WO2002078672A2 - Liposomal tumor necrosis factor compositions and methods - Google Patents

Abstract

Liposome compositions containing an entrapped TNF compound and a method of using the same for effective upregulation of immunological factors are described. Also disclosed is a method of combination therapy where a conventional anti-tumor therapy and the liposome-entrapped TNF compound are administered to achieve an enhanced anti-tumor effect.

Description

LIPOSO AL TUMOR NECROSIS FACTOR COMPOSITIONS AND METHODS

Field of the Invention

[0001] The present invention relates to liposomal compositions containing entrapped tumor necrosis factor compounds and to methods of using the same for upregulating immunological factors. The invention also relates to a method for enhancing anti-tumor effects of conventional anti-tumor treatments by providing for a combined treatment modality that includes administration of a liposome- entrapped tumor necrosis factor.

Background of the Invention

[0002] Tumor necrosis factor-α (TNF-α) plays an integral role in destroying tumors, in mediating responses to tissue injury, and in protecting hosts from infections by various microorganisms. TNF-α induces a variety of responses, including hemorrhagic necrosis of tumors in vivo through destruction or alteration of tumor vasculature, direct cytotoxicity against some tumor cells in vitro, inflammation, and activation of many cell types (Carswell, et al., Proc Natl Acad Sci U S A, 72:3666-70, 1975; Sugarman, et al., Science, 230:943-5, 1985). TNF-α may also sensitize the tumor cells or the host response, resulting in synergistic effects when combined with other agents. The addition of TNF-α as an adjuvant or neoadjuvant agent often produces a better than additive response for the combination therapies (Abbas, et al., CELLULAR AND MOLECULAR IMMUNOLOGY, 2nd Ed., W. B. Saunders Company, 1994, pp. 244-249). [0003] TNF-β shows anticellular activity on neoplastic cell lines but not on primary cell cultures and normal cell lines, suggesting that it has potent anti-tumor activity. TNF-β also plays an important role in lymphoid organ development. [0004] However, like other cancer chemotherapeutic agents, the TNFs are toxic drugs. Systemic injections of TNFs can lead to severe toxicities that can prevent the administration of even the minimal effective dose (Hersh, et al., J Immunother., 10:426-31 , 1991 ; Taguchi, et al., Gan To Kagakυ Ryoho., 13:3491-7, 1986; Zwaveling, et al., Crit. Care Med., 24:765-70, 1996; Tracey, et al., Science, 234:470-4, 1986). In addition, it is also known that the TNFs when administered in free form are quickly cleared from circulation, preventing them from accumulating within, or perhaps even reaching, the tumor target (Rathjen, et al., Mol Immunol., 28:79-86 1991 ).

[0005] Attempts to minimize the toxicity and enhance the circulation half-life of anti-tumor agents have included combination therapy (U.S. Patent No. 5,976,800) and entrapment in liposomes (Sur, B., et al, Oncology, 40:372-376, 1983; Weiss, R.B., et al., Drugs 46(3):360-377, 1993). It has also been shown that antineoplastic agents entrapped in liposomes have a reduced toxicity, relative to the agent in free form, while retaining anti-tumor activity (Steerenberg, P.A., et al, International Journal of Pharmaceutics, 40:51-62, 1987; Weiss, et al., 1993). However, the effect of liposome-entrapped TNF on the immune response has not been reported.

Summary of the Invention

[0006] In one aspect, the invention includes a liposomal composition containing an entrapped TNF compound. The composition includes liposomes which are composed of a vesicle-forming lipid and between about 1-20 mole percent of a vesicle-forming lipid derivatized with a hydrophilic polymer. The TNF compound,- either TNF-α or TNF-β, is entrapped in the liposomes.

[0007] The TNF compound, in one embodiment, is TNF-α. In another embodiment, TNF-α is entrapped in the liposomes at a ratio of 25 μg of TNF-α to

100 μmol of liposome lipid.

[0008] In another embodiment, the hydrophilic polymer is selected from the group consisting of polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, poly- hydroxypropylmethacrylamide, polymethacrylamide, poly-dimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol, and polyaspartamide. In a preferred embodiment, the hydrophilic polymer is polyethyleneglycol.

[0009] In a preferred embodiment, the vesicle-forming lipid is hydrogenated soy phosphatidylcholine and the derivatized vesicle-forming lipid is distearoylphosphatidylethanolamine derivatized with polyethyleneglycol. [0010] In another preferred embodiment, the vesicle-forming lipid is phosphatidylethanolamine and the derivatized vesicle-forming lipid is distearoylphosphatidylethanolamine derivatized with polyethyleneglycol. [0011] In another aspect, the invention includes a method of sustained stimulation of immune cells to induce immunological factors upregulation in a subject. The method includes administering the above-described liposomal composition to the test subject. The liposomal composition induces upregulation of one or more of the following: leukocyte, granulocyle, monocyte, or natural killer (NK) cells in the blood; or basal leukocyte, leukocyte, T cell, NK cell, or CD25⁺ activation marker expression in the spleen.

[0012] In yet another aspect, the invention includes a method for inhibiting tumor growth by administering to a subject, a TNF compound entrapped in a liposome, as described above, waiting for a selected period of time, typically for approximately between about 10 to about 40 hours, more typically from about 15- 40 hours, and most typically from about 18-36 hours, and then administering a conventional tumor treatment modality. In one embodiment, the waiting period is approximately about 24 hours.

[0013] Administering the TNF-α compound entrapped in liposomes is effective to achieve an increase in at least one of leukocyte, granulocyte, monocyte, or natural killer cells in the blood; and/or an increase in least one of basal leukocyte, leukocyte, T cell, natural killer cells, or CD25⁺ activation marker expression in the spleen.

[0014] In one embodiment, ionizing radiation is administered in combination with the liposomal composition. For example, ionizing radiation can be administered before, during, or after administration of the liposomes, but is preferably administered after, and more preferably is administered after a defined waiting period. In another embodiment a chemotherapeutic agent, such as doxorubicin, cisplatin, paclitaxel, or the like, is administered before, during, or after the liposomal composition, and in a preferred embodiment, the chemotherapeutic agent is administered after the liposome-entrapped TNF compound, and more preferably is administered after a defined waiting period. In one embodiment, the chemotherapeutic agent is also entrapped in liposomes. [0015] These and other objects and features of the invention will be more fully appreciated when the following detailed description of the invention is read in conjunction with the accompanying drawings.

Brief Description of the Drawings [0016] Fig. 1 is a schematic illustration of a liposome formed in accordance with the present invention.

[0017] Fig. 2 illustrates a general reaction scheme for derivatizing a vesicle- forming lipid with a polyalkylether.

[0018] Fig. 3 is a reaction scheme for preparing phosphatidylethanolamine derivatized with polyethyleneglycol via a cyanuric chloride linking agent. [0019] Fig. 4 illustrates a reaction scheme for preparing phosphatidylethanolamine derivatized with polyethyleneglycol by means of a diimidazole activating reagent.

[0020] Figs. 5A-5C are plots showing the amount, in pg/mg, of recombinant human TNF-α (rhu-TNF-α) in plasma (Fig. 5A), tumor (Fig. 5B), and liver (Fig. 5C) of tumor-bearing mice as a function of time, in hours, in untreated, control animals (•), and in animals after injection of placebo liposomes (T), TNF-α in free form (o), or liposome-entrapped TNF-α (v).

[0021] Figs. 6A-6D are plots showing normalized red blood cell (RBC) count (Fig. 6A), hematocrit (Fig. 6B), hemoglobin (Fig. 6C), and platelets (Fig. 6D) in tumor-bearing mice as a function of time following treatment with TNF-α in free form (•), liposome-entrapped-TNF-α (T), or placebo liposomes (o). Each point represents the normalized mean ± SEM for seven mice where the data are normalized with respect to untreated tumor-bearing mice. [0022] Figs. 7A-7F are plots showing normalized leukocyte (white blood cell, WBC) counts in the blood (Fig. 7A) and the spleen (Fig. 7D); spontaneous blastogenesis in the blood (Fig. 7B) and spleen (Fig. 7C), spleen weight relative to body weight (Fig. 7E) and lipopolysaccharide stimulation index (Fig. 7F) in tumor- bearing mice as a function of time, in hours, after injection of TNF-α in free form (•), liposome-entrapped TNF-α (T), or placebo liposomes (o). Each point represents the normalized mean ± SEM for seven mice where the data are normalized with respect to untreated tumor-bearing mice. [0023] Figs. 8A-8D show the non-lymphoid granulocyte and monocyte cell populations in the blood (Figs. 8A, 8B) and spleen (Figs. 8C, 8D) of tumor-bearing mice as a function of time, in hours, after injection of TNF-α in free form (•), liposome-entrapped TNF-α (T), or placebo liposomes (o). Each point represents the normalized mean ± SEM for seven mice where the data are normalized with respect to untreated tumor-bearing mice.

[0024] Figs. 9A-9F show normalized lymphoid cell populations in the blood (Figs. 9A, 9C, 9E) and spleen (Figs. 9B, 9D, 9F) of tumor-bearing mice as a function of time, in hours, after injection of TNF-α in free form (•), liposome- entrapped TNF-α (T), and placebo liposomes (o). Each point represents the normalized mean ± SEM for seven mice where the data are normalized with respect to untreated tumor-bearing mice.

[0025] Figs. 9G-9J show normalized NK and B cells in the blood (Figs. 9G, 91) and spleen (Figs. 9H, 9J) of tumor-bearing mice as a function of time, in hours, after injection of TNF-α in free form (•), liposome-entrapped TNF-α (Y), and placebo liposomes (o). Each point represents the normalized mean ± SEM for seven mice where the data are normalized with respect to untreated tumor-bearing mice.

[0026] Figs. 9K-9S show normalized T cell parameters in the blood (Figs. 9K, 9M, 9O, 9Q, 9S) and spleen (Figs. 9 , 9N, 9P, 9R) of tumor-bearing mice as a function of time, in hours, after injection of TNF-α in free form (•), liposome- entrapped TNF-α (T), and placebo liposomes (o). Each point represents the normalized mean ± SEM for seven mice where the data are normalized with respect to untreated tumor-bearing mice.

Detailed Description of the Invention

I. Liposome Composition

[0027] The liposomal composition of the present invention includes liposomes having an entrapped TNF compound. A "TNF compound" as used herein intends either TNF-α, TNF-β, or a mixture thereof in any proportion, captured within a liposome, such that the TNF compound is retained substantially within the liposome prior to administration. [0028] Fig. 1 illustrates a liposome 10, prepared in accordance with the invention, which includes an inner lipid bilayer 12 and an outer lipid bilayer 14. The inner and outer lipid bilayers are formed predominantly of vesicle-forming lipids, such as lipid 16, which include a polar head group 16a and a hydrophobic tail 16b. It will be appreciated that the liposome may include additional bilayers and that for simplicity only one bilayer is shown. Exemplary vesicle-forming lipids are listed below.

[0029] Liposome 10 also includes vesicle-forming lipids derivatized with a hydrophilic polymer, such as derivatized lipid 18 in Fig. 1. Derivatized lipid 18 includes a hydrophobic tail 18a, a polar head group 18b, and attached to the polar head group, by means described below, a hydrophilic polymer 18c. The hydrophilic polymer chains 18, which are preferably densely packed to form a brush-like outer surface coating 20.

[0030] Liposome 10 also includes a TNF compound, e.g., TNF-α, TNF-β, or a mixture thereof, in entrapped form. The drug is entrapped in the aqueous compartments in dissolved form or in precipitated form.

A. Vesicle-Forming Lipid Component [0031] The liposome composition of the present invention is composed primarily of vesicle-forming lipids. Such a vesicle-forming lipid is one which (a) can form spontaneously into bilayer vesicles in water, as exemplified by the phospholipids, or (b) is stably incorporated into lipid bilayers, with its hydrophobic moiety in contact with the interior, hydrophobic region of the bilayer membrane, and its head group moiety oriented toward the exterior, polar surface of the membrane.

[0032] The vesicle-forming lipids of this type are preferably ones having two hydrocarbon chains, typically acyl chains, and a head group, either polar or nonpolar. There are a variety of synthetic vesicle-forming lipids and naturally- occurring vesicle-forming lipids, including the phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI), and sphingomyelin (SM), where the two hydrocarbon chains are typically between about 14-22 carbon atoms in length, and have varying degrees of unsaturation. The above-described lipids and phospholipids whose acyl chains have varying degrees of saturation can be obtained commercially or prepared according to published methods. Other suitable lipids also include glycolipids and sterols such as cholesterol. [0033] Preferred diacyl-chain lipids for use in the present invention include diacyl glycerol, phosphatidyl ethanolamine and phosphatidylglycerol. These lipids are preferred for use as the vesicle-forming lipid, the major liposome component, and for use in the derivatized lipid described below.

[0034] Additionally, the vesicle-forming lipid is selected to achieve a specified degree of fluidity or rigidity, to control the stability of the liposome in serum and to control the rate of release of the entrapped agent in the liposome. Liposomes having a more rigid lipid bilayer, or a liquid crystalline bilayer, are achieved by incorporation of a relatively rigid lipid, e.g., a lipid having a relatively high phase transition temperature, e.g., up to 60 °C. Rigid, i.e., saturated, lipids contribute to greater membrane rigidity in the lipid bilayer. Other lipid components, such as cholesterol, are also known to contribute to membrane rigidity in lipid bilayer structures. On the other hand, lipid fluidity is achieved by incorporation of a relatively fluid lipid, typically one having a lipid phase with a relatively low liquid to liquid-crystalline phase transition temperature, e.g., at or below room temperature (20 - 25 °C).

[0035] The lipids forming the bilayer vesicle, i.e., liposome, can also be cationic lipids, which have a lipophilic moiety, such as a sterol, an acyl or diacyl chain, and where the lipid has an overall net positive charge. Preferably, the head group of the lipid carries the positive charge. Exemplary cationic lipids include 1 ,2- dioleyloxy-3-(trimethylamino) propane (DOTAP); N-[1-(2,3,-ditetradecyloxy)propyl]- N.N-dimethyl-N-hydroxyethylammonium bromide (DMRIE); N-[1-(2,3,- dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethylammonium bromide (DORIE); N- [1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA); 3β[N- (N'.N'-dimethylaminoethane) carbamoly] cholesterol (DC-Choi); and dimethyldioctadecylammonium (DDAB).

[0036] The cationic vesicle-forming lipid may also be a neutral lipid, such as dioleoylphosphatidylethanolamine (DOPE) or an amphipathic lipid, such as a phospholipid, derivatized with a cationic lipid, such as polylysine or other polyamine lipids. For example, the neutral lipid (DOPE) can be derivatized with polylysine to form a cationic lipid. [0037] The liposomes may include other lipids that can stabilize a vesicle or liposome composed predominantly of phospholipids. A frequently employed lipid for this purpose is cholesterol at between 25 to 40 mole percent. At between 0 to 20 mole percent cholesterol in a bilayer, separate domains exist containing cholesterol and phospholipids and pure phospholipid (Mabrey, S., et al., Biochem., 17:2464-2468 (1978)). These bilayers show an increased permeability to water (Tsong, T.Y., Biochem. 14:5409-5414, 5415-5417, 1975).

B. Derivatized Vesicle-forming Lipid Component [0038] As discussed above, the liposomes of the present invention contain a hydrophilic polymer surface coating 20 (Fig. 1 ). The surface coating is provided by including in the liposome composition between about 1-20 mole percent of a lipid derivatized with a hydrophilic polymer.

[0039] Hydrophilic polymers suitable for derivatization with a vesicle-forming lipid include polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol, and polyaspartamide. The polymers may be employed as homopolymers or as block or random copolymers. [0040] In a preferred embodiment, the hydrophilic polymer is polyethyleneglycol (PEG), preferably as a PEG chain having a molecular weight between about 500- 10,000 daltons, more preferably between about 2,000-10,000 daltons and most preferably between about 1 ,000-5,000 daltons. Methoxy or ethoxy-capped analogues of PEG are also preferred hydrophilic polymers, and are commercially available in a variety of polymer sizes, e.g., 120-20,000 daltons. [0041] Vesicle-forming lipids suitable for derivatization with a hydrophilic polymer include any of those lipids listed above, and, in particular phospholipids, such as distearoylphosphatidylethanolamine (DSPE). Preparation of DSPE derivatized with PEG is described below in Examples 1 and 2. [0042] The derivatized lipid is incorporated into the liposome by including an amphipathic lipid derivatized with hydrophilic polymer with vesicle-forming lipids during formation of the lipid vesicles. [0043] The surface coating on the liposome provided by the hydrophilic polymer chains provides colloidal stability and serves to protect the liposomes from uptake by the reticuloendothelial system, providing a long blood circulation lifetime for the liposomes to distribute in the subject. The extent of enhancement of blood circulation time is preferably several fold over that achieved in the absence of the polymer coating, as described in co-owned U.S. Patent No. 5,013,556.

C. Other Liposome Components

[0044] The liposomes in the composition of the present invention may include other components, such as targeting molecules, including antibodies, antibody fragments, or cell-surface recognition molecules, which are attached to the liposome by means of a hydrophilic polymer chains. For example, a vesicle- forming lipid is derivatized with a hydrophilic polymer chain, as described above, and the hydrophilic polymer is end-functionalized for coupling antibodies to its functionalized end. The functionalized end group may be a hydrazide or hydrazine group which is reactive toward aldehyde groups, although any of a number of PEG-terminal reactive groups for coupling to antibodies may be used. Hydrazides can also be acylated by active esters or carbodiimide-activated carboxyl groups. Acyl azide groups reactive as acylating species can be easily obtained from hydrazides and permit attachment of amino-containing molecules. The functionalized end group may also be 2-pyridyldithio-propionamide, for coupling an antibody or other molecule to the liposome through a disulfide linkage. A variety of targeting moieties are described in U.S. Patent Nos. 5,891 ,468 and 6,043,094 and the portions thereof relating to targeting ligands are incorporated by reference herein.

D. TNF Compound

[0045] As referred to herein, "TNF compound" refers to either one of two structurally and functionally related proteins, TNF-α and TNF-β. The two proteins are about 30% homologous at the amino-acid level, and bind to the same cell- surface receptors; both exist as homotrimers. Both TNF-α (cachectin) and TNF-β (lymphotoxin) were originally thought of as selective antitumour agents, but are now known to have a multiplicity of actions. In binding to their receptors, present on virtually all cells examined, they activate a large array of cellular genes and also multiple signal-transduction pathways, kinases, and transcription factors. Their genes are single-copy genes, closely linked with the major histocompatibility complex (MHC) cluster.

[0046] TNF-α, a multifunctional cytokine, is produced by macrophages, monocytes, endothelial cells, neutrophils, smooth muscle cells, activated lymphocytes, and astrocytes. It is a transmembrane glycoprotein and a cytotoxin with a variety of functions, including the ability to mediate the expression of genes for growth factors, cytokines, transcription factors, and receptors. It can cause cytolysis of certain tumour cell lines and has been implicated in the induction of cachexia. The protein is a potent pyrogen, causing fever by direct action or by stimulation of interleukin-1 secretion; and it can stimulate cell proliferation and induce cell differentiation under certain conditions. TNF-α induces a variety of responses, including hemorrhagic necrosis of tumors in vivo through destruction or alteration of tumor vasculature, direct cytotoxicity against some tumor cells in vitro, inflammation, and activation of many cell types (Carswell et al., 1975; Sugarman et al., 1985). TNF-α may also sensitize the tumor cells or the host response, resulting in synergistic effects when combined with other agents. The molecule is a homotrimer containing three 152 amino acid (16.82 kDa) subunits. The sequence can be obtained from well known sources, such as the NCBI GenBank databases (www.ncbi.nlm.nih.gov/GenBank), database code NRL_1TNFA. The 3- D structure is also known.

[0047] TNF-β, a cytokine, produced by T-lymphocytes, is cytotoxic for a wide range of tumor cells. TNF-β shows anticellular activity on neoplastic cell lines but not on primary cell cultures and normal cell lines, suggesting that it has potent anti- tumor activity. TNF-β also plays an important role in lymphoid organ development. TNF-β binds to the same cell surface receptors as TNF-α. The TNF-β molecule of a human consists of three 205 amino acid (22.27 kDa) subunits and the GenBank database code is TNFB_HUMAN.

[0048] Recombinant human TNF-α and TNF-β proteins are available commercially (R&D Systems Inc., Minneapolis, MN) as lyopilized powders. The TNF-α powder has a reported activity of 5 x 10⁶ units/mg (as determined by cytotoxicity assay using the murine L929 cell line). The lyopilized powder may be reconstituted in sterile PBS with 0.1% bovine serum albumin, aliquoted, and stored at -70°C until immediately before use. The drug may be, directly or encapsulated in liposomes, administered intravenously for a tumor adjuvant therapy, preferably given between 18-36 hours before traditional tumor therapy. In one embodiment, a stock solution of the TNF-α liposomal composition (liposome-entrapped TNF-α) is prepared at approximately 60 μmole/ml of lipid containing 25 μg TNF-α per 100 μmole liposome. The solution is diluted in PBS immediately before use.

II. Preparing the Liposome Composition

[0049] Section A below describes synthesis of vesicle-forming lipids derivatized with a hydrophilic polymer for use in forming the liposomes of the present invention. Section B describes a method of preparing liposomes including the derivatized lipids and an entrapped TNF compound.

A. Preparation of Derivatized Vesicle-Forming Lipids [0050] Fig. 2 shows a general reaction scheme for preparing a vesicle-forming lipid derivatized with a biocompatible, hydrophilic polymer, as exemplified by polyethylene glycol (PEG). The polymer can be capped by a methoxy, ethoxy or other unreactive group at one end, or can be a polymer having one end that is naturally or rendered to be more reactive than the other. For example, the polymer can activated at one end by reaction with a suitable activating agent, such as cyanuric acid, diimadozle, anhydride reagent, or the like, as described below. The activated compound is then reacted with a vesicle-forming lipid, such as phosphatidylethanol (PE), to produce the derivatized lipid. [0051] Alternatively, the polar group in the vesicle-forming lipid may be activated for reaction with the polymer, or the two groups may be joined in a concerted coupling reaction, according to known coupling methods. PEG capped at one end with a methoxy or ethoxy group can be obtained commercially in a variety of polymer sizes, e.g., 500-20,000 dalton molecular weights. [0052] The vesicle-forming lipid is preferably one having two hydrocarbon chains, typically acyl chains, and a polar head group, such as those listed above. [0053] Fig. 3 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 of sodium carbonate under conditions which produced the activated PEG compound in the figure. This material is purified to remove unreacted cyanuric acid. The activated PEG compound is reacted with PE in the presence of triethylamine to produce the desired PE-PEG compound, also shown in the figure.

[0054] The method just described may be applied to a variety of lipid amines, including PE, cholesteryl amine, and glycolipids with sugar-amine groups. [0055] A second method of coupling a polyalkylether, such as capped PEG to a lipid amine is illustrated in Fig. 4. Here the capped PEG is activated with a carbonyl diimidazole coupling reagent, to form the activated imidazole compound shown in Fig. 4. 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.

B. Liposome Preparation [0056] The liposomes may be prepared by a variety of techniques, such as those detailed in Szoka, F., Jr., et al., Ann. Rev. Biophys. Bioeng., 9:467, 1980. One method for preparing drug-containing liposomes is the reverse phase evaporation method, described by Szoka and in U.S. Patent No. 4,235,871. Reverse phase evaporation vesicles (REVs) have typical average sizes between 2-4 microns and are predominantly oligolamellar, that is, contain one or a few lipid bilayer shells.

[0057] In another method, multilamellar vesicles (MLVs) can be formed by simple lipid-film hydration techniques. In this procedure, a mixture of liposome- forming lipids of the type detailed above dissolved in a suitable organic solvent is evaporated in a vessel to form a thin film, which is then covered by an aqueous medium. The lipid film hydrates to form MLVs, typically with sizes between about 0.1 to 10 microns.

[0058] The lipid components used in forming the liposome composition of the present invention, are preferably present in a molar ratio of about 70-90 mole percent vesicle-forming lipids, and 1-20 mole percent polymer-derivatized lipid. One exemplary liposome formulation includes 80-90 mole percent phosphatidylethanolamine, 1-20 mole percent of PEG-DSPE. Cholesterol may be included in the formulation at between about 1-50 mole percent. Another exemplary liposome formulation includes hydrogenated soy phosphatidylcholine (HSPC) and cholesterol (Choi), in about a 1 :1 molar ratio, and between about 1-5 mole% of DSPE-PEG, added to form liposomes with an inner and outer bilayer surface coating of PEG. Preparation of an exemplary HSPC-Chol-DSPE-PEG liposome form is described in Example 3.

[0059] Another procedure suitable for preparation of the liposomes of the present invention is described by Uster, et al., FEBS Letters, 386:243-246, 1996. In this method, liposomes with an entrapped therapeutic agent are prepared from vesicle- forming lipids. The preformed liposomes are added to a solution containing a concentrated dispersion of micelles of PEG-derivatized lipid conjugates and incubated under conditions effective to achieve insertion of the micellular lipid conjugates into the preformed liposomes.

[0060] Still another liposome preparation procedure suitable for preparation of the liposomes of the present invention is a solvent injection method. In this procedure, a mixture of the lipids, dissolved in a solvent, preferably ethanol or DMSO, is injected into an aqueous medium with stirring to form liposomes. The solvent is removed by a suitable technique, such as dialysis, and the liposomes are then sized as desired. This method achieves relatively high encapsulation efficiencies. [0061] Generally, the TNF compound is incorporated into liposomes during liposome formation by adding a solution containing the TNF compound to a dried lipid film, as will be described below.

[0062] The liposomes are preferably prepared to have substantially homogeneous sizes in a selected size range, typically between about 0.03 to 0.5 microns. One effective sizing method for REVs and MLVs involves 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. Homogenization methods are also useful for down-sizing liposomes to sizes of 100 nm or less (Martin, F.J., in SPECIALIZED DRUG DELIVERY SYSTEMS-MANUFACTURING AND PRODUCTION TECHNOLOGY, (P. Tyle, Ed.) Marcel Dekker, New York, pp. 267-316, 1990). [0063] After final sizing, the liposomes can be treated, if necessary, to remove free (non-entrapped) TNF. Conventional separation techniques, such as centrifugation, diafiltration, and molecular-seive chromatography are suitable. The composition can be sterilized by filteration through a conventional 0.45 micron depth filter.

[0064] Preparation of an exemplary composition is described in Example 3. A lipid solution HSPC, cholesterol, and mPEG-DSPE in a molar ratio of 2:1 :0.1 was prepared by dissolving the lipids in an organic solvent. The organic solvent was evaporated to form a dried lipid film. An aqueous solution of TNF-α was separately prepared. The dried lipid film was hydrated with the TNF-α solution to form liposomes having a TNF to lipid ratio of approximately 25 μg TNF-α to approximately 100 μmole of lipid. In the present invention, TNF-α was entrapped in liposomes which were sized to a diameter suitable for intravenous administration, preferably between about 80-160 nm, more preferably between about 100-140 nm, most preferably between about 100-120 nm.

III. In Vivo Administration of Liposome-Entrapped TNF For Upregulation of Immunological Factors

[0065] In studies performed in support of the invention, a TNF compound, as exemplified by TNF-α, was administered in liposome-entrapped form to tumor- bearing mice to induce upregulation of various immunological factors. Liposomes containing TNF were prepared as described in Example 3 and compared to administration of TNF in free form, as will now be described. [0066] As detailed in Example 4, mice were injected with human LS174T colon adenocarcinoma to induce formation of tumors. By day 11 , all tumor-injected mice had detectable tumors (-200 mm³) and treatment was initiated. Tumor-bearing mice were divided into test groups for treatment with (1 ) phosphate buffered saline (control), (2) TNF-α in free form, (3) placebo liposomes, or (4) liposome-entrapped TNF-α. The test group receiving placebo liposomes was included to assess the immune response from placebo liposomes. The test substances were administered intravenously at either 30 μg of free TNF-α, 30 μg TNF-α entrapped in 120 μmol of liposome lipid, or 120 μmol of placebo liposome lipid, per kilogram body weight. A subset from each test group was euthanized at 6, 18, 36, and 96 hours following injection. Blood, tumor, and selected organs (spleen, liver, kidney) were assayed for the following immunological factors: splenic leukocyte counts; mitogen-induced blastogenesis,; blood leukocyte, erythrocyte, and thrombocyte enumeration; spontaneous blastogenesis of leukocytes in blood and spleen; lymphocyte populations in blood and spleen. The relative spleen weights and the amount of recombinant human TNF-α in tumor, liver, and plasma were also determined. The assays used are generally known to those skilled in the art and are also described in Example 4. The results of the assays are shown in Figs. 5 to 9.

[0067] Figs. 5A-5C show the amount (in pg/mg) of recombinant human TNF-α (rhu-TNF-α) in the plasma (Fig. 5A), tumor (Fig. 5B), and liver (Fig. 5C) of tumor- bearing mice treated with placebo liposomes (▼); TNF-α in free form (o), or liposome-entrapped-TNF-α (v). The results for untreated tumor-bearing animals as a control are also shown (•). The amount of rhu-TNF-α was measured at 6, 18, 36, and 96 hours after injection of the test substances. Six hours after administration, the level of rhu-TNF-α was elevated in all three tissues (plasma, tumor, and liver) in the animals treated with free TNF-α or with liposome-entrapped TNF-α. By 36 hours, the TNF levels returned to baseline in all tissues for animals treated with free TNF-α (o). However, in animals treated with TNF-α in liposome- entrapped form (v), the rhu-TNF-α level in the plasma remained elevated with respect to the control animals for the entire measured period and until the 96 hour time point in the tumor (Fig. 5B) and liver (Fig. 5C).

[0068] The hematological toxicity of the test substances was assessed by an analysis of the red blood cell count, the hematocrit, the hemoglobin number, and the platelet number in the blood of the animals in each test group. The results for samples taken at 6, 18, 36, and 96 hours after injection of TNF-α in free form (•), liposome-entrapped TNF-α (r) or placebo liposomes (o) are shown in Figs. 6A- 6D. The data for the treated groups was normalized to that obtained for the control group (untreated, tumor-bearing mice) to minimize any immunodulatory effects due to the presence of the tumor. Overall, there were no observable signs of toxicity. Blood analyses showed that red blood cell (RBC) counts (Fig. 6A), hematocrit (Fig. 6B), hemoglobin (Fig. 6C), and platelet number (Fig. 6D) were initially (at the six hour sample) decreased in all three test groups, but recovery occurred by 18 hours post-administration. However, the mice injected with free TNF-α (•) exhibited residual effects up to the 96 hour time point, as evidenced by low levels of RBC (Fig. 6A) and platelets (Fig. 6D) and significantly low hematocrit (Fig. 6B) and hemoglobin (Fig. 6C). Fig. 6D shows that animals treated with free TNF-α (•) or liposome-entrapped TNF-α (▼) had a significantly decreased platelet count at the initial six hour time point. However, the liposome-entrapped TNF-α treatment group rapidly returned to baseline platelet count (normalized platelet value of 1.0), whereas the animals treated with free TNF-α had a continued, significantly decreased normalized platelet level until the 96 hour time point. Other blood parameters, such as the volume of an average RBC (MCV), amount of hemoglobin in an average RBC, and mean corpusular hemoblobin concentration did not show significant differences among the test groups (data not shown). [0069] The toxicity of free TNF-α, even at the relatively low concentration used, is clearly reflected in the blood parameters. Anemia and other blood alterations are a few of the toxic effects of that limit the use of free TNF-α. Though thromobcytopenia was seen with both TNF-α in free form and in liposome- entrapped form, only free TNF-α had sustained thrombocytopenia. The initial decrease in platelet count with liposome-entrapped TNF-α may reflect the small release of TNF-α from the liposome that occurs within the first 12 hours after injection.

[0070] Figs. 7A-7F show the leukocyte counts (Figs. 7A, 7C), the spontaneous and mitogen-induced blastogenesis (Figs. 7B, 7D, 7F) and the spleen weight relative to body weight (RSW, Fig. E) in the tumor-bearing mice after treatment with free TNF-α (•), liposome-entrapped TNF-α (r), or placebo liposomes (o). Data were normalized to the data obtained for the control group (untreated, tumor- bearing mice) to minimize any immunodulatory effects due to the presence of the tumor. As seen in Fig. 7A, the leukocyte counts in the blood from mice treated with free TNF-α or with liposome-entrapped TNF-α were significantly greater than the leukocyte counts in the control group at six hours post administration of the test substance. By 36 hours post administration, the leukocyte count in the free TNF-α-treated group had substantially decreased, whereas the counts in the liposome-entrapped treated animals remained elevated (T). [0071] Fig. 7B shows the basal proliferation rates of circulating leukocytes (white blood cells), as determined by [³H]-TdR incorporation into DNA (see Example 4.C.3) for each of the test groups. As seen, the animals treated with liposome-entrapped TNF-α or with free TNF-α had a significantly elevated proliferation rate at the 18 hour time point.

[0072] In the spleen, animals treated with free TNF-α (•) or with liposome- entrapped TNF-α (T) had significant but opposite effects on leukocyte counts at 6, 18, and 36 hours post administration, as seen in Fig. 7C. The basal proliferation rates of the splenocytes in the animals treated with liposome-entrapped TNF-α was significantly increased at 18 and 96 hr when compared to the animals treated with free TNF-α, as seen in Fig. 7D.

[0073] Spleen weights in relation to body weights, as shown in Fig. 7E, were significantly increased in animals treated with TNF-α in either free form (•) or in liposome-entrapped form (Y) at the six hour time point, with a return to basal weight thereafter. As seen in Fig. 7F, there were no differences in splenocyte responsiveness to lipopolyaccharide (LPS) among the groups, except for the group treated with placebo liposomes, which had significantly high stimulation index (SI = (cpm with LPS - cpm without LPS)/cpm without LPS) values at the 96 hour time point.

[0074] Figs. 8A-8D show the effect of the treatments on non-lymphoid cell populations in the blood and spleen of mice in the various test groups. As before, the data were normalized to the data obtained for the control group (untreated, tumor-bearing mice) to minimize any immunodulatory effects due to the presence of the tumor. Blood and spleen samples were taken from animals at 6, 18, 36, and 96 hours after injection of TNF-α in free form (•), liposome-entrapped TNF-α (T) and placebo liposomes (o).

[0075] Figs. 8A-8B show the normalized granulocyte and monocyte numbers in the blood. Animals treated with free TNF-α or liposome-entrapped TNF-α had higher counts at six hours post treatment than did the animals treated with the placebo liposomes. By 18 hours post administration of TNF-α, the counts for both granulocytes and monocytes had returned to about a baseline level (normalized value of 1). However, at 36 hours post administration, blood granulocytes (Fig.. 8A) for the animals treated with TNF-α in free form were decreased in number when compared to the animals treated with liposome-entrapped TNF-α. At 96 hours, the number of granulocytes were significantly elevated compared to the groups receiving liposome-entrapped TNF-α or placebo liposomes. [0076] Granulocyte and monocyte/macrophage numbers in the spleen are shown in Figs. 8C-8D. Here, the cell numbers were increased at six hours after treatment in the group receiving free TNF-α (•). Although granulocyte number (Fig. 8C) was slightly increased in the group receiving liposome-entrapped TNF-α at six hours post injection, statistical significance was lacking. No statistically significant differences were observed in the monocyte/macrophage counts (Fig. 8D) at the six hour time point. By 96 hours post administration of the test substances, the granulocyte and monocyte numbers in all test groups were similar and were at about a baseline level. Over the 96 hour observation period, the two groups receiving a liposome-based treatment (liposome-entrapped TNF-α and placebo liposomes) had an increased monocyte and granulocyte count for a longer period of time relative to the animals treated with TNFα in free form. [0077] Figs. 9A-9S show the effect of the test substances on lymphoid cell populations in the blood and spleen of the tumor-bearing mice. As before, the data were normalized to the data obtained for the control group (untreated, tumor- bearing mice) to minimize any immunodulatory effects due to the presence of the tumor. Blood and spleen samples were taken from animals at 6, 18, 36, and 96 hours after injection of TNF-α in free form (•), liposome-entrapped TNF-α (T), and placebo liposomes (o).

[0078] Figs. 9A, 9C, and 9E show the normalized lymphocyte, CD25⁺, and CD71⁺ levels, respectively, in blood taken from the mice after treatment. Six hours after treatment, the total number of lymphocytes in the blood (Fig. 9A) from the group receiving free TNF-α (•) was significantly decreased, yet at the same time point their activation (i.e. CD25⁺ or CD71⁺ lymphocytes) was significantly increased (Figs. 9C, 9E). In contrast, mice receiving liposome-entrapped TNF-α (T) did not show any significant changes at the six hour time point in these counts. In the spleen, total lymphocyte numbers (Fig. 9B) at the 6 hour and 36 hour time points were elevated, in contrast to the decrease in the total lymphocyte number in animals treated with liposome-entrapped TNF-α. With respect to the number of lymphocytes expressing activation marker CD25⁺ in the spleen (Fig. 9D), an increase was observed in the liposome-entrapped TNF-α-treated group at 18 hours post treatment. However, lymphocytes expressing the CD71⁺ activation marker were increased for the free TNF-α group at six hours, while both groups receiving liposomes were activated at 18 hours, as seen in Fig. 9F. [0079] Fig. 9G shows that in the blood, the natural killer (NK) cell numbers were significantly elevated at six hours post administration for the groups receiving either free TNF-α or liposome-entrapped TNF-α. The animals treated with liposome-entrapped TNF-α had a more sustained NK cell level for an additional 12 hours, as seen by the continued elevation at the 18 hour time point. Fig. 9H shows the NK cell level in the spleen for each of the treatment groups. Here, the NK cells for the animals treated with free TNF-α or with liposome-entrapped TNF- α were elevated beginning at 18 hours post-treatment. In addition, the animals treated with liposome-entrapped TNF-α had significantly more NK cells than did the free TNF-α group at this time point.

[0080] The B cells in the blood and spleen are shown in Figs. 91 and 9J, respectively. Generally, the B cell numbers mirrored the pattern seen with total lymphocyte numbers. Administration of free TNF-α (•) resulted in significant B cell depletion in the blood by six hours post administration, followed by a significant increase at 18 hours, as seen in Fig. 91. The B cells in the animals treated with liposome-entrapped TNF-α (T) followed a similar pattern, although not to the same degree. In the spleen, (Fig. 9J) B cell counts were higher at all measured time points following treatment with TNF-α in free form compared to the group receiving liposome-entrapped TNF-α, and a statistically significant difference was obtained at the 6 hour and 18 hour time points.

[0081] Figs. 9K, 9M, 9O show level of CD3⁺ T cells, CD4⁺ T-helper (Th) cells, and CD8⁺ T-cytotoxic (Tc) cells, respectively, in the blood for each of the treatment groups as a function of time. As seen in Fig. 9K, the total T cell numbers were significantly increased in the animals treated with free TNF-α (•) at six hours post administration, followed by a decrease to baseline by 36 hours, and an increase by 96 hours post administration. The two groups receiving the liposome formulations, either as liposome-entrapped TNF-α (T) or as placebo liposomes, showed significant increases in the T cell levels at the 20 hour time point. [0082] Figs. 9Q and 9S shows the total number of CD3⁺/CD25⁺ in the blood (Fig. 9Q) and the total number of CD37CD71⁺ in the blood (Fig. 9S) for each of the treatment groups. A significant increase in the number of activated T cells (CD37CD25⁺ or CD37CD71⁺) within the total T cell population was apparent in both the free TNF-α and liposome-entrapped TNF-α treatment groups at the six hour time point. Thereafter, a return to baseline activation at all other measured time points was observed. A similar pattern was observed for the CD4⁺ Th and CD8⁺ Tc cells, shown in Figs. 9M and 90.

[0083] In the spleen (Figs. 9L, 9N, 9P, and 9R), total T cells were significantly increased only in the group receiving liposome-entrapped TNF-α at the 18 hour time point compared to the animals treated with free TNF-α (Fig. 9L). However, the total number of T cells expressing CD71 (CD37CD71⁺) was significantly increased at six hours following free TNF-α treatment, and was slightly (non- significantly) elevated in the liposome-entrapped TNF-α group at 18 hours (Fig. 9R). The T-helper cells in the spleen followed a similar pattern to the T-helper cells in the blood, as seen by comparing Figs. 9N and 9M. Splenic Tc cell counts, shown in Fig. 9P, were significantly elevated in the free TNF-α group at 36 hours post administration relative to the group treated with liposome-entrapped TNF-α. [0084] The data presented in Figs. 5-9 shows that entrapping a TNF compound in liposomes having a coating of hydrophilic polymer chains achieves at least the following, relative to the toxicity of a TNF compound in free form: (1 ) a decreased toxicity (Figs 6A-6D); (2) an elevated leukocyte count (Fig. 7A); (3) an increased proliferation rate of spleenocytes (Fig. 7D); (4) increased monocyte and granulocyte counts in the spleen (Figs. 8C-8D); (5) activation of lymphocytes at a time different than that observed by the free form of the compound (Figs 9A-9F); (6) sustained increased level of natural killer cells in the blood and spleen (Figs. 9G-9H); (7) alteration of spleen B cell counts (Fig. 9F); and (8) increased T cell counts in the blood and spleen at 20 hours post administration (Figs. 9K, 9L, 9M). [0085] More generally, the data presented in Figs. 5-9 show that TNF-α when administered in liposome-entrapped form achieves temporal and spatial immunological alterations when compared to that achieved by administration of TNF-α in free form. Administration of free TNF-α produced, in most leukocyte and lymphocyte populations, an increase in the population counts at six hours and a return to baseline counts or a decrease of the counts by 36 hours, in both blood and spleen. Any increase in cell numbers achieved by administration of TNF-α in free form generally did not correlate with greater spontaneous proliferation or an increase in activation markers. In contrast, animals treated with liposome- entrapped a TNF compound had leukocyte numbers and certain lymphocyte population numbers different from that observed for animals treated with free TNF- α, particularly at 18 hours after injection. In many cell populations, the liposome- entrapped TNF-α treatment group had significantly lower cell populations than all the other groups. Furthermore, the spontaneous proliferation and activation of these cells correlated well with the increase in number.

[0086] In summary, the data shows that administration of a TNF compound in the form of a long circulating liposome achieves upregulation of various immunological factors. This upregulation contributes to or augments any direct, non-immunological antitumor effects of TNF-α administered in free form. The increased blood residence time and tissue localization offered by long-circulating liposomes containing TNF-α alters immunological parameters relative to free TNF- α and contributes to an increased efficacy and decreased toxicity of the compound. Therefore, entrapping the TNF compound in liposomes having a long blood circulation time by virtue of a hydrophilic polymer coating increases the anti- tumor effectiveness of the TNF compound by increasing the accumulation of the TNF compound at the tumor site itself, and additionally contributes, by a directed and productive immune upregulation, to augment any direct TNF effect on the tumor or its immediate environment.

IV. Method of Use

[0087] In another aspect of the invention, the liposome-entrapped TNF compound is administered to achieve a sustained stimulation of immune cells to induce upregulation of immunological factors for improved therapy. As described above, administration of a TNF compound entrapped in liposomes having a surface coating of a hydrophilic polymer results in an upregulation of leukocyte and/or lymphocyte cell production for up to 18 hours post administration of the liposome-entrapped TNF compound. Despite the sustained stimulation of immune cells, the toxicity associated with the TNF compound is avoided. Accordingly, a method of prolonging the upregulation of immune cells, such as lymphocytes and leukocytes, is provided by administering to a subject suffering from a condition responsive to a TNF compound, a liposome-entrapped TNF compound. Upregulation of certain immunological factors is beneficial in the treatment of various cancers, such as solid tumors. For example, radiation treatment of tumors causes a drastic change in immune cell populations (Kajioka, et al., In Vivo, 13:525-33 (1999); Kajioka, et al., Radiat Res., 153:587-94 (2000), since natural killer (NK) cells are relatively radio-resistant but CD8⁺ cells, some of which are immunosuppressive, are moderately radiosensitive. Administration of, for example, TNF-α entrapped in liposomes causes an upregulation of NK cells in the spleen 20 hours after administration (see Fig. 9H). Thus, if radiation is applied after 24 hours, the sustained upregulation of NK cells may be further enhanced not only by its increase in number but also by the decrease in immunosuppressive cells.

[0088] In another aspect, the invention includes a method of inhibiting tumor growth in a subject. In this method, a TNF compound entrapped in liposomes is administered to a subject afflicted with a solid tumor. As noted above, the liposome-entrapped TNF compound is effective to upregulate one or more specific immunological factors. After administration of the liposome-entrapped TNF compound, a certain amount of time is allowed to pass, a so-called "waiting period", after which a conventional tumor treatment modality is initiated. The waiting period, or amount of time allowed to lapse, will vary according to the time suitable for upregulation of a desired immunological factor. Preferably, the liposome composition is administered to the subject about 10-40 hours, more preferably between about 15-40 hours, still more preferably between about 18-36 hours prior to initiating a conventional therapy, to induce an upregulation of immunological factors. In a most preferred embodiment, the liposome-entrapped TNF compound is administered approximately about 24 hours prior to a conventional tumor treatment therapy.

[0089] After the "waiting period", that after the time period post administration of the liposome-entrapped TNF compound while upregulation of one or more immunological factors is occurring, but during the time of upregulation of the one or more factors, the conventional tumor treatment modality is administered. Exemplary conventional tumor treatment modalities but are not limited to ionizing radiation and administration of chemotherapeutic drugs, such as doxorubicin, daunorubicin, cisplatin, paclitaxel. It will be appreciated that the chemotherapeutic agent can be in free form or in liposome-entrapped form. [0090] With respect to a dosage suitable for therapy, the studies described herein were performed with an internal liposomal concentration of TNF-α of about 25 μg TNF-α/100 μmol liposome. In Example 4, in a treatment against human colon cancer cell in a mouse model, TNF-α was administered at a dose of 30 μg in 120 μmol of liposome per kilogram body weight of the mouse and was effective in inducing upregulation of various immunological factors. The dosages may be adjusted to suit other models, e.g. human, and such an adjusted dosage can be readily determined by one of skill in the art. It will be appreciated that the liposome-entrapped TNF compound can be administered by any suitable mode of administration, including but not limited to injection (intravenous, subcutanous, intramuscular), oral, nasal, inhalation, buccal, and the like.

V. Examples

[0091] The following examples are intended to illustrate, but not limit, the scope of the invention.

Materials [0092] Male athymic nude mice were purchased from Charles River Breeding Laboratories, Inc. (Wilmington, MA). Recombinant human TNF-α protein was obtained from Boehringer, GmbH (Ingelheim, Germany) and encapsulated in STEALTH^® liposomes at AL2A Corporation (Mountain View, CA) using DSPE purchased from Avanti Polar Lipids (Birmingham, AL), methoxy-polyethyleneglycol (mPEG), MW 2000 Daltons, obtained from Fluka Chemie AG (Buchs, Switzerland), cholesterol obtained from Croda, Inc., (NY, NY) and HSPC made by Lipoid K.G. (Ludwigshafen, Germany) and mPEG-DSPE made by Sygena, Inc., (Liestal, Switzerland). Example 1 Preparation of PEG-Derivatized DSPE linked by Cyanuric Chloride

A. Preparation of Activated PEG [0093] 2-0-Methoxypolyethylene glycol 1900-4,6-dichloro-1 ,3,5 triazine previously called activated PEG was prepared as described in J. Biol. Chem. 252:3582 (1977) with the following modifications. [0094] Cyanuric chloride (5.5 g; 0.03 mol) was dissolved in 400 ml of anhydrous benzene containing 10 g of anhydrous sodium carbonate, and PEG- 1900 (19 g; 0.01 mol) was added and the mixture was stirred overnight at room temperature. The solution was filtered, and 600 ml of petroleum ether (boiling range, 35-60°C) was added slowly with stirring. The finely divided precipitate was collected on a filter and redissolved in 400 ml of benzene. The precipitation and filtration process was repeated several times until the petroleum ether was free of residual cyanuric chloride as determined by high pressure liquid chromatography on a column (250 x 3.2 mm) of 5-m "LiChrosorb" (E. Merck), developed with hexane, and detected with an ultraviolet detector. Titration of activated PEG-1900 with silver nitrate after overnight hydrolysis in aqueous buffer at pH 10.0, room temperature, gave a value of 1.7 mol of chloride liberated/mol of PEG. [0095] TLC analysis of the product was effected with TLC reversed-phase plates obtained from Baker using methanol: water, 4:1 (v/v) as developer and exposure to iodine vapor for visualization. Under these conditions, the starting methoxy polyglycol 1900 appeared at R,=0.54 to 0.60. The activated PEG appeared at R_f=0.41. Unreacted cyanuric chloride appeared at R_τ=0.88 and was removed.

[0096] The activated PEG was analyzed for nitrogen and an appropriate correction was applied in selecting the quantity of reactant to use in further synthetic steps. Thus, when the product contained only 20% of the theoretical amount of nitrogen, the quantity of material used in the next synthetic step was increased by 100/20, or 5-fold. When the product contained 50% of the theoretical amount of nitrogen, only 100/50 or a 2-fold increase was needed. B. Preparation of N-(4-Chloro-polyglycol 1900)- 1 ,3,5-Triazinyl Egg Phosphatidylethanolamine

[0097] In a screw-capped test tube, 0.74 ml of a 100 mg/ml (0.100 mmole) stock solution of egg phosphatidylethanolamine in chloroform was evaporated to dryness under a stream of nitrogen and was added to the residue of the activated PEG described in section A, in the amount to provide 205 mg (0.100 mmole). To this mixture, 5 ml anhydrous dimethyl formamide was added. 27 microliters (0.200 mmole) triethylamine was added to the mixture, and the air was displaced with nitrogen gas. The mixture was heated overnight in a sand bath maintained at 110°C.

[0098] The mixture was then evaporated to dryness under vacuum and a pasty mass of crystalline solid was obtained. This solid was dissolved in 5 ml of a mixture of 4 volumes of acetone and 1 volume of acetic acid. The resulting mixture was placed at the top of a 21 mm x 240 mm chromatographic absorption column packed with silica gel (Merck Kieselgel 60, 70-230 mesh) which had first been moistened with a solvent composed of acetone acetic acid, 80/20; v/v. [0099] The column chromatography was developed with the same solvent mixture, and separate 20 to 50 ml aliquots of eluent were collected. Each portion of eluent was assayed by TLC on silica gel coated plates, using 2-butanone/acetic acid/water; 40/25/5; v/v/v as developer and iodine vapor exposure for visualization. Fractions containing only material of R_f=about 0.79 were combined and evaporated to dryness under vacuum. Drying to constant weight under high vacuum afforded 86 mg (31.2 micromoles) of nearly colorless solid N-(4-chloro- polyglycol 1900)-1 ,3,5-triazinyl egg phosphatidylethanolamine containing phosphorous.

[0100] The solid compound was taken up in 24 ml of ethanol/chloroform; 50/50 and centrifuged to remove insoluble material. Evaporation of the clarified solution to dryness under vacuum afforded 21 mg (7.62 micromoles) of colorless solid.

Example 2 Preparation of PEG-Derivatized DSPE linked by Carbamate

A. Preparation of the Imidazole Carbamate of Polyethylene Glycol Methyl Ether 1900

[0101] 9.5 grams (5 mmoles) of polyethylene glycol methyl ether 1900 obtained from Aldrich Chemical Co. was dissolved in 45 ml benzene which had been dried over molecular sieves. 0.89 grams (5.5 mmoles) of pure carbonyl diimidazole was added. The purity was checked by an infra-red spectrum. The air in the reaction vessel was displaced with nitrogen. Vessel was enclosed and heated in a sand bath at 75°C for 16 hours.

[0102] The reaction mixture was cooled and a clear solution formed at room temperature. The solution was diluted to 50.0 ml with dry benzene and stored in the refrigerator as a 100 micromole/ml stock solution of the imidazole carbamate of PEG ether 1900.

B. Preparation of the Phosphatidylethanolamine Carbamate of Polyethylene Glycol Methyl Ether 1900

[0103] 10.0 ml (1 mmol) of the 100 mmol/ml stock solution of the imidazole carbamate of polyethylene glycol methyl ether 1900 was pipetted into a 10 ml pear-shaped flask. The solvent was removed under vacuum. 3.7 ml of a 100 mg/ml solution of egg phosphatidyl ethanolamine in chloroform (0.5 mmol) was added. The solvent was evaporated under vacuum. 2 ml of 1 ,1 ,2,2- tetrachloroethylene and 139 microliters (1.0 mmol) of triethylamine was added. The vessel was closed and heated in a sand bath maintained at 95°C for 6 hours. At this time, thin-layer chromatography was performed with fractions of the above mixture to determine the extent of conjugation on Si0₂ coated TLC plates, using butanone/acetic acid/water; 40/5/5; v/v/v; as developer. Iodine vapor visualization revealed that most of the free phosphatidylethanolamine of Rf=0.68, had reacted, and was replaced by a phosphorous-containing lipid at R_f=0.78 to 0.80. [0104] The solvent from the remaining reaction mixture was evaporated under vacuum. The residue was taken up in 10 ml methylene chloride and placed at the top of a 21 mm x 270 mm chromatographic absorption column packed with Merck Kieselgel 60 (70-230 mesh silica gel), which has been first rinsed with methylene chloride. The mixture was passed through the column, in sequence, using the following solvents.

[0105] 50 ml portions of eluent were collected and each portion was assayed by TLC on Si0₂ - coated plates, using l₂ 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 phosphates were found in fractions 11 , 12, 13 and 14.

[0106] These fractions were combined, evaporated to dryness under vacuum and dried in high vacuum to constant weight. They yielded 669 mg of a colorless wax of phosphatidyl ethanolamine carbamate of polyethylene glycol methyl ether. This represented 263 micromoles and a yield of 52.6% based on the phosphatidylethanolamine.

[0107] 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 methylene 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 of about 2000 for the polyethylene oxide portion of the molecule of the desired product polyethylene glycol conjugated phosphatidylethanolamine carbamate, M.W. 2,654.

Example 3 Liposome Preparation [0108] Liposomes were prepared according to standard procedures by dissolving in chloroform hydrogenated soy phosphatidylcholine (HSPC), cholesterol (Choi) and PEG₂₀oo derivatized distearoylphosphatidyl-ethanolamine, prepared as in Example 1 or 2, in about a 2:1 :0.01 molar ratio. The lipids were dried as a thin film by rotation under reduced pressure. The lipid film was hydrated by addition of an aqueous phase to form liposomes which were sized by sonication or by sequential extrusion through Nucleopore polycarbonate membranes with pore sizes of 0.4 μm, 0.2 μm, 0.1 μm, and 0.05 μm to obtain liposomes of 100-150 nm in size.

Example 4 Treatment of Tumor-Bearing Mice [0109] Liposomes containing entrapped TNF were prepared as described in Example 3 and were composed of HSPC, cholesterol and mPEG-DSPE in a 50.6/44.3/5/1 molar ratio. The TNF-α concentration was approximately 25 μg TNF-α/100 μmol liposome.

A. Human LS174T Adenocarcinoma Mice Model.

[0110] The human LS174T colon adenocarcinoma cell line (American Type Culture Collection, Rockville, MD), originally established by Tom, B.H. et al., In Vitro, 12:180, 1976, were cultured in complete RPMI-1640 medium (Sigma Chemical Co., St. Louise, MO) containing 10% bovine calf serum (BCS; Hyclone Laboratories, Logan, UT), 12 mM hepes buffer (Mediatech Inc., Herndon, VA), and 1 % antibiotic antimycotic solution (10,000 unit/ml penicillin, 10 mg/ml streptomycin, and 25 μg/ml amphotericin B; Sigma). The cells were harvested with 0.25% trypsin, washed, and adjusted to 2.5 x 10⁷/ml in phospate buffered saline (PBS). [0111] Male athymic nude mice (outbred background nu/nu; n=128) were purchased from Charles River Breeding Laboratories, Inc., Wilmington, MA as 6- week-old weanlings. The animals were acclimated for 2 weeks in microisolator cages (4-7 mice/cage) under conditions appropriate for immuno-compromised rodents. The mice were weighted twice each week, observed for signs of treatment-related toxicities, and euthanized with CO₂ as recommended by the American Veterinary Medical Association (AVAMA) Panel on Euthanasia. B. Treatment Protocol

[0112] At the time of tumor cell implantation, the mice were assigned to treatment groups (n=4 mice for non-tumor control groups; n=7 mice for each treatment group at each time point) as follows: (1 ) non-tumor control - PBS; (2) tumor control - PBS; (3) free TNF-α; (4) placebo liposomes; and (5) liposome- entrapped TNF-α. With the exception of the non-tumor control group, the mice in all other groups were injected subcutaneously in the right thigh with 5 x 10⁶ human LS174T colon adenocarcinoma tumor cells /0.2 ml.

[0113] At 11 days post-implantation, when the average volume of the tumors reached -200 mm³, the mice were injected i.v. into the heat-dilated tail vein with PBS, free TNF-α, placebo liposome, or liposome-entrapped TNF-α in the appropriate test groups. The free TNF-α group was administered a dose of 30 μg, the liposome-entrapped TNF-α group was administered 30 μg entrapped in 120 μmol of liposome, and the placebo liposome group was administered at 120 μmol of placebo liposomes, per kilogram body weight of the mice. A subset from these groups was euthanized at 6, 18, 36, and 96 hours following treatment. Blood, tumor, and selected organs (spleen, liver, kidney) were assayed for relative spleen weights, splenic leukocyte counts, and mitogen-induced blastogenesis; blood leukocyte, erythrocyte, and thrombocyte enumeration; spontaneous blastogenesis of leukocytes in blood and spleen; lymphocyte populations in blood and spleen, and the presence of recombinant human TNF-α in tumor, liver, and plasma.

C. Analytical methods.

[0114] 1. Relative spleen weights, splenic leukocyte counts, and mitogen- induced blastogenesis. Spleens were weighed at the time of euthanasia and relative spleen weight (RSW) values were calculated as follows: RSW = [spleen weight (g) x 10⁴] / body weight (g). Spleens were then aseptically dispersed into single-celled suspensions, filtered, washed, and centrifuged. After red blood cells (RBC) removal with cold lysing buffer, the remaining viable leukocytes were washed, suspended in 2 ml of medium, and counted using the Vet ABC-Diff Hematology Analyzer (HESKA Corporation, Waukesha, Wl). To quantify responsiveness to mitogen, the cells were adjusted to 2 x 10⁶/ml in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone), antibiotics, and mercaptoethanol. Aliquots (100 μl) of each cell suspension were dispensed in triplicate into 96-well microtiter plates and lipopolysaccharide (LPS; Sigma) from E. coli serotype 0111 :134 was added at 0.02 μg/100 μl/well; control wells received 100 μl medium with no mitogen. [³H]-thymidine ([³H]-TdR; specific activity = 46 Ci/μmol; ICN Radiochemicals, Irvine, CA) was added at 1 μCi/50 μl/well for the final 4 hr of a 48 hr incubation at 37°C in 5% CO₂. The cells were harvested and the amount of incorporated radioactivity (counts per minute; cpm) was quantified in a beta scintillation counter. The results were expressed as a stimulation index (SI): SI = (cpm with LPS - cpm without LPS)/cpm without LPS. [0115] 2. Blood leukocyte, erythrocyte, and thrombocyte enumeration. Samples of whole blood (12 μl), collected in K₂EDTA-containing syringes at the time of euthanasia, were evaluated using the Vet ABC-Diff Hematology Analyzer (Heska Corp.). The measurements included leukocytes counts (WBC), lymphocyte, monocyte, and granulocyte counts and percentages, red blood cell (RBC) and thrombocyte counts, hemoglobin concentration, hematocrit (percentage of whole blood composed of RBC), mean corpuscular volume (MCV; volume of an average RBC), mean corpuscular hemoglobin (MCH; amount of hemoglobin in an average RBC), and mean corpuscular hemoglobin concentration (MCHC; concentration of hemoglobin in the RBC component of blood). [0116] 3. Spontaneous blastogenesis of leukocytes in blood and spleen. To assay basal DNA synthesis, 50 μl of spleen leukocyte and whole blood samples were diluted with 150 μl of complete RPMI 1640 medium and 1 μCi [³H]-TdR in 50 μl of medium was immediately added. Each mouse was tested in triplicate in 96- well microculture plates. The cells were incubated for a total of 3 hr at 37°C in 5% CO₂, and the uptake of radioactivity was quantified as described above for the mitogen-induced blastogenesis assay. The leukocyte counts and the tested volume were used to convert the raw cpm into cpm/10⁶ leukocytes. [0117] 4. Flow cytometry analysis of lymphocyte populations in blood and spleen. Lymphocyte populations were quantified using standard direct-staining techniques and a FACSCalibur™ 4-channel flow cytometer (Becton Dickinson, Inc., Rutherford, NJ). The CD3⁺ T, CD4⁺ T helper (Th), CD8⁺ T cytotoxic (Tc), CD19⁺ B, and pan-NK⁺ natural killer (NK) cells were identified using 4-color, 2-tube mixtures of fluorescence-labeled monoclonal antibodies (MAb). Additional mixtures of the appropriate MAb were used to identify lymphocytes expressing CD25 and CD71 activation markers. The MAb (Pharmingen, San Diego, CA) were labeled with fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), allophycocyanin (APC) or peridinin chlorophyll protein (PerCP). Analysis of 10,000 lymphocyte events/sample was performed using CellQuest™ software version 3.1 (Becton Dickinson). The number of cells in each population was based on the total leukocyte count ml and the percentage of cells expressing the marker(s) of interest.

[0118] 5. Detection of recombinant human TNF-α. Tumor, liver, and plasma from each mouse were collected at euthanasia and stored at -70°C until immediately before assaying for cytokines. One to three representative tumors and livers were each weighed before processing. Frozen tissues were cut into small pieces and further dispersed in PBS. After centrifugation at 400 g for 10 min, supernatants were collected and volumes were measured. Thawed plasma from mice within each group was pooled. All samples were then assayed for cytokine content in enzyme-linked immunosorbent assays (ELISA). Quantikine™ High Sensitivity Human TNF-α Immunoassay kits were purchased from R&D Systems Inc. (Minneapolis, MN) and the tests were performed according to the manufacturers' instructions. The optical density of each sample was determined using a microplate reader set at 490 nm with a 620 nm wavelength correction. TNF-α concentration was calculated using the optical density and the generated standard curves. The total amount in plasma was expressed directly as pg/ml; for supernatants from tissues the following formula was used: pg/mg of tissue = [pg/ml x total volume of supernatant (ml)] ÷ tissue weight (mg).

D. Data Analysis. [0119] Data from the treated group was normalized to more readily identify any variations due exclusively to treatment and to minimize any immunomodulatory effects that may be due to tumor presence. Normalization was accomplished by first assigning a value of 1 to the means obtained in each assay for the control mice with tumor (injected i.v. with PBS). Values from tumor-bearing animals treated with SL-TNF-α, SL, or free TNF-α were then expressed in relation to the mean of the tumor control group from each assay. The control mice with no tumor were included in this study to ensure that assays were performed with no or minimal technical or other errors.

[0120] The data were subjected to one-way and two-way analysis of variance (ANOVA) and Tukey's pair-wise multiple comparison test using SigmaStat™ software version 2.03 (SPSS Inc., San Rafael, CA). Outlying values (greater than ± two standard deviations from the mean) were exclude from analysis. Differences giving a p value of <0.05 were considered significant.

E. Results [0121] The mean values of various assays of the tumor control group are listed in Tables 1 and 2 below. The assay results of the other treatment groups relative to the tumor control group results are plotted in Figs. 5 to 9.

TABLE 1 Blood parameters of the control group with tumor.

TABLE 2 Spleen parameters of the control group with tumor.

[0122] From the foregoing, it can be appreciated how various features and objects of the invention are met. The liposomal compositions of the present invention include liposomes having an entrapped TNF compound. TNF compounds entrapped in liposomes offer the advantages of reduced toxicity and of sustained stimulation of immune cells, relative to the that observed when the TNF compound is administered in free form. In addition, a liposome-entrapped TNF compound alters immunological factors differently from that observed from administration of free TNF-α and achieves enhanced a ntitumor effects, particularly when used in combination with other conventional antitumor therapies. [0123] Although the invention has been described with respect to particular embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the invention.

Claims

IT IS CLAIMED:

1. A liposomal composition containing an entrapped TNF compound, comprising liposomes comprised of a vesicle-forming lipid and between about 1-20 mole percent of a vesicle-forming lipid derivatized with a hydrophilic polymer, and a TNF compound entrapped in said liposomes.

2. The composition according to claim 1 , wherein said hydrophilic polymer is selected from the group consisting of polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol, and polyaspartamide.

3. The composition according to claim 1 , wherein said hydrophilic polymer is polyethylene glycol.

4. The composition according to claim 1 , wherein said vesicle forming lipid is phosphatidylethanolamine and said derivatized vesicle forming lipid is distearoylphosphatidylethanolamine derivatized with polyethylene glycol.

5. The composition according to claim 1 , wherein said vesicle forming lipid is hydrogenated soy phosphatidylcholine and said derivatized vesicle forming lipid is distearoylphosphatidylethanolamine derivatized with polyethylene glycol.

6. The composition according to any of the preceding claims, wherein said TNF compound is TNF-alpha or TNF-beta.

7. The composition according to any one of claims 1-5, wherein said TNF compound is TNF-alpha entrapped at a drug-to-liposome ratio of approximately 25 μg TNF-alpha to 100 μmole of liposome.

8. A composition for use in inducing upregulation of an immunological factor, comprising liposomes according to any one of claims 1-7.

9. The composition according to claim 8, wherein said immunological factor is an immunological factor in the blood and is selected from the group consisting of leukocyte, granulocyte, monocyte, and natural killer cells.

10. The composition according to claim 8, wherein said immunological factor is an immunological factor in the spleen and is selected from the group consisting of basal leukocyte, leukocyte, T cells, natural killer cells, and CD25⁺ activation marker.

11. A composition for use in inhibiting tumor growth, comprising liposomes according to any one of claims 1-7, in combination with a tumor treatment modality.

12. The composition according to claim 11 , wherein said liposomes are administered between about 10 to about 40 hours prior to initiation of said tumor treatment modality.

13. The composition according to claim 11 , wherein said liposomes are administered between about 24 hours prior to initiation of said tumor treatment modality.

14. The composition according to claim 11 , wherein said liposomes are effective to achieve an increase in at least one cell population in the blood selected from leukocyte, granulocyte, monocyte, and natural killer cells, and said tumor treatment modality is initiated during the increase in said at least one cell population.

15. The composition according to claim 11 , liposomes are effective to achieve an increase in at least one cell population in the spleen selected from basal leukocyte, leukocyte, T cell, natural killer cells, and CD25⁺ activation marker expression, and said tumor treatment modality is initiated during the increase in said at least one cell population.

16. The composition according to claim 11 , wherein said tumor treatment modality is ionizing radiation.

17. The composition according to claim 11 , wherein said tumor treatment modality is a chemotherapeutic agent.

18. The composition according to claim 17, wherein said chemotherapeutic agent is doxorubicin, cisplatin, or paclitaxel.

19. The composition according to claim 17, wherein said chemotherapeutic agent is entrapped in a liposome.

20. Use of a liposome composition according to any one of claims 1-7 for the manufacture of a medicament for treatment of a tumor, said liposome composition being administered prior to treatment with another tumor treatment modality.