homogeneous-size REV and MLV compositions described in the examples below. A more recent method involves extru¬ sion through an asymmetric ceramic filter. The method is detailed in U.S. patent No. 4,737,323 for Liposome Extru¬ sion issued April 12, 1988. Homogenization methods are also useful for down-sizing liposomes to sizes of lOOnm or less (Martin) . C Compound Loading In one embodiment, the composition of t^e invention is used for localizing an imaging agent, such as radio- isotopes including 67 Ga and U1 ln, or paramagnetic com¬ pounds at the tumor site. In this application, where the radiolabel can be detected at relatively low concentra¬ tion, it is generally sufficient to encapsulate the imaging agent by passive loading, i.e., during liposome formation. This may be done, for example, by hydrating lipids with an aqueous solution of the agent to be encap- sulated. Typically radiolabeled agents are radioisotopic metals in chelated form, such as 67 Ga-desferal, and are retained in the liposomes substantially in entrapped form. After liposome formation and sizing, non-encapsu¬ lated material may be removed by one of a variety of SUBSTITUTE SHEET methods, such as by ion exchange or gel filtration chro matography. The concentration of chelated metal whic can be achieved by passive loading is limited b th concentration of the agent in the hydrating medium. Active loading of radioimaging agents is also pos sible by entrapping a high affinity, water soluble chela ting agent (such as EDTA or desferoxa ine) within th aqueous compartment of liposomes, removing any unen trapped chelating agent by dialysis or gel exclusio column chromatography and incubating the liposomes in th presence of the metal radioisotope chelated to a lowe affinity, lipid soluble chelating agent such as 8-hydr oxyquinoline. The metal radioisotope is carried into th liposome by the lipid soluble chelating agent. Once i the liposome, the radioisotope is chelated by the en trapped, water soluble chelating agent - effectivel trapping the radioisotope in the liposome interior (Gabi zon) . Passive loading may also be employed for th amphipathic anti-tumor compounds, such as the alkaloid vinblastine and vincristine, which are the.rapeuticall active at relatively low drug doses, e.g., about 1-1 mg/m 2 . Here the drug is either dissolved in the aqueou phase used to hydrate the lipid or included with th lipids in liposome formation process, depending on th solubility of the compound. After liposome formation an sizing, free (unbound) drug can be removed, as above, fo example, by ion exchange or gel exclusion chro atographi methods. Where the anti-tumor compound includes a peptide o protein drug, such as interleukin-2 (IL-2) or tissu necrosis factor (TNF) , or where the liposomes are formu lated to contain a peptide immunomodulator, such a muramyl di- or tri-peptide derivatives or a protei SUBSTITUTE SHEET immunomodulator such as macrophage colony stimulatin factor (M-CSF) , the liposomes are preferably prepared b the above reverse phase method or by rehydrating a freez dried mixture of the protein and a suspension of smal unilamellar vesicles with water (Kirby) . Both method combine passive loading with relatively high encapsu lation efficiency, e.g., up to 50% efficiency. Nonencap sulated material can be readily removed from the liposom suspension, e.g., by dialysis, diafiltration or exclusio chromatography. The concentration of hydrophobic drug which can b accommodated in the liposomes will depend on drug/lipi interactions in the membrane, but is generally limited t a drug concentration of less than about 20 μg drug/m lipid. More specifically, for a variety of anthracyclin antibiotics, such as doxorubicin and epirubicin, th highest concentration of encapsulated material which ca be achieved by passive loading into the aqueous compart ment of the liposome is about 10-20 μg/μmoles lipid (du to the low intrinsic water solubility of thes compounds) . When 20-30 mole percent of an a.iionic phos pholipid such as PG is included in the membrane th loading factor can be increased to about 40 μg/μmol lipid because the anthracyclines are positively charge and form an "ion pair" complex with the negativel charged PG at the membrane interface. However, suc charged complexed anthracycline formulations have limite utility in the context of the present invention (whic requires that the drug be carried through the bloodstrea for the first 24-48 hours following IV administration i liposome entrapped form) because the drugs tend to b rapidly released from the liposome membrane when intro duced into plasma. SUBSTITUTE SHEET In accordance with another aspect of the inventio it has been found essential, for delivery of an therape tically effective dose of a variety of amphipathic ant tumor drugs to tumors, to load the liposomes to a hi drug concentration by active drug loading methods. F example, for anthracycline antibiotic drugs, such doxorubicin, epirubicin, daunorubicin, carcinomycin, acetyladriamycin, rubidazone, 5-imidodaunomycin, and acetyldaunomycin, a final concentration of liposom entrapped drug of greater than about 25 μg/μmole lip and preferably 50 μg/μmole lipid is desired. Intern drug concentrations as high as 100-200 μg/μmole lipid a contemplated. One method for active loading of amphipathic dru into liposomes is described in co-owned U.S. pate application Serial No. 413,037, filed September 28, 1988 In this method, liposomes are prepared in the presence a relatively high concentration of ammonium ion, such 0.125 M ammonium sulfate. After sizing the liposomes t a desired size, the liposome suspension is treated t create an inside-to-outside ammonium ion gradient acros the liposomal membranes. The gradient may be created dialysis against a non-ammonium containing medium, su as an isotonic glucose medium, or by gel filtration, su as on a Sephadex G-50 column equilibrated with 0.15M Na or KC1, effectively replacing ammonium ions in the ext rior phase with sodium or potassium ions. Alternativel the liposome suspension may be diluted with a non-a monium solution, thereby reducing the exterior-pha concentration of ammonium ions. The ammonium concentr tion inside the liposomes is preferably at least times, and more preferably at least 100 to 1000 tim that in the external liposome phase. SUBSTITUTE SHEET The ammonium ion gradient across the liposomes in turn creates a pH gradient, as ammonia is released across the liposome membrane, and protons are trapped in the internal aqueous phase of the liposome. To load lipo- somes with the selected drug a suspension of the lipo¬ somes, e.g., about 20-200 mg/ml lipid, is mixed with an aqueous solution of the drug, and the mixture is allowed to equilibrate over an period of time, e.g., several hours, at temperatures ranging from room temperature to 60°C - depending on the phase transition temperature of the lipids used to form the liposome. In one typical method, a suspension of liposomes having a lipid con¬ centration of 50 μmoles/ml is mixed with an equal volume of anthracycline drug at a concentration of about 5-8 mg/ml. At the end of the incubation period, the suspen¬ sion is treated to remove free (unbound) drug. One preferred method of drug removal for anthracycline drugs is by passage over an ion exchange resin, such S Dowex 50 WX-4, which is capable of binding the drug. Although, as noted above, the plant alkaloids such as vincristine do not require high loading factors in liposomes due to their intrinsically high anti-tumor activity, and thus can be loaded by passive ntrapment techniques, it also possible to load these drug by active methods. Since vincristine is amphipathic and a weak base, it and similar molecules can be loaded into lipo¬ somes using a pH gradient formed by entrapping ammonium sulfate as described above for the anthracycline antibio¬ tics. The remote loading method just described is illus¬ trated in Example 10, which describes the preparation of 0.1 micron MLVs loaded with doxorubicin, to a final drug concentration of about 80-100 μg/μmoles lipid. The lipo- SUBSTITUTE SHEET somes show a very low rate of drug leakage when stored a 4°C. III. Liposome Localization in Solid Tumors A. Extended Bloodstream Halflife One of the requirements for liposome localization i a target tumor, in accordance with the invention, is a extended liposome lifetime in the bloodstream followin IV liposome administration. One measure of liposom lifetime in the bloodstream in the blood/RES ratio deter mined at a selected time after liposome administration as discussed above. Blood/RES ratios for a variety o liposome compositions are given in Table 3 of Example 5 In the absence of PEG-derivatized lipids, blood/RE ratios were 0.03 or less. In the presence of PEG-deriva tized lipids, the blood/RES ratio ranged from 0.2, fo low-molecular weight PEG, to between 1.7-4 for several o the formulations, one of which lacks cholesterol, an three of which lack an added charged phospholipid (e.g. PG) . The data presented in Table 5 in Example 6 sho blood/RES ratios (excluding two points with low perce recovery) between about 1.26 and 3.27, consistent wit the data given in Table 3. As noted in Section II above the blood lifetime values are substantially independe of degree of saturation of the liposome lipids, presen of cholesterol and presence of charged lipids. The blood/RES values reported above can be compar with blood/RES values reported in co-owned U.S. Pate No. 4,920,016, which used blood/RES measurement metho identical to those used in generating the data present in Tables 3 and 5. The best 24-hour blood/RES rati which were reported in the above-noted patent .was 0. for a formulation composed of GM saturated PC, a SUBSTITUTE SHEET cholesterol. The next best formulations gave 24-hou blood/RES values of about 0.5. Thus, typical 24-hou blood/RES ratios obtained in a number of the curren formulations were more than twice as high as the bes formulations which have been reported to date. Further ability to achieve high blood/RES with GMj . or HPI lipid was dependent on the presence of predominantly saturate lipids and cholesterol in the liposomes. Plasma pharmacokinetics of a liposomal marker in th bloodstream can provide another measure of the enhance liposome lifetime which is achieved by the liposom formulations of the present invention. Figure 7 and discussed above show the slow loss of liposomal marke from the bloodstream over a 24 hour period in typica PEG-liposome formulations, substantially independent o whether the marker is a lipid or an encapsulated water soluble compound (Figure 8) . In both plots, the amoun of liposomal marker present 24 hours after liposom injection is greater than 10% of the originally injecte material. Figure 9 shows the kinetics of liposome- loss fro the blood stream for a typical PEG-liposome formulatio and the same liposomes in the absence of a I- ' G-deriva tized lipid. After 24 hours, the percent marker remain ing in the PEG-liposomes was greater than about 20% whereas the conventional liposomes showed less than 5 retention in the blood after 3 hours, and virtually n detectable marker at 24 hours. The results seen in Figures 7-9 are consistent wit 24 hour blood liposome values measured for a variety o liposome formulations, and reported in Tables 3 and 5- in Example 5-8 below. As seen in Table 3 in Example 5 the percent dose remaining at 24 hours was less than 1 for conventional liposomes, versus at least 5% for th SUBSTITUTE SHEET PEG-liposo es. In the best formulations, values betw about 20-40% were obtained. Similarly in Table 5 f Example 6, liposome levels in the blood after 24 ho (again neglecting two entries with low recovery valu were between 12 and about 25 percent of total dose giv Similar results are reported in Tables 6 and 7 of Exam 7. The ability of the liposomes to retain an amp pathic anti-tumor drug in the bloodstream over the 24 period required to provide an opportunity for the li some to reach and enter a systemic tumor has also b investigated. In the study reported in Example 11, plasma pharmacokinetics of doxorubicin loaded in P liposomes, doxorubicin given in free form, and doxoru cin loaded into liposomes containing hydrogenated ph phatidylinositol (HPI) was invested in beagle dogs. HPI liposomes were formulated with a predominantly sa rated PC lipid and cholesterol, and represents one of optimal formulations described in the above co-owned U. patent. The kinetics of doxorubicin in the blood up 72 hours after drug administration is shown ir. Figure Both liposomal formulations showed single-mode expon tial loss of drug, in contrast to free drug hl show bi-exponential pattern. However, the amount of d retained in the blood stream at 72 hours was about 8 times greater in the PEG-liposomes. For both blood/RES ratios, and liposome retent time in the bloodstream, the data obtained from a mo animal system can be reasonably extrapolated to hum and veterinary animals of interest. This is beca uptake of liposomes by liver and spleen has been found occur at similar rates in several mammalian speci including mouse, rat, monkey, and human (Gregoriad 1974; Jonah; Kimelberg, 1976; Juliano; Richards SUBSTITUTE SHEET Lopez-Berestein) . This result likely reflects the fac that the biochemical factors which appear to be mos important in liposome uptake by the RES — includin opsinization by serum lipoproteins, size-dependent uptak effects, and cell shielding by surface moieties — ar common features of all mammalian species which have bee examined. B. Extravasation into Tumors Another required feature for high-activity liposome targeting to a solid tumor, in accordance with the inven¬ tion, is liposome extravasation into the tumor through the endothelial cell barrier and underlying basement membrane separating a capillary from the tumor cells supplied by the capillary. This feature is optimized in liposomes having sizes between 0.07 and 0.12 microns. That liposome delivery to the tumor is required for selective drug targeting can be seen from the study reported in Example 12. Here mice were inoculated sub- cutaneously with the J-6456 lymphoma which formed a solid tumor mass of about 1 cm 3 after one-two weeks . The ani¬ mals were then injected either with free doxorubicin or doxorubicin loaded into PEG-liposomes at a do.«a of lOmg drug per kg body weight. The tissue distribution (heart, muscle, and tumor) of the drug was then assayed at 4, 24, and 48 hours after drug administration. Figure 12A shows the results obtained for free drug. No selective drug accumulation into the tumor occurred, and in fact, the highest initial drug levels were in the heart, where greatest toxicity would be produced. By contrast, drug delivery in PEG-liposomes showe increasing drug accumulation into the tumor between 4-24 hours, and high selective tumor levels between 24 and 48 hours. Drug uptake by both heart and muscle tissue was, SUBSTITUTE SHEET by contrast, lower than with free drug. As seen from t data plotted in Figure 12B, the tumor contained 8 ti more drug compared with healthy muscle and 6 times t amount in heart at 24 hours post injection. To confirm that the PEG-liposomes deliver more ant tumor drug to a intraperitoneal tumor, groups of mi were injected IP with 10 6 J-6456 ly phoma cells. Aft five days the IP tumor had been established, and t animals were treated IV with lO g/kg doxorubicin, eit in free drug form or entrapped in PEG-containing li somes. Tissue distribution of the drug is tabulated Table 9, Example 12. As shown, the tumor/heart ratio about 272 greater for liposome delivery than for f drug at 24 hours, and about 47 times greater at 48 hour To demonstrate that the results shown in Table 9 due to the entry of intact liposomes into the extrav cular region of a tumor, the tumor tissue was separat into cellular and supernatant (intercellular flui fractions, and the presence of liposome-associated free drug in both fractions was assayed. Figure 13 sh the total amount of drug (filled diamonds) anc' the amo of drug present in tumor cells (solid circles) and sup natant (solid diamonds) over a 48-hour post injecti period. To assay liposo e-associated drug, the sup natant was passed through an ion-exchange resin to rem free drug, and the drug remaining in the supernatant assayed (solid triangles) . As seen, most of the drug the tumor is liposome-associated. Further demonstration of liposome extravasation i tumor cells was obtained by direct microscopic obser tion of liposome distribution in normal liver tissue in solid tumors, as detailed in Example 14. Figure shows the distribution of liposomes (small, . dar stained bodies) in normal liver tissue 24 hours after injection of PEG-liposomes. The liposomes are confine exclusively to the Kupfer cells and are not presen either in hepatocytes or in the intercellular fluid o the normal liver tissue. Figure 14B shows a region of C-26 colon carcinom implanted in the liver of mice, 24 hours after injectio of PEG-liposomes. Concentrations of liposomes are clear ly evident in the region of the capillary in the figure on the tumor tissue side of the endothelial barrier an basement membrane. Liposomes are also abundant in th intercellular fluid of the tumor cells, further eviden cing passage from the capillary lumen into the tumor The Figure 14C photomicrograph shows another region o the tumor, where an abundance of liposomes in the inter cellular fluid is also evident. A similar finding wa made with liposome extravasation into a region of C-2 colon carcinoma cells injected subcutaneously, as seen i Figure 14D. IV. Tumor Localization Method As detailed above, the liposomes of the inventio are effective to localize specifically in a solid tumo region by virtue of the extended lifetime of the lipo somes in the bloodstream and a liposome size which allow both extravasation into tumors, a relatively high dru carrying capacity and minimal leakage of the entrappe drug during the time required for the liposomes to dis tribute to and enter the tumor (the first 24-48 hour following injection) . The liposomes thus provide a effective method for localizing a compound selectively t a solid tumor, by entrapping the compound in such lipo somes and injecting the liposomes IV into a subject. I this context a solid tumor is defined as one that grow in an anatomical site outside the bloodstream (in con trast, for example, to blood-born tumors such as leuke mias) and requires the formation of small blood vessel and capillaries to supply nutrients, etc. to the growin tumor mass. In this case, for an IV injected liposo (and its entrapped anti-tumor drug) to reach the tumo site it must leave the bloodstream and enter the tumor. In one embodiment, the method is used for tumor treatmen by localizing an anti-tumor drug selectively in th tumor. The anti-tumor drug which may be used is an compound, including the ones listed below, which can b stably entrapped in liposomes at a suitable loadin factor and administered at a therapeutically effectiv dose (indicated below in parentheses after eac compound) . These include amphipathic anti-tumor com pounds such as the plant alkaloids vincristine (1. mg/m 2 ) , . vinblastine (4-18 mg/m 2 ) and etoposide (35-10 mg/m 2 ) , and the anthracycline antibiotics including doxo rubicin (60-75 mg/m 2 ), epirubicin (60-120 mg/m 2 ) an daunorubicin (25-45 mg/m 2 ) . The water-soluble anti-meta bolites such as methotrexate 3 mg/m 2 ) , cytosine arabino side (100 mg/m 2 ), and fluorouracil (10-15 mg/kg), th antibiotics such as bleomycin (10-20 units/m 2 ) , mitomyci (20 mg/m 2 ) , plicamycin (25-30 μg/m 2 ) and dactincycin (1 μg/m 2 ) , and the alkylating agents including cyclophospha mide (3-25 mg/kg), thiotepa (0.3-0.4 mg/Kg) and BCN (150-200 mg/m 2 ) are also useful in this context. noted above, the plant alkaloids exemplified by vincris tine and the anthracycline antibiotics including doxoru bicin, daunorubicin and epirubicin are preferably active ly loaded into liposomes, to achieve drug/lipid ratio which are several times greater than can be achieved wit passive loading. Also as noted above, the liposomes ma contain encapsulated tumor-therapeutic peptides a protein drugs , such as IL-2, and/or TNF, and/or immun modulators, such as M-CSF, which are present alone or i combination with anti-tumor drugs, such as an anthracy cline antibiotic drug. The ability to effectively treat solid tumors, i accordance with the present invention, has been shown i a variety of in vivo systems. The method reported i Example 15 compares the rate of tumor growth in animal with implanted subcutaneously with a C-26 colon carci noma. Treatment was with epirubicin, either in fre form, or entrapped in PEG-liposomes, in accordance wit the invention, with the results shown in Figures 15A- As seen, and discussed more fully in Example 15, treat ment with epirubicin loaded PEG-liposomes produced marked supression of tumor growth and lead to long ter survivors among groups of animals inoculated with normally lethal dose of tumor cells. Moreover, delaye treatment of animals with the epiribicin loaded PEG lipo somes resulted in regression of established subcutaneou tumors, a result not seen with free drug treatment. Similar results were obtained for treatment of lymphoma implanted interperitoneally in mice, as detaile in Example 16. Here the- animals were treated with doxo rubicin in free form or entrapped in PEG-liposomes Percent survivors over a 100-day period following tumo implantation and drug treatment is shown in Figure 16 The results are similar to those obtained above, showin marked increase in the median survival time and percen survivors with PEG-liposomes over free drug treatment. Since reduced toxicity has been observed in mode animal systems and in a clinical setting in tumor treat ment by doxorubicin entrapped in conventional liposome (as reported, for example, in U.S. Patent No. ) it is of interest to determine the degree of toxicit protection provided in the tumor treatment method of th present invention. In the study reported in Example 1 animals were injected IV with increasing doses of doxor bicin or epirubicin in free form or entrapped in conve tional or PEG-liposomes. The maximum tolerated do (MTD) for the various drug formulations is given in Tab 10 in the Example. For both drugs, entrapment in PE liposomes approximately doubled the MTD of the dru Similar protection was achieved with conventional lip somes. Although reduced toxicity may contribute to t increased efficacy of tumor treatment reported abov selective localization of the drug by liposomal extrav sation is also important for improved drug efficac This is demonstrated in the drug treatment method d scribed in Example 18. Here conventional liposom containing doxorubicin (which show little or no tum uptake by extravasation when administered IV) were co pared with free drug at the same dose (10 mσ/kg) reduce reduce the rate of growth of a subcuatrneous implanted tumor. Figure 17 plots tumor size with time days following tumor implantation for a saline contr (solid line) , free drug (filled circles) and convention liposomes (filled triangles) . As seen convertional lip somes do not supress tumor growth to any greater exte than free drug at the same dose. This finding stands stark contrast to the results shown in Figures 15A-C a 16 where improved survival and tumor growth supression seen compared to free drug when tumor-bearing animals a treated with anthracyclines anti-tumor drugs entrapped PEG liposomes. Thus, the tumor-treatment method allows both high levels of drug to be administered, due to reduced dr toxicity in liposomes, and greater drug efficacy, due selective liposome localization in the intercellul fluid of the tumor. It will be appreciated that the ability to locali a compound selectively in a tumor, by liposome extravas tion, can also be exploited for improved targeting of imaging agent to a tumor, for tumor diagnosis. Here t imaging agent, typically a radioisotope in chelated for or a paramagnetic molecule is entrapped in liposome which are then administered IV to the subject bei examined. After a selected period, typically 24- hours, the subject is then monitored, for example gamma scintillation radiography in the case of radiois tope or by NMR in the case of the paramagnetic agent, detect regions of local uptake of the imaging agent. The following examples illustrate methods preparing liposomes with enhanced circulation times, a for accessing circulation times in vivo and in vitr The examples are intended to illustrate specific liposo compositions and methods of the invention, but are in way intended to limit the scope thereof. Materials Cholesterol (Choi) was obtained from Sigma (S Louis, MO) . Sphingomyelin (SM) , egg phosphcλti ylcholi (lecithin or PC), partially hydrogenated PC having t composition IV40, IV30, IV20, IV10, and IV1, phosphati dylglycerol (PG) , phosphatidylethanolamine (PE) , dipalmi toyl-phosphatidyl glycerol (DPPG) , dipalmitoyl PC (DPPC) dioleyl PC (DOPC) and distearoyl PC (DSPC) were obtain from Avanti Polar Lipids (Birmingham, AL) or Aust Chemical Company (Chicago, IL) . [ 125 I]-tyraminyl-inulin was made according to pu lished procedures. 67 Gallium-8-hydroxyquinoline was su plied by NEN Neoscan (Boston, MA) . Doxorubicin HC1 a Epirubicin HCL were obtained from Adria Laboratori (Columbus. OH) or Farmitalia Carlo Erba (Milan, Italy) . Example 1 Preparation of PEG-PE Linked by Cyanuric C h 1 ride A. Preparation of activated PEG 2-0-Methoxypolyethylene glycol 1900-4, 6-dichl ro-1,3,5 triazine previously called activated PEG prepared as described in J. Biol. Chem., 252:3582 (197 with the following modifications. Cyanuric chloride (5.5 g; 0.03 mol) was dissolved 400 ml of anhydrous benzene containing 10 g of anhydr sodium carbonate, and PEG-1900 (19 g; 0.01 mol) was ad and the mixture was stirred overnight at room tempe ture. The solution was filtered, and 600 ml of petrol ether (boiling range, 35-60°) was added slowly with st ring. The finely divided precipitate was collected on filter and redissolved in 400 ml of benzene. The pre pitation and filtration process was repeated seve times until the petroleum ether was free of resid cyanuric chloride as determined by high presεure liq chromatography on a column (250 x 3.2 mm) of 5-m "LiCh sorb" (E. Merck), developed with hexane, and detec with an ultraviolet detector. Titration of activa PEG-1900 with silver nitrate after overnight hydroly in aqueous buffer at pH 10.0, room temperature, gave value of 1.7 mol of chloride liberated/mol of PEG. TLC analysis of the product was effected with reversed-phase plates obtained from Baker using methan water, 4:1; v/v, as developer and exposure to iod vapor for visualization. Under these conditions- starting methoxy polyglycol 1900 appeared at R f =0.54 0.60. The activated PEG appeared at R £ =0.41. Unreac cyanuric chloride appeared at R f =0.88 and was removed. The activated PEG was analyzed for nitrogen and a appropriate correction was applied in selecting th quantity of reactant to use in further synthetic steps Thus, when the product contained only 20% of the theore tical amount of nitrogen, the quantity of material use in the next synthetic step was increased by 100/20, o 5-fold. When the product contained 50% of the theore tical amount of nitrogen, only 100/50 or a 2-fold in crease was needed. B. Preparation of N-(4-Chloro-polyglycol 1900)-l,3,5-triazinyl egg phosphatidylethanolamine. In a screw-capped test tube, 0.74 ml of a 100 mg/m (0.100 mmole) stock solution of egg phosphatidylethanol amine in chloroform was evaporated to dryness under stream of nitrogen and was added to the residue of th activated PEG described in section A, in the amount t provide 205 mg (0.100 mmole). To this mixture, 5 ml an hydrous dimethyl formamide was added. 27 microliter (0.200 mmole) triethylamine was added to the mixture, an the air was displaced with nitrogen gas. The nixture wa heated overnight in a sand bath maintained at 110°C The mixture was then evaporated to αryness unde vacuum and a pasty mass of crystalline solid was ob tained. This solid was dissolved in 5 ml of a mixture o 4 volumes of acetone and 1 volume of acetic acid. Th resulting mixture was placed at the top of a 21 mm X 24 mm chromatographic absorption column packed with silic gel (Merck Kieselgel 60, 70-230 mesh) which had firs been moistened with a solvent composed of acetone aceti acid, 80/20; v/v. The column chromatography was developed with th same solvent mixture, and separate 20 to 50 ml aliquot of effluent were collected. Each portion of effluent wa assayed by TLC on silica gel coated plates, using 2-buta none/acetic acid/water; 40/25/5; v/v/v as developer an iodine vapor exposure for visualization. Fraction containing only material of R f =about 0.79 were combine and evaporated to dryness under vacuum. Drying to con stant weight under high vacuum afforded 86 mg (31. micrσmoles) of nearly colorless solid N-(4-chloro-poly glycol 1900) -1,3,5-triazinyl egg phosphatidylethanolamin containing phosphorous. The solid compound was taken up in 24 ml of etha nol/chloroform; 50/50 chloroform and centrifuged t remove insoluble material. Evaporation of the clarifie solution to dryness under vacuum afforded 21 mg (7.6 micromoles) of colorless solid. Example 2 Preparation of Carbamate and Amide Linked Hydrophilic Polymers with PE A. Preparation of the imidazole carbamate of poly ethylene glycol methyl ether 1900. 9.5 grams (5 mmoles) of polyethylene glycol methy ether 1900 obtained from Aldrich Chemical Cc. was dis solved in 45 ml benzene which has been dried ov-=r mole cular sieves. 0.89 grams (5.5 mmoles) of pure carbony diimidazole was added. The purity was checked by a infra-red spectrum. The air in the reaction vessel wa displaced with nitrogen. Vessel was enclosed and heate in a sand bath at 75°C for 16 hours. The reaction mixture was cooled and the clear solu tion formed at room temperature. The solution was dilu ted to 50.0 ml with dry benzene and stored in the refri gerator as a 100 micromole/ml stock solution of th imidazole carbamate of PEG ether 1900. B. Preparation of the phosphatidylethanolamine ca bamate of polyethylene glycol methyl ether 1900. 10.0 ml (lmmol) of the 100 mol/ml stock solution the imidazole carbamate of polyethylene glycol meth ether 1900 was pipetted into a 10 ml pear-shaped flas The solvent was removed under vacuum. 3.7 ml of a 1 mg/ml ^ solution of egg phosphatidyl ethanolamine in chl roform (0.5 mmol) was added. The solvent was evaporat under vacuum. 2 ml of 1,1,2,2-tetrachloroethylene a 139 microliters (1.0 mmol) of triethylamine VI was adde The vessel was closed and heated in a sand bath mai tained at 95°C for 6 hours. At this time, thin-lay chromatography was performed with fractions of the abo mixture to determine an extent of conjugation on Si coated TLC plates, using butanone/acetic acid/wate 40/5/5; v/v/v; was performed as developer. 12 vapo visualization revealed that most of the free phosphatid ethanolamine of Rf=0.68, had reacted, and was replaced a phosphorous-containing lipid at R f =0.78 to 0.80. The solvent from the remaining reaction mixture w evaporated under vacuum. The residue was taken up in 1 ml methylene chloride and placed at the top of a 21 mm 270 mm chromatographic absorption column pached wit Merck Kieselgel 60 (70-230 mesh silica gel) , which h been first rinsed with methylene chloride. The mixtu was passed through the column, in sequence, using t following solvents. Table 1 50 ml portions of effluent were collected and eac portion was assayed by TLC on Si02 - coated plates, usin 12 vapor absorption for visualization after development with chloroform/methanol/water/concentrated ammonium hydroxide; 130/70/8/0.5%; v/v/v/v. Most of the phos¬ phates were found in fractions 11, 12, 13 and 14. These fractions were combined, evaporated to dryness under vacuum and dried in high vacuum to constant weight. They yielded 669 mg of colorless wax of phosphatidyl etha-nolamine carbamate of polyethylene glycol methyl ether. This represented 263 micromoles and a yield of 52.6% based on the phosphatidyl ethanolamine. An NMR spectrum of the product dissolved in deutero— chloroform showed peaks corresponding to the spectrum for egg PE, together with a strong singlet due to the methy¬ lene groups of the ethylene oxide chain at Delta = 3.4 ppm. The ratio of methylene protons from the ethylene oxide to the terminal methyl protons of the PE acyl groups was large enough to confirm a molecular weight o about 2000 for the polyethylene oxide portion of th molecule of the desired product polyethylene glyco conjugated phosphatidyethanolamine carbamate, M.W. 2,654. C. Preparation of polylactic acid amide of phosphoti- dyletanolamine. 200 mg (0.1 mmoles) poly (lactic acid), m. wt. = 2,00 (ICN, Cleveland, Ohio) was dissolved in 2.0 ml dimethy sulfoxide by heating while stirring to dissolve th material completely. Then the solution was cooled imme diately to 65°C and poured onto a mixture of 75 m (0.1 mmoles) of distearylphosphatidyl-ethanolamine (Cal Biochem, La Jolla) and 41 mg (0.2 mmoles) dicyclohexyl carbodiimide. Then 28 ml (0.2 mmoles) of triethylamin was added, the air swept out of the tube with nitroge gas, the tube capped, and heated at 65°C for 48 hours. After this time, the tube was cooled to room tempera ture, and 6 ml of chloroform added. The chlorofor solution was washed with three successive 6 ml volumes o water, centrifuged after each wash, and the phases sepa rated with a Pasteur pipette. The remaining chlorofor phase was filtered with suction to remove suspende distearolyphosphatidylethanolamine. The filtrate wa dried under vacuum to obtain 212 mg of semi-crystallin solid. This solid was dissolved in 15 ml of a mixture of volumes ethanol with 1 volume water and passed through 50 mm deep and 21 mm diameter bed of H + Dowex 50 catio exchange resin, and washed with 100 ml of the sn e sol vent. The filtrate was evaporated to dryness to obtain 131 m colorless wax. 291 mg of such wax was dissolved in 2.5 ml chlorofor and transferred to the top of a 21 mm x 280 mm column o silica gel wetted with chloroform. The chromatogram wa developed by passing through the column, in sequence, 10 ml each of: 100% chloroform, 0% (1% NH 4 OH in methanol) ; 90% chloroform, 10% (1% NH 4 OH in methanol) ; 85% chloroform, 15% (1% NH 4 OH in methanol) ; 80% chloroform, 20% (1% NH 4 OH in methanol); 70% chloroform, 30% (1% NH 4 OH in methanol); Individual 25 ml portions of effluent were saved an assayed by TLC on SF0 2 -coated plates, using CHC1 3 , CH 3 OH H 2 0, con. NH 4 OH, 130, 70, 8, 0.5 v/v as developer and vapor absorption for visualization. The 275-325 ml portions of column effluent contained single material, P0 4 +, of R £ = 0.89. When combined and evaporated to dryness, these afforde 319 mg colorless wax. Phosphate analysis agrees with a molecular weight o possibly 115,000. Apparently, the polymerization of the poly (lacti acid) occurred at a rate comparable to that at which i reacted with phosphatidylethanolamine. This side-reaction could probably be minimized b working with more dilute solutions of the reactants. D. Preparation of poly (glycolic acid) amide of DSPE A mixture of 266 mg. (3.50 mmoles) glycolic acid, 74 mg (3.60 mmoles) dicyclohexyl carbodiimide, 75 mg. (0.1 mmoles) distearoyl phosphatidyl ethanolamine, 32 micro liters (0.23 mmoles triethyl amine, and 5.0 ml dry dim ethyl sulfoxide was heated at 75° C, under a nitroge atmosphere, cooled to room temperature, then diluted wit an equal volume of chloroform, and then washed with thre successive equal volumes of water to remove dimethy sulfoxide. Centrifuge and separate phases with a Pasteu pipette each time. Filter the chloroform phase with suction to remove small amount of suspended material and vacuum evaporat the filtrate to dryness to obtain 572 mg. pale amber wax Re-dissolve this material in 2.5 ml chloroform an transfer to the top of a 21 mm X 270 mm column of silic gel (Merck Hieselgel 60) which has been wetted wit chloroform. Develop the chro atogram by passing through the column, in sequence, 100 ml each of: 100% chloroform, 0 % (1% NH 4 OH in methanol) ; 90% chloroform, 10% (1% NH 4 OH in methanol) ; 85% chloroform, 15% (1% NH 4 OH in methanol) ; 80% chloroform, 20% (1% NH 4 OH in methanol) ; 70% chloroform, 30% (1% NH 4 OH in methanol) . Collect individual 25 ml portions of effluent and assa each by TLC on Si) 2 -coated plates, using CH Cl 3 , CH 3 OH, H 2 0, con-NH 4 OH; 130, 70, 8, 0.5 v/v as developer. Almost all the P04 + material will be in the 275-300 ml portion of effluent. Evaporation of this to dryness under vacuum, followed by high-vacuum drying, affords 281 mg of colorless wax. Phosphate analysis suggests a molecular weight o 924,000. Manipulation of solvent volume during reaction and molar ratios of glycolic acid and dicyclohexyl carbodi¬ imide would probably result in other sized molecules. Example 3 Preparation of Ethylene-Linked PEG-PE A. Preparation of I-trimethylsilyloxy-polyethylene glycol is illustrated in the reaction scheme shovrn in Figure 3. 15.0 gm (10 mmoles) of polyethylene glycol) M.Wt. 1500, (Aldrich Chemical) was dissolved in 80 ml benzene. 1.40 ml (11 mmoles) of chlorotrimethyl silane (Aldrich Chemi cal Co.) and 1.53 ml (Immoles) of triethylamine was added. The mixture was stirred at room temperature unde an inert atmosphere for 5 hours. The mixture was filtered with suction to separa crystals of triethylammonium chloride and the crysta were washed with 5 ml benzene. Filtrate and benzene wa liquids were combined. This solution was evaporated dryness under vacuum to provide 15.83 grams of colorle oil which solidified on standing. TLC of the product on Si-C 18 reversed-phase plat using a mixture of 4 volumes of ethanol with 1 volume water as developer, and iodine vapor visualizatio revealed that all the polyglycol 1500 (R f =0.93) has be consumed, and was replaced by a material of R £ =0.82. infra-red spectrum revealed absorption peaks characteri tic only of polyglycols. Yield of I-trimethylsilyoxypolyethylene glycol, M. 1500 was nearly quantitative. B. Preparation of trifluoromethane sulfonyl ester ltrimethylsilyloxy-polyethylene glycol. 15.74 grams (10 mmol) of the crystalline I-trimethy silyloxy polyethylene glycol obtained above was dissolv in 40 ml anhydrous benzene and cooled in a bath crushed ice. 1.53 ml (11 mmol) triethylamine and 1.85 (11 mmol) of trifluoromethanesulfonic anhydride obtain from Aldrich Chemical Co. were added and the mixture w stirred over night under an inert atmosphere until t reaction mixture changed to a brown color. The solvent was then evaporated under reduced pressu and the residual syrupy paste was diluted to 100.0 with methylene chloride. Because of the great reactivi of trifluoromethane sulfonic esters, no further purific tion of the trifluoromethane sulfonyl ester of I-tr methylsilyloxy polyethylene glycol was done. C. Preparation of N-1-trimethylsilyloxy polyethyle glycol 1500 PE. 10 ml of the methylene chloride stock solution of th trifluoromethane sulfonyl ester of 1-trimethylsilylox polyethylene glycol was evaporated to dryness unde vacuum to obtain about 1.2 grams of residue (approxi mately 0.7 mmoles). To this residue, 3.72 ml of a chlo roform solution containing 372 mg (0.5 mmoles) egg PE wa added. To the resulting solution, 139 microliters (1. mmole) of triethylamine was added and the solvent wa evaporated under vacuum. To the obtained residue, 5 m dry dimethyl formamide and 70 microliters (0.50 mmoles) triethylamine (VI) was added. Air from the reactio vessel was displaced with nitrogen. The vessel wa closed and heated in a sand bath a 110°C for 22 hours. The solvent was evaporated under vacuum to obtain 1.5 grams of brownish colored oil. A 21 X 260 mm chromatographic absorption column fille with Kieselgel 60 silica 70-230 mesh, was prepared an rinsed with a solvent composed of 40 volumes of butanone, 25 volumes acetic acid and 5 volumes of water. The crud product was dissolved in 3 ml of the same solvent an transferred to the top of the chromatography column. Th chromatogram was developed with the same solvent an sequential 30 ml portions of effluent were assayed eac by TLC. The TLC assay system used silica gel coated glas plates, with solvent combination butanone/acetic acid/wa ter; 40/25/5; v/v/v. Iodine vapor absorption served fo visualization. In this solvent system, the N-i-tri methylsilyloxy polyethylene glycol 1500 PE appeared a R f =0.78. Unchanged PE appeared at R f =0.68. The desired N-1-trimethylsilyloxy polyethylene glyco 1500 PE was a chief constituent of the 170-300 ml por tions of column effluent. When evaporated to drynes under vacuum these portions afforded 111 mg of pal yellow oil of compound. D. Preparation of N-polyethylene glycyl 1500: phospha tidyl-ethanolamine acetic acid deprotection. Once-chromatographed, PE compound was dissolved in 2 m of tetrahydrofuran. To this, 6 ml acetic acid and 2 m water was added. The resulting solution was let to stan for 3 days at 23°C. The solvent from the reaction mix ture was evaporated under vacuum and dried to constan weight to obtain 75 mg of pale yellow wax. TLC on Si-C1 reversed-phase plates, developed with a mixture of volumes ethanol, 1 volume water, indicated that some fre PE and some polyglycol-like material formed during th hydrolysis. The residue was dissolved in 0.5 ml tetrahydrofuran an diluted with 3 ml of a solution of ethanol water; 80:20 v:v. The mixture was applied to the top of a 10 mm X 25 mm chromatographic absorption column packed with octade cyl bonded phase silica gel and column was developed wit ethanol water 80:20% by volume, collecting sequential 2 ml portions of effluent. The effluent was assayed b reversed phase TLC Fractions containing only product o Rf=0.08 to 0.15 were combined. This was typically th 20-100 ml portion of effluent. When evaporated to dry ness, under vacuum, these portions afforded 33 mg o colorless wax PEG-PE corresponding to a yield of only 3% based on the starting phosphatidyl ethanolamine. NMR analysis indicated that the product incorporate both PE residues and polyethylene glycol residues, bu that in spite of the favorable-appearing elemental analy sis, the chain length of the polyglycol chain has bee reduced to about three to four ethylene oxide residues SUBSTITUTE SHEET The product prepared was used for a preparation of PEG-P liposomes. E. Preparation of N-Polyethylene glycol 1500 P.E. b fluoride deprotection. 500 mg of crude N-1-trimethylsilyloxy polyethylen glydol PE was dissolved in 5 ml tetrahydrofuran and 18 mg (0.600 millimoles) of tetrabutyl ammonium fluoride wa added and agitated until dissolved. The reactants wer let to stand over night at room temperature (20°C) . The solvent was evaporated under reduced pressure an the residue was dissolved in 10 ml chloroform, washe with two successive 10 ml portions of water, and centri fuged to separate chloroform and water phases. Th chloroform phase was evaporated under vacuum to obtai 390 mg of orange-brown wax, which was determined to be impure N-polyethylene glycol 1500 PE compound. The wax was re-dissolved in 5 ml chloroform and trans¬ ferred to the top of a 21 X 270 mm column of silica gel moistened with chloroform. The column was developed by passing 100 ml of solvent through the column. The Table 2 solvents were used in sequence: Table 2 Volume % Volume % Methanol Containing Chloroform 2% Cone. Ammonium Hydroxide/methanol 100% 0% 95% 5% 90% 10% 85% 15% 80% 20% 70% 30% 60% 40% 50% 50% 0% 100% Separated 50 ml fractions of column effluent w saved. The fractions of the column were separated by on Si-C18 reversed-phase plates. TLC plates were de loped with 4 volumes of ethanol mixed with 1 volume water. Visualization was done by exposure to iodi vapor. j Only those fractions containing an iodine-absorbi lipid of R f about 0.20 were combined and evaporated dryness under vacuum and dried in high vacuum to const weight. In this way 94 mg of waxy crystalline solid obtained of M.W. 2226. The proton NMR spectrum of t material dissolved in deuterochlorofor showed the e pected peaks due to the phosphatidyl ethanolamine porti of the molecule, together with a few methylene proto attributable to polyethylene glycol. (Delta = 3.7) . Example 4 Preparation of REVs and MLVs A. Sized REVs A total of 15 μmoles of the selected lip components, in the mole ratios indicated in the exampl below, were dissolved in chloroform and dried as a t film by rotary evaporation. This lipid f lm was di solved in 1 ml of diethyl ether washed with distill water. To this lipid solution was added 0.34 ml of aqueous buffer solution containing 5 mM Tris, 100 NaCl, 0.1 mM EDTA, pH 7.4, and the mixture was emulsifi by sonication for 1 minute, maintaining the temperat of the solution at or below room temperature. Where t liposomes were prepared to contain encapsulated [ 1Z tyraminyl-inulin, such was included in the phosph buffer at a concentration of about 4 μCi/ml buffer. The ether solvent was removed under reduced pr sure at room temperature, and the resulting gel was ta up in 0.1 ml of the above buffer, and shaken vigorousl The resulting REV suspension had particle sizes, determined by microscopic examination, of between abo 0.1 to 20 microns, and was composed predominantly relatively large (greater than 1 micron) vesicles havi one or only a few bilayer lamellae. The liposomes were extruded twice through a pol carbonate filter (Szoka, 1978), having a selected po size of 0.4 microns or 0.2 microns. Liposomes extrude through the 0.4 micron filter averaged 0.17± (0.05 micron diameters, and through the 0.2 micron filter, 0.1 (0.05) micron diameters. Non-encapsulated [ 12S I] tyr aminyl-inulin was removed by passing the extruded lipo somes through Sephadex G-50 (Pharmacia) . B. Sized MLVs Multilamellar vesicle (MLV) liposomes were pre pared according to standard procedures by dissolving mixture of lipids in an organic solvent containing prima rily CHC1 3 and drying the lipids as a thin film by rota tion under reduced pressure. In some cases a radioactiv label for the lipid phase was added to the lipid solutio before drying. The lipid film was hydrated by additio of the desired aqueous phase and 3 mm glass beads fol lowed by agitation with a vortex and shaking above th phase transition temperature of the phospholipid com ponent for at least 1 hour. In some cases a radioactiv label for the aqueous phase was included in the buffer In some cases the hydrated lipid was repeatedly froze and thawed three times to provide for ease of the follow ing extrusion step. The size of the liposome samples was controlled b extrusion through defined pore polycarbonate filter using pressurized nitrogen gas. In one procedure, th liposomes were extruded one time through a filter w pores of 0.4 μm and then ten times through a filter w pores of 0.1 μm. In another procedure, the liposo were extruded three times through a filter with 0.2 pores followed by repeated extrusion with 0.05 μm po until the mean diameter of the particles was below 100 as determined by DLS. Unencapsulated aqueous compone were removed by passing the extruded sample through a permeation column separating the liposomes in the v volume from the small molecules in the included volume. C Loading 67 Ga Into DF-Containing Liposomes The protocol for preparation of Ga 67 -DF labe liposomes as adapted from known procedures (Gabiz 1989). Briefly, liposomes were prepared with the chelator desferal mesylate encapsulated in the inter aqueous phase to bind irreversibly Ga transported thro the bilayer by hydroxyquinoline (oxine) . D. Dynamic Light Scattering Liposome particle size distribution measureme were obtained by DLS using a NICOMP Model 200 *-*ith Brookhaven Instruments BI-2030AT autocorrelator attach The instruments were operated according to the manuf turer's instructions. The NICOMP results were expres as the mean diameter and standard deviation of a Gauss distribution of vesicles by relative volume. Example 5 Liposome Blood Lifetime Measurements A. Measuring Blood Circulation Time and Bloo RES Ratios In vivo studies of liposomes were performed two different animal models: Swiss-Webster mice at 25 each and laboratory rats .at 200-300 g each. The studi in mice involved tail vein injection of liposome sampl at 1 μM phospholipid/mouse followed by animal sacrifi after a defined time and tissue removal for label qua titation by gamma counting. The weight and percent the injected dose in each tissue were determined. T studies in rats involved establishment of a chron catheter in a femoral vein for removal of blood sampl at defined times after injection of liposome samples in catheter in the other femoral artery at 3-4 μM phosph lipid/rat. The percent of the injected dose remaining the blood at several time points up to 24 hours w determined. B. Time Course of Liposome Retention in t Bloodstream PEG-PE composed of methoxy PEG, molecular weig 1900 and l-palmitoyl-2-oleyl-PE (POPE) was prepared as Example 2. The PEG-POPE lipid was combined with a partially hydrogenated egg PC (PHEPC) in a lipid:lip mole ratio of about 0.1:2, and the lipid mixture w hydrated and extruded through a 0.1 micron polycarbona membrane, as described in Example 4, to produce MLV with average size about 0.1 micron. The MLV lipi included a small amount of radiolabeled lipid marker 14 cholesteryl oleate, and the encapsulated marker 3 H-i ulin. The liposome composition was injected and the perce initial injected dose in mice was determined as describ in Example 4, at 1, 2, 3, 4, and 24 after injection. T time course of loss of radiolabeled material is seen Figure 7 which is a plot of percent injected dose f encapsulated inulin (solid circles) , inulin marker cor rected to the initial injection point of 100% (ope circles) , and lipid marker (closed triangles) , over a 24 hour period post injection. As seen, both lipid an encapsulated markers showed greater than 10% of origina injected dose after 24 hours. C 24 Hour Blood Liposome Levels Studies to determine percent injected dose in th blood, and blood/RES ratios of a liposomal marker, 2 hours after intravenous liposome injection, were carrie out as described above. Liposome formulations having th compositions shown at the left in Table 3 below wer prepared as described above. Unless otherwise noted, th lipid-derivatized PEG was PEG-1900, and the liposome siz was 0.1 micron. The percent dose remaining in the bloo 24 hours after intravenous administration, and 24-hou blood/RES ratios which were measured are shown in th center and right columns in the table, respectively. Table 3 Lipid Composition* 24 Hours After IV Dose % Injected Dose in Blood B/RE PG:PC:Chol (.75:9.25:5) 0.2 0.01 PC:Choi (10:5) 0.8 0.03 PEG-DSPE:PC:Chol 23.0 3.0 PEG-DSPE:PC:Chol (250 nm) 9.0 0.5 PEG S00() -DSPE:PC:Choi 21.0 2.2 PEGu o -DSPE : PC : Choi 5.0 2.0 PEG-DSPE :PC (0 . 75 : 9 .25) 22.0 0.2 PEG-DSPE :PG :PC : Chol 40.0 4.0 (0 . 75 :2 .25 : 7 : 5) PEG-DSPE :NaCholSO, :PC : Chol 25.0 2.5 (0 .75 : 0 .75 : 9.25 : 4.25) *A11 formulations contain 33% cholesterol and 7.5% charged componen and were 100 nm mean diameter except as noted. PEG-DSPE consiste of PEG uc except as noted. As seen, percent dose remaining in the blood 24 hour after injection ranged between 5-40% for liposomes con taining PEG-derivatized lipids. By contrast, in bot liposome formulations lacking PEG-derivatized lipids less than 1% of liposome marker remained after 24 hours Also as seen in Table 3, blood/RES ratios increased fro 0.01-0.03 in control liposomes to at least 0.2, and a high as 4.0 in liposomes containing PEG-derivatized lipo somes. C. Blood lifetime measurements with polylactic aci derivatized PE. Studies to determine percent injected dose in the bloo at several times after intravenous liposome injectio were carried out as described above. Typical result with extruded MLV liposome formulation having the com position Polylactic Acid-PE:HSPC:Chol at either 2:3.5: or 1:3.5:1 weight% is shown in Fig 10 (solid squares) The percent dose remaining normalized at 15 min. is show over 24 hours. These data indicate that the clearance of the polylac tic acid-coated liposomes is severalfold greater tha similar formulations without polylactic acid deriv-sti.ze PE. D. Blood lifetime measurements with polyglycolic aci Derivatized PE. Studies to determine percent injected dose in the bloo at several times after intravenous liposome injectio were carried out as described above. Typical result with extruded MLV liposome formulation having the com position Polyglycolic Acid-PE:HSPC:Chol at 2:3.5: weight% are shown in Fig 10 (open triangles) . The per cent dose remaining normalized at 15 min. is shown ove SUBSTITUTESHEET 24 hours. These data indicate that the clearance of the polygl colic acid-coated liposomes is severalfold greater th similar formulations without polyglycolic acid deriv tized PE. Example 6 Effect of Phospholipid Acyl-Chain Saturation on Blood/RES Ratios in PEG-PE Liposomes PEG-PE composed of methoxy PEG, molecular weight 19 and distearylPE (DSPE) was prepared as in Example 2. T PEG-PE lipids were formulated with selected lipids fr among sphingomyelin (SM) , fully hydrogenated soy PC (PC cholesterol (Choi) , partially hydrogenated soy (PHSPC) , and partially hydrogenated PC lipids identifi as PC IV1, IV10, IV20, IV30, and IV40 in Table 4. T lipid components were mixed in the molar ratios shown the left in Table 5, and used to form MLVs sized to 0 micron as described in Example 4. Table 4 24 hours after injection, the percent material injected (as measured by percent of 14 C-cholesteryl ole- ate) remaining the blood and in the liver (L) and spleen (S) were determined, and these values are shown in the two data columns at the left in Table 4. The blood and L+S (RES) values were used to calculate a blood/RES τ-slue for each composition. The column at the right in Table 4 shows total amount of radioactivity recovered. The two low total recovery values in the table indicate anomalous clearance behavior. The results from the table demonstrate that t blood/RES ratios are largely independent of the fluidit or degree of saturation of the phospholipid componen forming the liposomes. In particular, there was systematic change in blood/RES ratio observed among lip somes containing largely saturated PC components (e.g IV1 and IV10 PCs), largely unsaturated PC componen (IV40), and intermediate-saturation components (e.g IV20) . In addition, a comparison of blood/RES ratios o tained using the relatively saturated PEG-DSPE compou and the relatively unsaturated PEG-POPE compound (Examp 5) indicates that the degree of saturation of the deriv tized lipid is itself not critical to the ability of t liposomes to evade uptake by the RES. Example 7 Effect of Cholesterol and Ethoxylated Cholesterol on Blood/RES Ratios in PEG-PE Liposomes A. Effect of added cholesterol PEG-PE composed of methoxy PEG, molecular weig 1900 and was derivatized DSPE as described in Example The PEG-PE lipids were formulated with selected lipi from among sphingomyelin (SM) , fully hydrogenated soy (PC), and cholesterol (Choi), as indicated in the colu at the left in Table 5 below. The three formulatio shown in the table contain about 30, 15, and 0 mo percent cholesterol. Both REVs (0.3 micron size; a MLVs (0.1 micron size) were prepared, substantially in Example 4, with encapsulated tritium-labeled inulin. The percent encapsulated inulin remaining in t blood 2 and 24 hours after administration, given at t right in Table 6 below, show no measurable effect cholesterol, in the range 0-30 mole percent. 1) 2) 3) B. Effect of ethoxylated cholesterol Methoxy-ethyoxy-cholesterol was prepared by coupling methoxy ethanol to cholesterol via the trifluorosulfonate coupling method described in Section I. PEG-PE composed of methoxy PEG, molecular weight 1900 and was derivatized DSPE as described in Example 6. The PEG-PE lipids were formulated with selected lipids from among distearylPC (DSPC) , partially hydrogenated soy ,PC (PHSPC) , choleste¬ rol, and ethoxylated cholesterol, as indicated at the right in Table 7. The data show that (a) ethoxylated cholesterol, in combination with PEG-PE, gives about the same degree of enhancement of liposome lifetime in th blood as PEG-PE alone. By itself, the ethoxylated chol esterol provides a moderate degree of enhancement o liposome lifetime, but substantially less than tha provided by PEG-PE. 1.85: 1: 0.15 Example 8 Effect of Charged Lipid Components on Blood/RES Ratios in PEG-PE Liposomes PEG-PE composed of methoxy PEG, molecular v:<e.igh 1900 and was derivatized DSPE as described in Example 6 The PEG-PE lipids were formulated with lipids selecte from among egg PG (PG) , partially hydrogenated egg P (PHEPC) , and cholesterol (Choi) , as indicated in th Figure 7. The two formulations shown in the figur contained about 4.7 mole percent (triangles) or 14 mol percent (circles) PG. The lipids were prepared as MLVs sized to 0.1 micron as in Example 4. The percent of injected liposome dose present 0.25 1, 2, 4, and 24 hours after injection are plotted fo both formulations in Figure 7. As seen, the percent P SUBSTITUTESHEET in the composition had little or no effect on liposom retention in the bloodstream. The rate of loss of encap sulated marker seen is also similar to that observed fo similarly prepared liposomes containing no PG. Example 9 Plasma Kinetics of PEG-Coated and Uncoated Liposomes PEG-PE composed of methoxy PEG, molecular weight 1900 and distearylPE (DSPE) was prepared as in Example 2. The PEG-PE lipids were formulated with PHEPC, and choles¬ terol, in a mole ratio of 0.15:1.85:1. A second lipid mixture contained the same lipids, but without PEG-PE. Liposomes were prepared from the two lipid mixtures as described in Example 5, by lipid hydration in the pre- sence of desferal mesylate, followed by sizing to 0.1 micron, and removal of non-entrapped desferal by gel filtration with subsequent loading of 67 Ga-oxine into the liposomes. The unencapsulated 67 Ga was removed during passage through a Sephadex G-50 gel exclusion cloumn. Both compositions contained 10 μmoles/ml in 0.15 M NaCl, 0.5 mM desferal. The two liposome compositions (0.4 ml) were injected IV in animals, as described in Example 6. At time 0.25, 1, 3 or 5 and 24 hours after injection, blood samples. were removed and assayed for amount inulin remaining in the blood, expressed as a percentage of the amount mea¬ sured immediately after injection. The results are shown in Figure 9. As seen, the PEG-coated liposomes have a blood halflife of about 11 hours, and nearly 30% o the injected material is present in the blood after 24 hours. By contrast, uncoated liposomes showed a halflife in the blood of less than 1 hour. At 24 hours, the amount of injected material was undetectable. Example 10 Preparation of Doxorubicin Liposomes Vesicle-forming lipids containing PEG-PE, PG, PHEP and cholesterol, in a mole ratio of 0.3: 0.3: 1.4: 1 we dissolved in chloroform to a final lipid concentration 25 μmol phospholipid/ml. Alpha-tocopherol (α-TC) in fr base form was added in chloroform.-methanol (2:1) soluti to a final mole ratio of 0.5%. The lipid solution w dried to a thin lipid film, then hydrated with a wa (60°C) solution of 125 mM ammonium sulfate containing mM desferal. Hydration was carried out with 1 ml aqueous solution per 50μmole phospholipid. The lip material was hydrated with 10 freeze/thaw cycles, usi liquid nitrogen and a warm water bath. Liposome sizing was performed by extrusion throu two Nuclepore polycarbonate membranes, 3 cycles throu 0.2 microns filters, and ten cycles through 0.05 micr filters. The final liposome size was 100 nm. The siz liposomes were then dialyzed against 50-100 volumes of glucose three times during a 24 hour period. A four cycle was carried out against 5% glucose titered to 6.5-7.0 for 1 hour. A solution of doxorubicin, 10 mg/ml in 0.9% NaCl a 1 mM desferal, was prepared and mixed with an equ volume of the dialyzed liposome preparation. The co centration of drug in the mixture was about 5 mg/ml dr 50 μmoles/ml phospholipid. The mixture was incubated f 1 hours at 60°C in a water bath with shaking. Untrapp drug was removed by passage through a Dowex 50 WX .es packed in a small column. The column was centrifuged a bench top centrifuge for 5 minutes to completely elu the liposome suspension. Sterilization of the mixtu was by passage through a 0.45 micron membrane, and t liposomes were stored at 5°C Example 11 Plasma Kinetics of Free and Liposomal Doxorubicin PEG-PE composed of methoxy PEG, molecular weigh 1900 and distearylPE (DSPE) was prepared as in Example 2. The PEG-PE lipids were formulated with hydrogenated so bean PC (HSPC) and cholesterol, in a mole ratio o 0.15:1.85:1 (PEG-Dox) . A second lipid mixture containe hydrogenated phosphatidylinositol (HPI), HSPC choleste rol, in a mole ratio of 1:10:5 (HPI-Dox) . Each lipi formulation was used in preparing sized MLVs containin an ammonium ion gradient, as in Example 10. The liposomes were loaded with doxorubicin, by mixing with an equal volume of a doxorubicin solution, 10 mg/ml plus 1 mM desferal, as in Example 15. The two compositions are indicated in Figure 11 and Table 7 belo as PEG-DOX and HPI-DOX liposomes, respectively. A doxo¬ rubicin HC1 solution (the marketed product. Free Dox) was obtained from the hospital pharmacy. Free DOX, PEG-Dox and HPI-Dox were diluted to the same concentration (1.8 mg/ml) using unbuffered 5% glucose on the day of injec¬ tion. Dogs were randomized into three groups (2 females, 1 male) and weighed. An 18 gauge Venflon IV catheter was inserted in a superficial limb vein in each animal. The drug and liposome suspensions were injected by quick bolus (15 seconds) . Four ml bllod samples were before injection and at 5, 10, 15, 30, 45 min, 1, 2, 4, 6, 8, 10, 12, 24, 48 and 72 hours post injection. In the lipo¬ some groups blood was also drawn after 96, 120, 144,. and 168 hours. Plasma was separated from the formed elements of the whole blood by centrifugation and doxorubicin concentrations assayed by standard fluorescence tech¬ niques. The amount of doxorubicin remaining in the bloo was expressed as a percentage of peak concentration o labeled drug, measured immediately after injection. Th results are plotted in Figure 11, which shows that bot the PEG-DOX and HPI-DOX compositions give linear loga rithmic plots (single-mode exponential) , and free dru give a bimodel exponential curve, as indicated in Table below. The halflives of the two liposome formulation determined from these curves are indicated in Table 8. Also shown in Table 8 is the area under the curv (AUC) determined by integrating the plasma kinetic curv over the 72 hour test period. The AUC results indicat that the total availability of drug from PEG-DOX lipo somes, for the 72 hour period following injection, wa nearly twice that of HPI-DOX liposomes. This is consis tent with the approximately twofold greater halflife o the PEG-DOX liposomes. The "CL" entry in Table 9 indi cates ... Table 8 Free DOX HPI-DOX PEG-DOX Kinetic Pattern Bi-exp. Mono-exp. Mono-ex Peak Cone. (mg/1) 0.4-2.2 4.3-6.0 4.5-5. AUC (mg/1) 7.1-10.0 73.9-97.5 132.9-329. tl/2 hr 1.9-3.3 11.1-12.0 19.6-45. CL (mg/hr) 0.6-0.9 1.1-1.6 1.3-2. Example 12 Tissue Distribution of Doxorubicin A. Subcutaneous Tumor PEG-liposomes loaded with doxorubicin were prepare as in Example 11 (PEG-DOX liposomes) . Free drug used wa clinical material obtained from the hospital pharmacy. Two groups of twelve mice were injected subcutane ously with 10 6 J-6456 tumor cells. After 14 days th tumors had grown to about 1 cm 3 in size in the subcu¬ taneous space and the animals were injected IV (tail vein) with 10 mg/kg doxorubicin as free drug (group 1) or encapsulated in PEG liposomes (group 2) . At 4, 24, and 48 hours after drug injection, four animal in each group were sacrificed, and sections of tumor, heart, and muscle tissue were excised. Each tissue was weighed, then homo¬ genized and extracted for determination of doxorubicin concentration using a standard florescence assay proce- dure (Gabizon, 1989) . The total drug measured in each homogenate was expressed as μg drug per gram tissue. The data for drug distribution in heart, muscle, and liver are plotted in Figures 12A and 12B for free and liposome-associated doxorubicin, respectively. In Figure 12A it is seen that all three tissue types take up about the same amount of drug/g tissue, although initially the drug is taken up preferentially in the heart. By con¬ trast, when entrapped in PEG-liposomes, the drug shows a strong selective localization in the tumor, with reduced levels in heart and muscle tissue. B. Ascites Tumor Two groups of 15 mice were injected interperitoneal- ly with 10 β J-6456 lymphoma cells. The tumor was allowed to grow for one-two weeks at which time 5 ml of ascites fluid had accumulated. The mice were then injected IV with 10 mg/kg doxorubicin either in free drug form (group 1) or entrapped in PEG liposomes as described in Example 11 (group 2) . Ascites fluid was withdrawn from three animals in each group at 1, 4, 15, 24 and 48 hours post treatment. The ascites tumor was further fractionated into cellular and fluid components by centrifugation (15 min. 5000 rpm) . Free and liposome-bound drug in the supernatant was determined by passing the fluid through a Dowex WX resin, as above, to remove free drug. The doxorubicin concentrations in the ascites fluid, tumo cells, supernatant, and resin-treated supernatant wer then determined, and from these values, μg doxorubicin/ gram tissue was calculated. The values for total ascite fluid supernatant (solid diamonds) , supernatant afte removal of free drug (solid triangles) , and isolate tumor cells (solid circles) are plotted in Figure 13. A seen, the total doxorubicin in the ascites fluid in creased steadily up to about 24 hours, then droppe slightly over the next 24 hours. Most of the doxorubici in the tumor is in liposome-entrapped form, demonstratin that liposomes are able to extravasate into solid tumor in intact form. In a similar experiment two groups of twelve mic were implanted IP with the J-6456 lymphoma and the tumo was allowed to establish as described above. Once th ascites tumor had reached about 5 ml, one group of ani mals was injected with 10 mg/kg free doxorubicin and th other group with 10 mg/kg doxorubicin entrapped in PE liposomes. At 4, 24 and 48 hours post treatment ascite fluid and blood samples were withdrawn from four animal in each group and the animals were sacrificed. Section of liver and heart tissue were excised from each animal homogenized and drug concentration assayed as describe above. Plasma was separated from whole blood by centri fugation and drug concentration assayed as stated above Doxorubicin concentration in the ascites fluid was als measured. The results are presented in Table 9. Plasm and ascites fluid levels are expressed as μg doxorubici per ml and liver and heart tissue values as μg doxoru bicin per gram tissue. The standard deviations for eac measurement is shown in parentheses. As shown, there i considerably more doxorubicin in plasma for the grou receiving the drug in PEG liposome entrapped form at al time points. Ascites tumor levels are also higher in the liposome group, particularly at the longer time points (24 and 48 hours) . These data confirm the selective delivery of the drug to the tumor by the PEG liposomes. Example 13 Tumor Uptake of PEG Liposomes Compared with Conventional Liposomes Two groups of 6 mice were injected subcutaneously with 10 s -10 s C-26 colon carcinoma cells and the tumor was allowed to grow in the subcutaneous space until it reached a size of about 1 cm (about two weeks following injection) Each group of animals was then injected with 0.5 mg of eithe conventional liposomes (100 nm DSPC/Chol, 1:1) or PEG lipo somes (100 nm DSPC/Chol/PEG-DSPE, 10:3:1) which had bee loaded with radioactive gallium as described in Example Three mice from each group were sacrificed at 2, 24 and 4 hours post treatment, the tumors excised and weighed and th amount of radioactivity quantified using a gamma counter The results are presented in the following table and ar expressed as the percent of the injected dose per gra tissue. *Expressed as amount of PEG Liposomes divided by amount of con ventional liposomes localized in the tumor Example 14 Liposome Extravasation into Intact Tumors: Direct Microscopic Visualization PEG-PE composed of methoxy PEG, molecular weigh 1900 and distearylPE (DSPE) was prepared as in Example 2 The PEG-PE lipids were formulated with HSPC, and choles terol, in a mole ratio of 0.15:1.85:1. PEG-liposome were prepared to contain colloidal gold particles (Hong) The resulting MLVs were sized by extrusion, as above, t an average 0.1 micron size. Non-entrapped material wa removed by gel filtration. The final concentration o liposomes in the suspension was about 10 μmol/ml. SUBSTITUTESHEET In a first study, a normal mouse was injected IV with 0.4 ml of the above liposome formulation. Twenty four hours after injection, the animal was sacrificed, and sections of the liver removed fixed in a standard water-soluble plastic resin. Thick sections were cut with a microtome and the sections stained with a solution of silver nitrate according to instructions provided with the "Intense 2" System kit supplied by Jannsen Life Sciences, Inc. (Kingsbridge, Piscataway, N.J.). The sections were further stained with eosin and hemotoxylin. Figure 14A is a photomicrograph of a typically liver section, showing smaller, irregularly shaped Kupfer cells, such as cells 20, among larger, more regular shaped hepatocytes, such as hepatocyes 22. The Kupfer cells show large concentrations of intact liposomes, seen as small, darkly stained bodies, such at 24 in Figure 14A. The hepatocytes are largely free of liposomes, as would be expected. In a second study, a C-26 colon carcinoma (about 10 6 cβU) was implanted in a mouse liver. Fourteen days post implantation, the animal was injected IV with 0.5 mg of the above liposomes. Twenty four hours later, the animal was sacrificed, and the liver was perfuseα, embeded, sectioned, and stained as above. The sections were examined for a capillary-fed tumor region. One exemplary region is seen in Figure 14B, which shows a capillary 26 feeding a region of carcinoma cells, such as cells 28. These cells have characteristic staining patterns, and often include darkly stained nuclii in various stages of mitosis. The capillary in the figure is lined by an endothelial barrier 30, and just below that, a basement membrane 32. It can be seen in Figure 14B that liposomes, such as liposomes 34, are heavily concentrated in the tumor re¬ gion, adjacent the capillary on the tumor side of the endothelial barrier and basement membrane, and many lipo- somes are also dispersed throughout the intercellular fluid surrounding the tumor cells. Figure 14C shows another region of the liver tumor from the above animal. Liposomes are seen throughout the intercellular fluid bathing the carcinoma cells. In a third study, C26 colon carcinoma cells were injected subcutaneously into an animal, and allowed to grow in the animal for 28 days. Thereafter, the animal was injected IV with 0.5 mg of the above liposomes. Twenty four hours later, the animal was sacrificed, and the tumor mass was excised. After embeding, tumor mass was sectioned on a microtome and stained as above. Figure 14D shows a region of the tumor cells, including a cell 36 in the center of the figure which is in late stage mitosis. Small, darkly stained liposomes are seen throughout the intercellular fluid. Example 15 Tumor Treatment Method Vesicle-forming lipids containing PEG-PE, PG, PHEPC, and cholesterol and α-TC in a mole ratio of 0.3: 0.3: 1.4: 1: 0.2 were dissolved in chloroform to a final lipid concentration of 25 μ ol phospholipid/ml. The lipid mix¬ ture was dried into a thin film under reduced pressure. The film was hydrated with a solution of .125M ammcnium sulfate to form MLVs. The MLV suspension was frozen in a dry ice acetone bath and thawed three times and sized to 80-100 nm. An ammonium ion gradient was created substan¬ tially as described in Example 10. The liposomes were loaded with epirubicin, and free (unbound drug) removed also as described in Example 10 for doxorubicin. The final concentration of entrapped drug was about 50-100 μg drug/μmol lipid. Epirubicin HC1 and doxorubicin HCL, the commercial products, were obtained from the hospital pharmacy. About 10 6 cells C-26 colon carcinoma cells were injected subcutaneously into three groups of 35 mice. The groups were subdivided into 5 7-animal subgroups. For the tumor suppression experiment shown in Figure 15A each subgroup was injected IV with 0.5 ml of either saline vehicle control (open circles) , 6 mg/kg epirubicin (open triangles) , 6 mg/kg doxorubicin (filled circles) , or the drug-loaded liposomes (PEG-DOX liposomes) at two doses, 6mg/kg (filled triangles) and 12 mg/kg (open squares) on days 1, 8 and 15 following tumor cell implan¬ tation. Each group was followed for 28 days. Tumor size was measured for each animal on days 5,7,12,14,17,21,24 and 28. The growth of the tumor in each subgroup (ex¬ pressed as the mean tumor size of the individual animals) at each time point is plotted in Figure 15A. With reference to this figure, neither free doxoru¬ bicin nor free epirubicin at .6 mg/kg significantly sup¬ pressed tumor growth compared with the saline control. In contrast, PEG liposome entrapped epirubicin both dose.'; significantly suppresses tumor growth. With respect to survival of the animals at 120 days following tumor implantation, none of the animals in the saline, epiru¬ bicin or doxorubicin groups survived whereas 5 out of the seven and seven out of seven survived in the 6 mg/kg liposome epirubicin and 12 mg/kg liposome epirubicin groups, respectively. The results of delayed treatment experiments using the same tumor model are presented in Figure 15B and 15C The same number of animals were inoculated with the same number of tumor cells as described above. The treatment groups in Figures 15B and 15C consisted of saline (solid line), 6 mg/kg epirubicin (filled triangles), 6 mg/kg free epirubicin plus empty PEG liposomes (open circles) and two doses of epirubicin entrapped in PEG liposomes, 6 mg/kg (filled triangles) and 9 mg/kg (open squares) . In contrast to the results presented in Figure 15A, only two treatments were given in these experiments: days 3 and 10 for the results plotted in Figure 15B; and days 10 and 17 for the results plotted in Figure 15C Importantly, in the case of the PEG liposome entrapped drug, both delayed treatment schedules at both dose levels result in tumor regression whereas the free drug and free drug plus empty liposome treatment groups show only a modest retar- dation in the rate of tumor growth. Example 16 Tumor Treatment Method PEG-DOX liposomes were prepared as in Example 15 except that doxorubicin was loaded in the liposomes to a final level of 60-80 μg/μmoles total lipid. A doxorubi¬ cin HC1 solution to be used as the free drug control was obtained from a hospital pharmacy. A total of 30 mice were injected IP with 10 6 J-6456 lymphoma cells. The animals were divided into three 10-animal groups, each of which was injected IV with 0.4 ml of either saline vehi¬ cle, 10 mg/kg doxorubicin solution or the doxorubicin- loaded liposomes at 10 mg/kg. Each group was followed for 100 days for number of surviving animals. The per- cent survivors for each treatment group is plotted in Figure 16. As can be seen, free drug (filled circles) provided little improvement in survival over the saline group (filled squares) . In the animals treated with dόxorubi- cin loaded PEG-liposomes (filled triangles) , however, about 50% of the animals survived over 40 days, 20% over 70 days, and 10% survived until the experiment was ter¬ minated at 100 days. Example 17 Reduced Toxicity of PEG-Liposomes Solutions of free doxorubicin HC1, epirubicin HC1 were obtained as above. PEG-liposome formulations con- taining either doxorubicin or epirubicin, at a drug concentration of 70-90 μg compound/μmole liposome lipid, were prepared as described in Example 16. Conventional liposomes (no PEG-derivatized lipid) were loaded with doxorubicin to a drug concentration of 40 μg/μmole lipid using standard techniques. Each of the five formulations was administered to 35 mice, at a dose between 10 and 40 mg drug/kg body weight, in 5 mg/kG increments, with five receiving each dosage. The maximum tolerated dose given in Table 11 below is highest dose which did not cause death or dramatic weight loss in the injected animals within 14 days. As seen from the data, both DOX-liposomes and PEG-DOX liposomes more than doubled the tolerated dose of doxorubicin over the drug in free form, with the PEG-DOX liposomes giving a slightly higher tolerated dose. A similar result was obtained for doses of tolerated epirubicin in free and PEG-liposomal form. Table 11 Maximum Tolerated Dose of DXN (mg/Kg in mice) Example 18 Tumor Treatment Method Conventional doxorubicin liposomes (L-DOX) were pre pared according to published methods. Briefly, a mixtur of eggPG, Egg,PC, cholesterol and a-TC in a mole ratio o 0.3: 1.4: 1: 0.2 was made in chloroform. The solvent wa removed under reduced presssure and the dry lipid fil hydrated with a solution of 155 mM NaCl containing 2-5 m doxorubicin HC1. The resulting MLV preparation was down sized by extrusion through a series of polycarbonat membranes to a final size of about 250 nm. The fre (unentrapped) drug was removed by passing the suspensio over a bed of Dowex resin. The final doxorubicin con centration was about 40 per μmole lipid. Three groups of 7 mice were inoculated subcutaneous ly with 10 5 - 10 6 C-26 colon carcinoma cells as detaile in Example 15. The animals were divided into three, 7 animal treatment groups, one of which receivd 0.5 ml o saline vehicle as a control. The other two groups wer treated with doxorubicin either as a free drug solutio or in the form of L-DOX liposomes at a dose of 10 mg/kg. The treatments were given on days 8, 15 and 22 afte tumor cell inoculation. Tumor size was measured on th days treatments were given and day 28. As shown i Figure 17, the free drug (filled circles) suppresse tumor growth to a modest extent compared with the salin control (solid line) . The tumor in the L-Dox-treated group (filled triangles) grew slightly faster than the free-drug-treated group and slightly more slowly than in the untreated group. These results indicate that the anti-tumor activity of the L-DOX preparation is about the same, and certainly no better than the same dose of free drug. This stands in marked contrast to the results presented in Example 15 (and Figures 15A-C) which show that at comparable doses epirubicin entrapped in PEG- liposomes has dramatically better anti-tumor activity than free drug in this same tumor model. Although the invention has been described and illu¬ strated with respect to particular derivatized lipid com- pounds, liposome compositions, and use, it will be ap¬ parent that a variety of modifications and changes may be made without departing from the invention.

Claims: IT IS CLAIMED: 1. A liposome composition for use in localizing compound in a solid tumor via the bloodstream comprising, liposomes (i) composed of vesicle-forming lipids an between 1-20 mole percent of an amphipathic vesicle- forming lipid derivatized with a hydrophilic polymer, an (ii) having a selected mean particle diameter in the size range between about 0.07-0.12 microns, and the compound in liposome-entrapped form. 2. The composition of claim 1, wherein the hydro¬ philic polymer is polyethyleneglycol having a molecular weight between about 1,000-5,000 daltons. 3. The composition of claim 2, wherein the hydro¬ philic polymer is selected from the group of poly lactic acic and poly glycolic acid. 4. The composition of claim 1, wherein the compoun is an anti-tumor agent, and at least about 80% of the compound is in liposome-entrapped form. 5. The composition of claim 4, wherein the anti- tumor agent is an anthracycline antibiotic, and the con centration of compound which is entrapped in the lipo¬ somes is greater than 50 μg compound/μmole liposome li pid. 6. The composition of claim 4, wherein the anthra cycline is selected from the group consisting of doxoru bicin, epirubicin, and daunorubicin, including pharmaco logically acceptable salts and acids thereof. 7. A liposome composition for use in localizing a anthracycline anti-tumor drug in a solid tumor via th bloodstream comprising, liposomes (i) composed of vesicle-forming lipids an between 1-20 mole percent of an amphipathic vesicle forming lipid derivatized with polyethyleneglycol, an (ii) having an average size in a selected size rang between about 0.07-0.12 microns, and the drug, at least about 80% in liposome-entrappe form, and having a concentration in the liposomes is greater than 50 μg agent/μmole liposome lipid. 8. The composition of claim 7, wherein the drug is selected from the group consisting of doxorubicin, epiru- bicin, and daunorubicin, including pharmacologically acceptable salts and acids thereof. 9. For use in localizing a compound in a solid tumor by IV administration of the agent, a liposome composition characterized by: (a) liposomes composed of vesicle-forming lipids and between 1-20 mole percent of an amphipathic 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 intravenous 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. 10. The composition of claim 9, wherein the hydr philic polymer is polyethyleneglycol having a molecul weight between about 1,000-5,000 daltons. 11. The composition of claim 9, for use in treati such tumor, wherein the compound is an anthracycli antibiotic, and the concentration of compound entrapp in the liposomes is greater than about50 μg compound μmole liposome lipid. 12. The composition of claim 11, wherein the a thracycline is selected from the group consisting doxorubicin, epirubicin, and daunorubicin, includi pharmacologically acceptable salts and acids thereof. 13. For use in treating a solid tumor by intrave ous administration of an anthracycline antibiotic drug, liposome composition characterized by: (a) liposomes composed of vesicle-forming lipids a between 1-20 mole percent of an amphipathic vesicl forming lipid derivatized with a polyethyleneglycol, (b) a blood lifetime, as measured by the percent a liposomal marker present in the blood 24 hours after administration which is several times greater than th of liposomes in the absence of the derivatized lipids; (c) an average liposome size in a selected si range between about 0.07-0.12 microns, (d) at least about 80% of the drug in liposom entrapped form, and (c) a concentration of drug in the liposomes of least about 50 μg drug/μmole lipid. 14. A method of preparing an agent for localizatio in a solid tumor, when the agent is administered by I injection, comprising entrapping the agent in liposomes which are charac- terized by: (a) a composition which includes between 1-20 mole percent of an amphipathic vesicle-forming lipid deriva¬ tized with a hydrophilic polymer, and (b) an average liposome size in a selected size range between about 0.07-0.12 microns. 15. The method of claim 14, wherein the agent is an anthracycline antibiotic drug, and said entrapping in¬ cludes loading the agent into preformed liposomes by remote loading across an ion or pH gradient, to a final concentration of liposome-entrapped material of greater than about 50 μg agent/umole liposome lipid. 16. The method of claim 15, wherein the drug is selected from the group consisting of doxorubicin, epiru¬ bicin, and daunorubicin, including pharmacologically acceptable salts and acids thereof. 17. A method of localizing a compound in a solid tumor in a subject comprising, preparing a composition of liposomes (i) composed of vesicle-forming lipids and between 1-20 mole percent of an amphipathic 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 r and (iii) containing the compound in liposome-entrapped form, and injecting the composition intravenously in th subject in an amount effective to localize a therapeuti cally effective quantity of the agent in the solid tumor. 18. The method of claim 17, wherein the hydrophili polymer is polyethyleneglycol having a molecular weigh between about 1,000-5,000 daltons. 19. A method of treating a subject having a soli tumor, comprising preparing a composition of liposomes (i) composed o vesicle-forming lipids and between 1-20 mole percent o an amphipathic vesicle-forming lipid derivatized with hydrophilic polymer, (ii) having an average size in selected size range between about 0.07-0.12 microns, an (iii) containing an anthracycline antibiotic drug en trapped in the liposomes, at a concentration of entrappe agent of greater than about 50 μg agent/μmole liposom lipid, with at least about 80% of the agent entrapped i the liposomes, and injecting the composition intravenously in th subject in an amount effective to localize a therapeuti cally effective quantity of the agent in the solid tumor. 20. The method of claim 19, wherein the hydrophili polymer is polyethyleneglycol having a molecular weigh between about 1,000-5,000 daltons, and the agent i selected from the group consisting of doxorubicin, epiru bicin, and daunorubicin, including pharmacologicall acceptable salts and acids thereof.

Inventors: MARTIN FRANCIS J, REDEMANN CARL, WOODLE MARTIN C, YAU-YOUNG ANNIE, MARTIN, FRANCIS, J., REDEMANN, CARL, WOODLE, MARTIN, C., YAU-YOUNG, ANNIE
Applicants: LIPOSOME TECHNOLOGY INC, LIPOSOME TECHNOLOGY, INC.
Application year: 1990
Main classification: A61K9
IPC codes: A61K9, A61K31, A61K38, A61K39, A61K47, A61K49, A61P31, A61P35