MXPA01003796A - Liposome-entrapped topoisomerase inhibitors - Google Patents

Abstract

A composition for administration of a therapeutically effective dose of a topoisomerase inhibitor I or topoisomerase I/II inhibitor is described. The composition includes liposomes having an outer surface and an inner surface defining aqueous liposome compartment, and being composed of a vesicle-forming lipid and of a vesicle-forming lipid derivatized with a hydrophilic polymer to form a coating of hydrophilic polymer chains on both the inner and outer surfaces of the liposomes. Entrapped in the liposomes is the topoisomerase inhibitor at a concentration of at least about 0.10&mgr;mole drug per&mgr;mole lipid.

Description

INHIBITORS OF TOPOISOMERASE ENTRAMPADA IN LIPOSOMAS FIELD OF THE INVENTION The present invention relates to a liposome composition having a trapped topoisomerase inhibitor.

BACKGROUND OF THE INVENTION After heart disease, cancer is the leading cause of death in the United States, causing approximately 500,000 deaths annually (Katzung, B., "Basic and Clinical Pharmacology", 7th Edition, Appleton &Lange, Stamford CT, 1988, p.882). With treatment methods present, one third of patients are cured with local measurements, such as surgery or radiation therapy, which are quite effective when the tumor has not metastasized by the time of treatment. Early diagnosis can lead to increased cure of patients undergoing such local treatments. However, in many cases, early micrometastasis is an essential feature of the neoplasm, which indicates that a systemic approach such as chemotherapy, often together with a local treatment method, for effective cancer administration may be required. Cancer chemotherapy may be curative in some disseminated neoplasms that have undergone massive or microscopic dissemination by the time of diagnosis. These include testicular cancer, diffuse large cell lymphoma, Hodgkm's disease, and cocpocarcinoma as well as childhood tumors such as acute nonoblastic leukemia. For other forms of disseminated cancer, chemotherapy provides a palliative rather than curative therapy. Effective curative therapy It results in a temporary elimination of symptoms and signs of cancer and prolongation of life. The advances in cancer therapy have recently provided evidence that chemical control is possible for a certain number of patients. Cancers A category of drugs used for cancer therapy are topoisomerase inhibitors. These compounds inhibit the action of topoisomerase enzymes which play a role in replication, repair, genetic recombination and transcription of DNA. of a topoisomerase inhibitor is the camotecin, a natural compound that interferes with n the activity of topoisomerase I, an enzyme involved in DNA replication and RNA transcription. Cam ptothecin and the camotechin analogs, topotecan and ipnotecan, are suitable for clinical use. Camptotecma and its analogs are effective in cancer chemotherapy by interfering with breakage / fusion actions of topoisomerase I Compounds stabilize and form a ternary complex of reversible DNA-coding ptothecin-cam which prevents the fusion step of the breakage / binding cycle of the topoisomerase reaction A problem with camotec is its insolubility to water, which inhibits the supply of the drug. Numerous camptothem analogues have been prepared to improve the water solubility of the compound. Another problem with camptotecma and its analogues is that the compounds are susceptible. in aqueous environments for hydrolysis in the a-hydroxy lactone ring The lactone ring opens to the carboxylate form of the dr oga, a form exhibiting little activity against topoisomerase I Various approaches have been described to improve the stability of camptothecin and its analogues One approach has been to entrap the compounds in Burke liposomes (US Patent No. 5,552,156) discloses a liposome composition intended for to overcome the instability of camptothecin and its analogs by entrapping the compounds in liposomes having a double bilayer membrane which allows the compound to penetrate, or intercalate, into the lipid bilayer With the compound intercalated in the bilayer membrane, it is extracted from the aqueous environment in the liposome nucleus and is consequently protected from hydrolysis. A problem with this approach is that liposomes are rapidly extracted from the bloodstream by reticuloendothelial systems (RES), avoiding the supply, and preferably the accumulation, in the Subramanian and Muller tumor site (Oncology Research, 7 (9) 461-469 (1995)) describe a formulation of topotecan liposomes and report that in the entrapped form of liposomes, the topotecan is stabilized from inactivation by hydrolysis of the lactone ring. However, the biological activity of the drug is affected by hposomes m vitro has only 60% of the activity of the free drug Lundberg (Anti-Cancer Drug Design, 1 3 453 (1 998)) describes two derivatives of oleic acid ester of camotecma analogues which are trapped in liposomes and interspersed in the bilayer for the stabilization of the lactone ring Daoud (Anti-Cancer Drugs, 6 83-93 (1995)) describes a composition of the iposomes that is used in the treatment of pheocetoma, where the drug is also intercalated the liposomes liposomes both in these two references are prepared conventionally, where the drug is passively entrapped in the liposomes to separate the drug in the membrane of bilayer lipid to its stable. Using this method of preparation, it is difficult to achieve a sufficient drug load in the posomes for clinical efficacy According to the above, there is still a need in the art for a formulation of liposomes which (i) includes an inhibitor of topoisomerase, such as camptothecin and its analogues, (n) permutes in the bloodstream for a prolonged period of time, (n) retains antitumor activity, and (iv) includes a sufficient drug load for clinical relevance.

BRIEF DESCRIPTION OF THE INVENTION According to the foregoing, it is an object of the present invention to provide a topoisomerase inhibitor composition for improved cancer therapy. It is another object of the invention to provide a liposome composition for the administration of a Topoisomerase inhibitor for antitumor therapy In one aspect, the invention includes a composition for treating a tumor in a patient, comprising liposomes composed of a vesicle-forming lipid and between about 1-20 mole percent of a lipid-forming lipid. vesicle derived with a hydrophilic polymer Liposomes are formed under conditions that distribute the polymer on both sides of the bilayer membranes of the liposomes Trapped in the hposomes is a topoisomerase I inhibitor or a topoisomerase I / II inhibitor at a concentration of less about 010 μmol of drug per μmol of lipid The liposomes ti have an inner / outer ion gradient sufficient to maintain the topoisomerase I inhibitor or topoisomerase I / ll inhibitor within the liposomes at the specified concentration. In one embodiment, the topoisomerase inhibitor is a topoisomerase I inhibitor selected from the group which consists of camptothecin and camptothecin derivatives. For example, the camptothecin derivative may be 9-am? nocamptotec? na, 7-ethylcamptotecma, 10-hydro? camptotec? na, 9-n? trocamptotec? na, 10.11 -methylenedioxicamptothecin, 9-am? no-10,11-met? lend? ox? camptotec? na, or 9-chloro-10,11-met? lend? ox? camptotec? na In other embodiments, the camptothecin derivatives are ipnotecan, topotecan, (7- (4-met? lp? peraz? nomet? len) -10,11-et? lend? ox? -20 (S) -camptotec? na, 7- (4-met? lp? peraz? nomet? len) -10,11-met? lend? ox? -20 (S) -camptotec? na or 7- (2- (N-? soprop? lam? no) et? l) - (20S) -camptotecna In another embodiment, the topoisomerase inhibitor is an inhibitor of topoisomerase I / II inhibitor, such as dichloride of 6 - [[2- (d? met? lam? no) -et? l] am? no] -3-h? drox? -7H-? ndeno [2,1-c] qu? nol? n- 7-on, azotoxm or 3-rnetox? -11H-p? R? Do [3 ', 4'-4,5] p? Rrolo [3,2-c] qu? Nol? N-1,4-d ? ona The hydrophilic polymer included in the composition of hposomes can be polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazole, polyhydroxypropylxazoline, polyhydroxypropylmethacrylamide, polymetamide, polydimethylampramide, polyhydroxypropylmethacrylate, polyhydroxyethylplate, hydroxymethylcellulose, hydroxyethylcellulose, polyethylene glycol and pohaspartamide., the hydrophilic polymer is polyethylene glycol having a molecular weight between 500-5,000 daltons In yet another embodiment, the liposomes further include a vesicle forming lipid having a phase transition temperature at about 37 ° C. In yet another embodiment, the vesicle-forming lipid is hydrogenated soy phosphatidylcholine, distearoyl phosphatidylcholine, or sphingomyelin A preferred liposome composition is comprised of 20-94 mole percent hydrogenated soy phosphatidylcholine, 1-20 percent distearoyl phosphatidylcholine derivative with polyethylene glycol and 5-60 mole percent cholesterol Another preferred composition is 30-65 mole percent of hydrogenated soy phosphatidylcholine, 5-20 mole percent of distearoyl phosphatidylcholine derived with polyethylene glycol and 30-50 percent by weight. 100 mole of cholesterol In another aspect, the invention includes a composition for the administration of an inh Topoisomerase I or a topoisomerase I / II inhibitor, which comprises liposomes composed of vesicle formation lipids and which have an effective interior / exterior ion gradient to maintain the drug within the liposomes. liposomes, the topoisomerase I inhibitor or the topoisomerase I / ll inhibitor is found at a concentration of at least about 20 μmol of drug per μmol of 11. In another aspect, the invention includes a method for treating a thy mor in a patient , which comprises preparing liposomes composed of vesicle formation lipids that include between 1-20% mol of a vesicle-forming lipid derivative with a hydrophobic polymer chain, the liposomes being formed under conditions that distribute the polymer on both sides of the bilayer mem branes of the liposomes The liposomes contain a topoisomerase I inhibitor or a topoisomerase I / II inhibitor in a At a concentration of at least about 0.1 mole per μmole, the liposomes have a sufficient inner / outer ion gradient to maintain the topoisomerase I inhibitor or topoisomerase I / II inhibitor within the hposome at the specified concentration. Liposomes are administered. then to the patient In an embodiment of this aspect, the method further includes entrapping the topoisomerase I inhibitor or topoisomerase I / II inhibitor in the liposomes by remote loading, for example, through a gradient of ammonia sulfate. These and other objectives of the invention will be more fully appreciated when reading the following detailed description of the invention in conjunction with the accompanying drawings BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A is a graphic representation of the duration of blood circulation of trapped MPE-camptothecin from liposomes (solid circles), taken as the percentage of dose injected as a function of time, compared to the free form of the drug (solid squares), Figure 1B shows the blood concentration of the MPE-camptothecin, as a function of time, in hours, after the administration of the trapped MPE-camptothecin of liposomes (solid circles) and free MPE-camptothecin (non-hposomal) (solid squares) to rats, Figure 2A is a graphic representation showing the body weight of the mice, in grams, based on the days after tumor inoculation with a HT29 colon tumor. The animals were treated. on days 10, 16, and 23 after tumor inoculation with encapsulated MPE-camptothecin liposomes at a dose of 24 mg / kg (closed circles), 15 mg / kg (triangles rrados) and 6 mg / kg (closed squares) and free MPE-camptotecma in doses of 24 mg / kg (open circles), 15 mg / kg (open triangles) and 6 mg / kg (open squares), Figure 2B is a graphic representation showing the tumor volume, in mm3, as a function of the days after inoculation with a HT29 colon tumor. The animals were treated on days 10, 16 and 23 after the tumor inoculation with trapped MPE-ca ptotecin. of liposomes in a dose of 24 mg / kg (closed circles), 15 mg / kg (closed triangles), and 6 mg / kg (closed squares) and with a free drug in a dose of 24 mg / kg (open circles), 15 mg / kg (open triangles) and 6 mg / kg (open squares), Figure 3A is a graphic representation showing the body weight of the mice, in grams, as a function of the days after the tumor inoculation with a colon tumor HT29 Animals were treated on days 9, 16, and 23 after tumor inoculation with MPE-camptothecin trapped liposomes in doses of 5 mg / kg (open triangles), 3 mg / kg (inverted open triangles), 1 mg / kg (open diamonds), 05 mg / kg (open circles) and 01 mg / kg (open squares) ) and with free MPE-camptothecin in a dose of 20 mg / kg (closed squares), Figure 3B is a graphic representation showing the tumor volume, in mm3, as a function of the days after the tumor inoculation with a tumor of HT29 colon Animals were treated on days 9, 16, and 23 after tumor inoculation with trapped MPE-camptothecin of hposomes at doses of 5 mg / kg (open triangles), 3 mg / kg (inverted open triangles), 1 mg / kg (open diamonds), 05 mg / kg (open circles) and 01 mg / kg (open squares) and with free MPE-camptotecma in a dose of 20 mg / kg (closed squares), Figures 4A-4B are graphical representations showing the plasma concentration of the topotecan as a function of time, in hours, after the admin liposome-entrapped topotecan (solid triangles) and free topotecan (non-liposomal) (solid squares) istration to rats in doses of 2 mg / kg (Figure 4A) and 5 mg / kg (Figure 4B), Figure 5A is a graphic representation of the body weight of the mice, in grams, based on the days after inoculation with a colon tumor HT29 The animals were treated on days 9, 16, and 23 after the tumor inoculation with topotecan entrapped liposomes in doses of 2 mg / kg (diamonds), 5 mg / kg (circles), 8 mg / kg (closed squares), MPE-camptothecin entrapped from posomes to 4 mg / kg (triangles), topotecan free in a dose of 25 mg / kg (inverted triangles) and saline (closed squares), Figure 5B is a graphical representation showing the tumor volume, mm3, as a function of the days after inoculation with a colon tumor HT29 The animals were treated in the 9, 16 and 23 days after tumor inoculation with topotecan entrapment of liposomes in doses of 2 mg / kg (diamonds), 5 mg / kg (circles), 8 mg / kg (open squares), MPE-camptothecin entrapped from hposomes at 4 mg / kg (triangles), topotecan free in a dose of 25 mg / kg (inverted triangles) and saline (closed squares), Figure 6 is a graphic representation of the plasma concentration of CKD602 as a function of time, in hours, after the administration of CKD602 entrapped liposomes (solid circles) and free topotecan (non-liposomal) (solid squares) to rats in a dose of 1 mg / kg, Figure 7A is a graphical representation showing the body weight of the mice, in grams, as a function of the days after inoculation with a colon HT29 tumor Animals were treated on days 9, 16, and 23 after tumor inoculation with CKD602 entrapped liposomes at a dose of 4 mg / kg (diamonds), 2 mg / kg circles ), 1 mg / kg (open squares), MPE-camptothecin entrapped from liposomes at 4 mg / kg (triangles), CKD602 free at a dose of 20 mg / kg (inverted triangles) and saline (closed squares), and Figure 7B is a graphic representation showing the tumor volume, in mm3, as a function of the days after inoculation with a colon HT29 tumor. The animals were treated on days 9, 16 and 23 after tumor inoculation with CKD602 trapped liposomes in a dose of 4 mg / kg (diamonds), 2 mg / kg (circles ), 1 mg / kg (open squares), MPE-camptotecma entrapped from liposomes at 4 mg / kg (triangles), free CKD602 at a dose of 20 mg / kg (inverted triangles) and saline (closed squares) DETAILED DESCRIPTION OF THE INVENTION I Definitions Unless otherwise indicated, the following terms have the following meaning: "Effective amount" or "effective dose" refers to the amount necessary or sufficient to inhibit undesirable cell growth, for example, avoiding undesirable cell growth or reduce existing cell growth, such as tumor cell growth. The effective amount may vary depending on factors known to those skilled in the art, such as the type of cell growth, the mode and mode of administration, the size of the patient, the severity of cell growth, etc. An expert in the field should be able to consider such factors and make the determination regarding the effective amount "Therapeutically effective antitumor therapy" refers to a therapy which is effective in maintaining or decreasing the size, e.g., volume, of a primary tumor or metastatic tumor. "Topoisomerase I inhibitor" refers to any compound that inhibits or reduces the action of the topoisomerase I enzyme "Topoisomerase I / M inhibitor" refers to any compound that inhibits or reduces the action of both the topoisomerase I enzyme and the topoisomerase II enzyme "Topoisomerase inhibitor" refers to a Topoisomerase I inhibitor or a topoisomerase I / II inhibitor "MPE-camptothecin" refers to 7- (4-met? lp? peraz? no-met? len) -10,11-et? lend? ox? -20 (S) -camptotec? Na "Topotecan" refers to 9-d? Met? L-am? Nomet? -10-hydroxycamptothecin "CKD602" refers to 7- (2- (N-? Soprop? Lam? No ) et? l) - (20S) -camptothecin II Liposome Composition The present invention is directed to a composition of hposomes for the administration of a topoisomerase I inhibitor or a topoisomerase I / II inhibitor. In studies conducted in support of the invention, three topoisomerase inhibitors were entrapped in liposomes and characterized vivo vivo topotecano, 7- (4-met? lp? peraz? no-met? len) -10,11-et? lend? ox? -20 (S) -camptotec? na (referred to herein as "MPE -camptothecin ") and 7- (2- (N-? soprop? lam? no) et? l) - (20S) -camptotecma (referred to herein as" CKD-602") The drugs were entrapped in liposomes by remote loading to achieve a high drug load maintained stably in the hposomes, as will be described. In vivo studies with the formulations demonstrated that the liposome composition achieves a surprising and unexpected degree of improvements in therapeutic activity when compared to the therapy with the topoisomerase inhibitor in free form More specifically, and as described below, the MPE-camptothecin dose of the entrapped liposome topoisomerase I inhibitor required to achieve therapeutic antitumor therapy is approximately 20 times lower than the dose required when the drug is administered in free form. In this section, the Liposome composition, including methods for preparing liposomes A Liposome Components Liposomes suitable for use in the composition of the present invention include those composed primarily of 11 vesicle formation peptides. Vesicle formation lipids can spontaneously form in bilayer vesicles in water, as exemplified by phospholipids. The liposomes can also include other lipids incorporated in the lipid bilayers, with the hydrophobic residue in contact with the interior, the hydrophobic region of the bilayer membrane, and the residue of the main group oriented towards the polar, outer surface of the membrane bilayer The vesicle formation lipids are preferably those having two hydrocarbon chains, typically acyl chains, and a major group, whether polar or non-polar There is a variety of synthetic vesicle formation lipids and vesicle formation lipids of natural generation, which include phospholipids, ta such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylmositol, and sphingomyelin, wherein the two hydrocarbon chains are typically between about 14-22 carbon atoms in length, and have varying degrees of non-saturation The lipids and phospholipids described above whose chains acyl have varying degrees of saturation can be obtained commercially or prepared according to published methods. Other suitable lipids include glycolipids and sterols such as cholesterol. Cationic lipids are also suitable for use in the liposomes of the invention, wherein the cationic lipid can be included as a minor component of the lipid composition or as a main or single component. Such cationic lipids typically have a lipophilic residue, such as a sterol, an acyl or diacyl chain, and wherein the lipid has a general net positive side. Preferably , the main group of the lipid carries the positive charge Examples of cationic lipids include 1,2-d? ole? lox? -3- (tpmet? lam? no) propane (DOTAP), N- [1- (2,3-bromide , -d? tetradec? lox?) prop? l] -N, Nd? met? lNh? drox? et? lamon? aco (DMRIE), bromide of N- [1- (2,3, -d? oe? lox?) prop? l] -N, Nd? met? lNh? drox? et? lamon? aco (DORIE), N- [1- (2,3-d? ole? lox?) prop?] -N, N, N-tr? Met? Lamon? Aco (DOTMA), cholesterol of 3 [N- (N ', N'-d? Met? Lam? Noethane) carbamoly] (DC-Col), and dimethyldioctadecylammoniaco ( DDAB) The cationic vesicle formation lipid may also be a neutral lipid, such as dioleoylphosphatidyl ethanolamine (DOPE) or an antipathetic lipid, such as a phospholipid, derived with a cationic lipid, such as polylysine or other polyamine lipids. For example , the neutral lipid (DOPE) can be derived with po lysm to form a cationic lipid. In another embodiment, the vesicle formation lipid is selected to achieve a specified degree of fluidity or stiffness, 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 incorporating a relatively rigid, example, a lipid having a relatively high transition phase temperature for example, higher than room temperature, more preferably higher than body temperature and up to 80 ° C. Rigid, ie saturated, lipids contribute to greater rigidity Other membrane lipid components, such as cholesterol, are also known to contribute to membrane rigidity in lipid bilayer structures. Moreover, lipid fluidity is achieved by the incorporation of a relatively liquid lipid, typically one having a lipid phase with a liquid to liquid crystalline phase transition temperature relatively low, for example, at a temperature of or lower than room temperature, more preferably, at or below body temperature Vesicle formation lipids having phase transition temperatures from about 2 ° C-80 ° C are suitable for use as the primary liposome component of the present composition In a preferred embodiment of the invention , a vesicle formation step having a main phase transition temperature greater than about 37 ° C is used as the primary lipid component of the liposomes. In another preferred embodiment, a lipid having a phase transition temperature is used. between about 37-70 ° C By way of example, the disteararoyl lipid phosphatidylcholine (DSPC) has a main phase transition temperature of 551 ° C and the hydrogenated soy phosphatidylcholine (HSPC) has a phase transition temperature of 58 ° C. ° C The phase transition temperatures of many lipids are tabulated in a variety of sources, such as the Avanti catalog Polar Lipids and in Lipid Thermotropic Phase Transition Datábase (LIPIDAT, NIST Standard Reference Datbase 34) Liposomes also include a vesicle-forming lipid derivatized with a hydrophilic polymer As described, for example in U.S. Patent No. 5,013,556, and in WO 98/07409, which are incorporated herein by reference, such a hydrophilic polymer provides a surface coating of hydrophilic polymer chains on both the inner and outer surface of the bilayer lipid membranes of liposomes. The outermost surface coating of The hydrophilic polymer chains are effective to provide a hposome with a long duration of the bloodstream. The inner lining of the hydrophilic polymer chains extends into the aqueous compartments in the liposomes, ie, between the lipid bilayers and in the compartment. of central core, and is in contact with the compound e Trapping after the compound is loaded by remote loading As will be illustrated below, the liposome formulation having a surface coating of hydrophobic polymer chains distributed over the inner and outer liposome surfaces provides for a topoisomerase I inhibitor or a topoisomerase I / II inhibitor wherein the compound is maintained in the posomes for improved therapeutic activity. The vesicle formation lipids suitable for derivatization with a hydrophilic polymer include any of those drugs described above, and, in particular, the phospholipids, such as distearoyl phosphatidylethanolamine (DSPE) Hydropylic polymers suitable for derivatization with a vesicle-forming lipid include polyvinylpyrrolidine, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl-oxazoline, polyhydroxypropyl-1-metacp-lamide, meta-p-lamide, polydimethylac LAMIDE, POLYHYDROXYPROPYL METAMYL PLATE, POLYHYDROXYTYL PLATE, HYDROXYMETHYL CARCELLULOSE, HYDROXYETHYL CHLUCULULOSE, POLYLENEGENOLIC, AND POLYASPARAMIDE Polymers can be used as homopolymers or as block or random copolymers A preferred hydrophilic polymer chain is polyethylene glycol (PEG), preferably as a PEG chain having a molecular weight of between 500-10,000 daltons, more preferably between 500-5,000 daltons, most preferably between 1,000-2,000 Metoxi daltons or the PEG ethoxy analogues are also preferred hydrophobic polymers, commercially available in a variety of cellulose sizes. polymer, for example, 120-20,000 daltons The preparation of vesicle-forming lipids derived with hydrophobic polymers has been described, for example in U.S. Patent No. 5,395,619. The preparation of liposomes including such derivatized lipids has also been described, in US Pat. where typically, between 1-20 percent in mol of such a lipid derivative is included in the liposome formulation. It will be appreciated that the hydrophobic polymer can be stably coupled to the lipid., or coupling through an unsuitable bond which allows the coated liposomes to pour the coating of the polymer chains as they circulate in the bloodstream or in response to a stimulus B Topoisomerase Inhibitor The liposomes of the invention include a topoisomerase inhibitor entrapped in the liposome. Trapping is intended to include the encapsulation of an agent in the aqueous core and aqueous spaces of the hposomes. It will be appreciated that for compounds having some hydrophobicity, it may be trapping occurs in the b) layer (s) of the liposome (s) Topoisomerases catalyze the introduction and relaxation of superhelicity in DNA It is known that several types of enzymes with variable specificities are important in the DNA replication, as well as in repair, genetic recombination and DNA transcription The simplest topoisomerases, designated topoisomerase I, relaxed superhelical DNA, a process that is energetically spontaneous Gyrases, which are known as topoisomerase II, catalyze the dependent introduction of ATP and energy claimants of the superhelical negative braids in DNA In the DNA rep- resentation, the topoisomerases I and II have the function of relaxing the positive superhelicity that is introduced beyond the repurposing forks in the action of the helicases. Moreover, the gyrases introduce negative forks in the DNA segments that allow to appear simple braid regions Next, topoisomerase inhibitors are compounds that inhibit topoisomerase activity. Compounds known as topoisomerase I inhibitors have activity against topoisomerase I, and topoisomerase II inhibitors have activity against topoisomerase II. Some compounds have activity against both topoisomerase I and against topoisomerase II and are known as topoisomerase I / II inhibitors. Preferred topoisomerase I inhibitors for use in the present invention are camptothecin and camptothecin analogues. Camptothecin is a pentacyclic alkanoid initially isolated from wood and bark of Camptotheca acummata, a tree native to China (Wall, ME et al, J Am Chem Soc, 94388 (1966)) Camptothecin exerts its pharmacological effects by irreversibly inhibiting topoisomerase I Methods for the synthesis of camptothecin and camptothem analogs or derivatives are known, and are summarized and set forth in US Patent No. 5,244,903 which is incorporated herein by reference in its entirety Camptothec analogs include SN-38 ((+) - (4S) -4,11-d? Et? L-4,9-d? H? Drox? -1H-p? Rano [3 ', 4', 6,7] -? Ndol? Z? No [1, 2- b) qu? nol? n-3 14 (4H 12 H) -d? ona), 9-am? nocamptotec? na, topotecan (hicamtma, 9-d? met? l-am? nomet? l-10-h? drox? camptotec? na), ipnotecano (CPT-11, 7-et? l-10- [4- (1-p? per? d? no) -1-p? per? d? no] -carbon? lox ? -camptotec? na), which is hydrolyzed m vivo for SN-38) 7-et? lcamptotec? na and its derivatives (Sawada, S et al, Chem Pharm Bull, 41 (2) 310-313 (1993)) , 7-chloromet? L-10,11-met? Len-d? Ox? -camptotec? Na, and others (SN-22, Kunimoto, T et al, J Pharmacobiodyn, 10 (3) 148-151 (1987), N-formylamino-12,13, d? H? Dro-1,11-d? H? Drox? -13- (beta-D-glucoprans? L? ) -5H-? Ndolo [2,3-a] pyrrolo [3,4-c] carbazole-5,7 (6H) -d? On (NB-506, Kanzawa, F, er, Cancer Res, 55 (13) 2806-2813 (1995), DX-8951f and lurtotecan (GG-211 or 7- (4-met? Lp? Peraz? No-met lien) - 10,11-et? Lend? Ox? -20 (S) -camptotec? Na) (Rothenberg, ML Ann Oncol, 8 (9) 837-855 (1997)) and 7- (2- (N-? Soprop? Lam? No) et? L) - (20S) -camptotec? Na (CKD602, Chong Kun Dang Corporation, Seoul, Korea) Topoisomerase inhibitors that have activity against both topoisomerase I and topoisomerase II include 6 - [[2- (d? Methylamine) -et? L] dihydrochloride. am? no] -3-h? drox? -7H-? ndeno [2,1-c] qu? nol? n-7-on, (TAS-103, Utsugí, T, et al, Jpn J Cancer Res, 88 (10) 992-1002 (1997)) and 3-methox? -11 Hp? R? Do [3 ', 4'- 4,5] p? Rrolo [3,2-c] qu? Nol? N- 1,4-dαone (AzalQD, Riou, JF, et al, Mol Pharmacol, 40 (5) 699-706 (1991)) In one embodiment of the invention, the topoisomerase I inhibitor administered is the pharmacologically active enantiomer of a camptothecin analog having a chiral center The enantiomer can be resolved from the racemic mixture using techniques known to those skilled in the art C Method for preparing the liposome composition Liposomes can be prepared by a variety of techniques, such as those detailed in Szoka, F, Jr, et al, Ann Rev Biophys Bioeng 9467 (1980), and specific examples of the liposomes prepared in BACKGROUND OF THE INVENTION Typically, liposomes are multilamellar vesicles (MLVs), which can be formed by simple lipid hydration techniques. In this procedure, a phase transition temperature mixture of hydrophilic polymer lipids is dissolved. liposome formation and including a vesicle formation lipid derivatized with a hydrophilic polymer in a suitable organic solvent which is evaporated in a vessel in order to form a dry thin film. Then the film is covered by an aqueous medium to form a thin film. MLVs, typically with sizes between about 01 to 10 microns have been described exemplary methods for preparing lipids derived from polymer-coated liposome formation in U.S. Patent Nos. 5,013,556, 5,631,018 and 5,395,619, which are incorporated herein by reference. The therapeutic agent of choice can be incorporated into liposomes by standard methods, which include (i) passive entrapment of a water-soluble compound by hydrating a lipid film with an aqueous solution of the agent, (ii) passive entrapment of a lipophilic compound by hydrating a lipid film containing the agent, and (iii) loading a drug. ionizable against a gradient of interior / exterior liposome ion, which acquires the term of remote charge. Other methods are also suitable, such as the preparation of reverse evaporation phase liposomes. In the present invention, a preferred method for preparing the liposomes is by remote charging. In the studies conducted in support of the invention, three exemplary topoisomerase I inhibitors were loaded into preformed liposomes by remote charging against an ion concentration gradient, as described in the art (US Patent No. 5,192,549) and as described in Example 1. In a remote loading procedure, drug is accumulated in the central compartment of the liposomes at concentration levels much higher than those achieved with other loading methods. In a preferred embodiment of the invention, the topoisomerase I inhibitor or topoisomerase I / ll inhibitor is loaded into the liposomes at a concentration of at least about 0.10 μmol of drug per μmol of lipid, more preferably of at least about 0.15 μmol of drug per μmol of lipid, most preferably of at least about 0.20 μmol of drug per μmol of lipid. The liposomes prepared in support of the invention contained MPE-camptothecin, topotecan or CKD602. As set forth in Example 1, these compounds were loaded into the liposomes by remote loading, described below, at a drug concentration level greater than 0 20 μmol of drug per μmol of lipid (see the table in Example 1). ) Liposomes that have an ion radiant bond through the bilayer for use in remote loading can be prepared by a variety of techniques. A typical procedure is as described above, wherein a mixture of liposomes forming lipids it is dissolved in a suitable organic solvent and evaporated in a container in the form of a thin film. The film is then covered with an aqueous medium containing the dissolved species that will form the aqueous phase in the interior spaces of the liposome. After the formation of liposomes , the vesicles can be dimensioned to achieve a size distribution of liposomes within a selected range, according to the known methods. The liposomes are preferably dimensioned uniformly at a range of size selected from 0 04 to 0 25 μm. You see small unilamellar particles (S UVs), typically in the range of 0 04 to 0 08 μm, can be prepared by sonification or extensive homogenization of the liposomes. Homogeneously sized liposomes having sizes in a range selected from about 0 08 to 0 4 m can be produced, for example, by extrusion through polycarbonate membranes or other defined pore size membranes that have selected uniform pore sizes ranging from 0 03 to 0 5 m icrons, typically, 0 05, 0 08, 0 1 or 0 2 m icrones The pore size of the membrane corresponds only to the largest size of pores produced by extrusion through that membrane, particularly where the preparation is extruded two or more times through the same membrane. It preferably performs in the original lipid hydrating regulator, so that the interior spaces of the liposome maintain this medium through the steps of initial hposome processing After sizing, the outer medium of the liposomes is treated to produce a radient of ion through the liposome membrane, which is typically a higher / lower exterior concentration gradient. be made in a variety of ways, for example, by (i) diluting the external medium, (n) dialysis against the desired final medium, (ni) molecular sieve chromatography, for example, using Sephadex G-50, against the desired medium, or (iv) high-speed centrifugation and resuspension of comonomic pores in the desired final medium. The external medium that is selected will depend on the mechanism of gradient formation and the desired external pH, as will now be considered. In the simplest approach to generate an ion g, the hydrated, sized liposomes have a medium-selected intepor pH. The suspension of the liposomes is titrated until a final pH is reached desired, or it is treated as explained above in order to change the external phase regulator with one having the desired external pH. For example, the original medium can have a pH of 5 5, in a selected regulator, for example. , regulator of glutamate or phosphate, and the final external medium can have a pH of 8 5 in the same or different regulator. The inner and outer media are preferably selected to contain approximately the same osmolarity, for example, by suitable adjustment of the concentration of the regulator, salt, or reduced molecular weight solute, such as sucrose. In another general approach, the gradient occurs when a selected ionophore is included in the liposomes. To illustrate, liposomes prepared to contain valinomycin in the bilayer of liposomes are prepared in a potassium regulator, sized, then exchanged with a sodium regulator, creating an exterior sodium / inner potassium gradient. The movement of the potassium ions in an interior to exterior direction in turn generates a greater / lower interior pH gradient presumably due to the movement of the protons towards the liposomes in response to the net electronegative charge through the liposome membranes (Deamer, et al., 1972). In another more preferred approach, the proton gradient used for drug loading occurs by creating an ammonia ion gradient across the liposome membrane, as described, for example, in the U.S. Patent. No. 5,192,549. Here the liposomes are prepared in an aqueous buffer containing an ammonia salt, typically 0.1 to 0.3 M ammonia salt, such as an ammonia sulfate, at a suitable pH, for example, 5.5 to 7.5. The gradient can also be produced by using sulfate polymers, such as dextran ammonia sulfate or heparin sulfate. After the formation of liposomes and sizing, the external medium is exchanged for ammonia-lacking ions, for example, the same regulator but one in which the ammonium sulfate is replaced by NaCl or a sugar that gives the same osmolapdad to the interior and exterior of the liposomes. After the formation of liposomes, the ammonium ions Within the liposomes are found in equilibrium with the ammonia and protons. The ammonia is able to penetrate the liposome bilayer and escape from the interior of the liposome. The ammonia leak continuously changes the equilibrium within the liposome to the right, to proton production The topoisomerase inhibitor is loaded into the liposomes by watering the drug to a suspension of the ionic radicals, and the suspension is treated under conditions effective to allow the passage of the compound from the medium outside the cells. liposomes The incubation conditions suitable for drug loading are those which (i) allow the diffusion of the derivative compound, with such an uncharged form, as s liposomes, and (n) preferably lead to a high concentration of drug loading, for example, 5-500 mM of encapsulated drug, more preferably from 20-200 mM, most preferably from 50-300 M The loading is carried out preferably at a temperature higher than the temperature of liposome lipids. Therefore, for liposomes formed predominantly from saturated phospholipids, the loading temperature may be as high as 60 ° C or more. The charging period is typically between 15 ° C and more. -120 minutes, depending on the permeability of the drug to the liposome bilayer membrane, temperature, and the relative concentrations of the liposome lipid and drug With the appropriate selection of the liposome concentration, the outside concentration of the added compound, and the ion gradient, essentially all of the compound can be loaded into the liposomes For example, with a pH gradient of 3 units (or the potential of such a gradient that employs an ammonia ion gradient), the final exterior indoor concentration of the drug will be approximately 1000 1 Knowing the calculated inner liposomal volume, and the maximum concentration of the loaded drug, one can then select a quantity of drug in the outside medium that lead to substantially complete loading in the liposomes. Alternatively, if the drug loading is not effective to substantially impoverish the external environment of the free drug, the liposome suspension can be treated, followed by a drug loading, to extract the non-encapsulated drug. Free drug can be extracted, for example, by molecular sieve chromatography, dialysis or centrifugation. In another embodiment of the invention, the topoisomerase inhibitor is loaded into the preformed liposomes which include within the liposome an effective entrapment agent for the complex with the topoisomerase inhibitor and improve the retention of the compound In a Preferred embodiment, the entrapment agent is a pohanionic polymer, for example, a molecule consisting of repetitive units of preferably similar chemical structure and having group extendable, ie, chemical functional groups capable of exhibiting electrolyte dissociation resulting in the formation of ionic charge, and preferably an ammonia charge Polymers having a molecular weight over a wide range, from 400-2,000,000 Daltons are suitable Polyanionic polymer is entrapped in liposomes during lipid vesicle formation After loading a drug into the preformed liposomes, the polymer serves to entrap or maintain the drug within the hposomes In the studies described herein, sulfate dextran was used as an exemplary polyanionic polymer Dextran sulfate is an anhydroglucose polymer with approximately 23 sulfate groups per glucose residue is found The compound is composed of approximately 95% alpha-D- (1-6) bonds and the rest of the bonds (1-3) represent the dextran branching. The polymer is readily available in molecular weights ranging from, 000 to 500,000 Daltons However, other polymers including phosphatic or carboxylated, sulphonated, sulphonated hydrophobic polymers are suitable. For example, sulphated proteoglycans, such as sulfated hepapine, sulphated polysaccharides, such as sulfated cellulose or cellulose derivatives, carrageenan , mucma, sulfated polypeptides, such as polysilva with sulfated amine groups, glycopeptides with saccharide subunits or peptides derived with sulfonate, and hyaluronic acid Also contemplated are chondroitme sulfates A, B and C, keratin sulfates, dermatan sulfates also be a modified neutral polymer to include an ammonium functional group. For example, amylose, pectin, amylopectin, celluloses, and dextran can be modified to include an ammonium subunit. Polymers that support a sulfo group such as polyvinyl sulfate, polyvinyl sulfonate, polystyrenesulfonate and sulfated rosin too The preparation of the liposomes including such entrapment agent is described with respect to Example 4 In this example, the dextran sulfate of polyanionic polymer is entrapped in the liposomes by adding the lipids of liposomes, which are dissolved first in ethanol, until an ammonia salt solution of dextran sulfate is obtained and mixed to form liposomes having ammonia salt of dextran sulfate entrapped within the hposomes. The outside media were exchanged to establish an ammonia ion gradient across the Liposomes for remote drug loading lll Live Administration of the Composition Liposomes were prepared in support of the invention as described in Example 1 Topoisomerase I (7- (4-met? lp? peraz? no) -met? len) -10 inhibitors, 11-et? Lend? Ox? -20 (S) -camptotec? Na, referred to herein as "MPE-camptothecin", topotecan, and 7- (2- (N-? Soprop? Lam? No) et? L ) - (20S) -camptotecna, referred to herein as "CKD-602", were loaded into the liposomes under a concentration gradient of ammonium sulfate ion. The liposomes were composed of hydrogenated soy phosphatidylcholine, cholesterol and pol Lettilyne derivatized to obtain distearoyl phosphatidylethanolamm (PEG-DSPE) in a molar ratio 5543956 The table in Example 1 summarizes the drug-to-lipid proportions for the liposome formulations prepared. The liposomal drug concentrations calculated for the three compounds, based on in a captured volume of extruded liposome of 09 μl-μmol of lipid, are 284 mM for MPE-camptothecin , 264 mM for the topotecan and 298 for CKD-602 Based on the captured volume of extruded liposome of 15 μl / μmol of lipid, the posomal drug concentrations calculated are 189 mM for the MPE-camptothecin, 174 mM for the topotecan and 198 for CKD-602 The in vivo studies performed with each drug will now be described 1 In vivo administration of MPE-camptotec The long-circulating, PEG-coated liposomes containing MPE-camptothecin were administered to rats to determine the duration of the bloodstream of drugs in the entrapped form of liposomes. The pharmacokinetic profile of the drug entrapped with liposome and the free drug are shown in Figure 1A as the percentage of the dose injected as a function of time As can be seen, the bloodstream time of the topoisomerase I inhibitor in the entrapped form of liposomes (solid circles) is significantly greater than the free form of the drug (solid squares). For the MPE-camptotecma, the half-life of the bloodstream of the entrapped drug of liposomes was 14 hours, compared to approximately 50 minutes for the free drug. The elimination of blood from the entrapped drug of liposomes in the rats was approximately 35 times less and the area under the curve was approximately 1250 times more than that of the free drug The analytical results indicate that essentially all the drug remains trapped in the hposomes in the bloodstream Figure 1B shows the concentration of MPE-camptothecin in the whole blood after the administration of the liposome formulation (solid circles) and from the free drug to rats The longer the average life of the torrent, the higher the a will be the concentration of the drug in the blood The antitumor efficacy of the liposome formulation of MPE-camptothecin was determined in xenograft tumor models, where the homozygous nude mice were inoculated with human tumor cells of colon, of HT29 origin. Surprisingly, these studies of toxicity and antitumor efficacy showed that the MPE-camptothecin liposomal was significantly more toxic that the free form of the drug in equivalent doses These studies and the results will now be described Liposomes were prepared as set forth in Example 1 to include MPE-camptothecin Nude mice with HT29 colon xenografts were treated with trapped MPE-camptothecin of liposomes in doses of 24 mg / kg, 15 mg / kg and 6 mg / kg or with free MPE-camptothecin in the same doses The treatment started 10 days after the tumor inoculation and doses were administered on days 10, 16 and 23 The tumor volume in each animal was evaluated during the next treatment as described in Example 2. The body weight of each animal is shown. test animal and tumor volume of each animal, respectively in Figures 2A and 2B, where the animals were treated with MPE-camptothecin entrapped liposomal in a dose of 24 mg / kg (closed circles), 15 mg / kg (closed triangles) and 6 mg / kg (closed squares) and with free MPE-camptothecin at a dose of 24 mg / kg (open circles), 15 mg / kg (open triangles) and 6 mg / kg (open circles) open squares) With respect to animals treated with the trapped MPE-camptothecin liposomes, all animals dosed with 15 mg / kg and 24 mg / kg died after two doses due to drug-related toxicity, with most deaths on day five after the first dose All animals treated with 6 mg / kg trapped MPE-camptothecin liposomes survived until the administration of the third dose on day 23, after which five of the ten animals died in about how many days The toxicity of trapped MPE-camptothecin liposomes is reflected in greater losses of body weight, as seen in Figure 2A In contrast, all animals treated with the free form of the drug survived the study, with the exception of one animal in the dosing group of 24 mg / kg that died a few days after the third dosage on day 23 Table 1 With respect to the antitumor activity of the formulations, the trapped MPE-camptothecin of liposomes was more effective than the free form of the drug in inhibiting tumor growth, despite its greater toxicity. This can be seen in Figure 2B, where the 6 mg / kg MPE-encapsulated camptothecin dose of liposomes was significantly more effective in inhibiting tumor growth (logarithmic growth rate of -0026) than even the highest dose level of free MPE-camptothecin (24 mg / kg , logarithmic growth rate 00048) Complete and partial remission of tumors in the test animals was monitored and is presented in Table 2 Complete remission of a tumor is defined as removal of the tumor mass until the end of the experiment. Partial remission is defined as tumor volume less than 50% of the peak tumor volume for an individual animal Table 2 complete remission defined as removal of the tumor mass until completion of the experiment partial remission defined as a tumor volume less than 50% of the peak tumor volume for an individual animal all 10 animals in test groups died after the second dose on day 16 = not applicable As can be seen in Table 2, the trapped MPE-camptothecin of liposomes in a dose of 6 mg / kg was effective to cause a complete remission of the tumors in the 10 test animals. This effect was observed five days after the second Treatment on day 16 As noted above, five of the test animals in the entrapped liposome test group of 6 mg / kg died shortly after the third dose. In the five surviving animals, the tumors did not recur by the end of the study, approximately 30 days after the final treatment on day 23 Data are not available for animals treated with 15 mg / kg and 24 mg / kg of MPE-camptothecin entrapped posomes, because all the animals in the test groups died due to the drug-related toxicity, as previously noted. The administration of MPE- in free form at a dose of 24 mg / kg camptothecin gave as a result 3 animals with complete tumor remission and 1 animal with partial tumor remission, as can be seen in Table 2 The comparison of the results observed for the drug administered in free form and in trapped form of liposomes indicates that the drug is more potent when In fact, the entrapped drug of liposomes is at least four times more potent than the free form of the drug, as can be seen when comparing the results obtained for a dose of 6 mg / kg of trapped MPE-camptothecin of hposomes up to a dose of 24 mg / kg of free MPE-camptotheme (Figure 2B, Table 2) It is clear from these results that the dose of trapped MPE-camptotecma of liposomes required for therapeutically effective antitumor therapy is four times smaller that the dose required when the drug is administered in free form Example 2 describes the details of a second study to determine the maximum tolerated dose and a minimum effective dose of the encapsulated MPE-camptothecin liposomes In this study, the liposomes were prepared as described in Example 1 and the liposome formulation was administered to test animals at a drug dose of 01 mg / kg, 05 mg / kg, 1 mg / kg, 3 mg / kg and 5 mg / kg Free drug was administered at 20 mg / kg as a comparison Table 3 summarizes the number of test animals in each group, specifying the number of surviving animals in each phase of the study dosage As can be seen in the table, all the control animals, treated with saline, and all the animals treated with MPE-free camptothecin survived the duration of the study Of the ten animals treated with 5 mg / kg of MPE-camptotecma entrapped liposomes, four of the animals died of drug-related toxicity and one additional animal died of seemingly non-specific causes after the third dose One of the ten animals in the test group receiving 3 mg / kg of MPE-camptothecin entrapped from liposomes died after the second dose, but death was not considered due to drug treatment due to the absence of any signs of correlated toxicity. Other animals treated with MPE-camptothecin entrapped from posomes survived the duration of the complete study Table 3 The results of the study are shown in Figures 3A-3B, where Figure 3A shows the body weight of the mice, in grams, as a function of the days after inoculation with the HT29 colon tumor. The animals were treated. on days 9, 16 and 23 after tumor inoculation with topoisomerase I inhibitor, liposomal entrapment at a dose of 5 mg / kg (open triangles), 3 mg / kg (inverted open triangles), 1 mg / kg (open diamonds) ), 0 5 mg / kg (open circles) and 0 1 mg / kg (square to open) and with free drug in a dose of 20 mg / kg (closed squares) As can be seen in Figure 3A, the changes in body weight were related to the dosage and, these changes were correlated with other observations of toxicity Figure 3B is a similar graphic representation showing the tumor volume, in mm3, as a function of the days after the tumor inoculation, where the doses are represented p or the same symbols as in Figure 3A Figure 3B shows that both levels of 5 mg / kg and 3 mg / kg of MPE-camptothecin entrapped liposomes were more therapeutically effective to inhibit tumor growth than the dose of 20 mg / kg of free drug Treatment with 20 mg / kg free MPE-camptothecin (logarithmic growth rate of 0011) was approximately equivalent in antitumor activity at the dose level of 1 mg / kg of the drug in the trapped form of liposomes ( logarithmic growth rate of 0017) Table 4 summarizes complete and partial remission in the test animals Table 4 Complete remission defined as removal of the tumor mass until the end of the experiment Partial remission defined as a tumor volume less than 50% of the peak tumor volume for an individual animal na = not applicable There were no complete tumor remissions in the animals treated with 20 mg / kg free MPE-camptothecin. In contrast, the ten animals treated with MPE-camptothecin entrapped from hposomes at the dose level of 5 mg / kg completed the remissions at the level of dose of 3 mg / kg, seven of the ten animals completed the remission of their tumors The results derived from the study of Example 3 show that the antitumor activity of the MPE-camptothecin of topoisomerase inhibitor entrapped by liposomes is significantly better When compared to the free form of the drug, indicating that the entrapped form of liposomes was approximately 20 times more potent because the antitumor activity of the free drug at a dose of 20 mg / kg was more comparable to the activity of a dose of 1 mg / kg of the entrapped form of liposomes of the drug The dose of 3 mg / kg and 5 mg / kg of MPE-camptothecin entrapped liposomes was significantly more effective In antitumor therapy, the dose of 20 mg / kg of the drug in a free-flowing form indicates that the therapeutic index of the drug entrapped in liposomes is approximately four times greater than the drug in free form. 2 Topotecan administration of vivo In another study carried out in support of the invention, topotecan was introduced into liposomes containing DSPC and m PEG-DSPE at a molar ratio of 95 5, as described in Example 4 Initial studies, no reported here, indicated that the topotecan was not easily maintained in the hposomes. The bilayer lipid was selected to use a simple phosphid as a single component that has an acyl chain length close to the DSPE in the m component PEG-DSPE. The bilayer has minimal packaging defects, which arises from imperfections in the closest neighborhood interactions in a solid phase bilayer, which have reduced rotational and lateral mobility in relation to the liquid bilayers. In addition, a charge battery was used. dextran-sulphate to achieve the precipitation of the topotecan in the inner zone. Other polymers, in particular the pohanionic polymersare suitable for this purpose, such as sulfate A, polyvinyl sulfuric acid and polyphosphocid acid. Preformed liposomes containing ammonium sulfate dextran in the central compartment loaded with topotecan as described in Example 4. The liposomes were loaded in a lipid drug ratio of 0 238 and the liposomes had an average particle diameter of 87 nm. The hposomes that contained topotecan were loaded, the non-incoming sample was extracted by diafiltration and the liposomes were characterized. rat were administered to rats to determine the duration of the bloodstream Figures 4A-4B show the plasma concentration of the topotecan as a function of time after the administration to the rats. Fig. 4A compares the concentration of topotecan enteric liposomes entered at 2 mg / kg (solid triangles) at the concentration of free topotene administered a in the same dose (solid squares) Figure 4B for the two forms of the dose in a dose of 5 mg / kg The calculated pharmacokinetic parameters are determined in Table 5 Table 5 The data in Table 5 show that the entrapped liposome drug has a significantly longer duration of circulation than the form of the drug. The efficacy of the liposomes was determined in another study. As described in Example 4, liposomes were administered to mice bearing a subcutaneous xenograft tumor. Tumor-bearing mice were randomized into six treatment groups of 1 2 mice per treatment with one of the following saline, M PE-camptothecin entrapped posomes 4 mg / kg , topotecan free 25 mg / kg, topotecan entrapped posomes in drug doses of 2 mg / kg, 5 mg / kg or 8 mg / kg. All the treatments were administered as intravenous bolus injections performed weekly for 3 treatments, specifically on days 9, 16 and 23. The tumor size in each animal was measured twice weekly during the study to evaluate the therapeutic efficacy. The body weight of each animal was monitored twice weekly to evaluate the toxicity of the formulations. The results are shown in Tables 6 and 7 and in Figures 5A-5B.

Table 6 Complete remission defined as removal of the tumor mass until the end of the experiment Partial remission defined as a tumor volume less than 50% of the peak tumor volume for an individual animal Non-sensitive was defined as a tumor volume equal to or greater than the initial tumor volume As can be seen from Figures 5A and Table 6, the tumors that were left untreated were developed at a rate of 178 mm3 per day for the duration of the study. The animals treated with MPE-camptotecma entrapped liposomes (control animals positive) experienced a tumor growth rate of -1 2 mm3 per day for the duration of the study Animals treated with non-encapsulated topotecan, which was administered at 25 mg / kg somewhat less than the maximum tolerated dose (MTD) of 40 mg / kg, had a tumor growth of 141 mm3 per day Animals treated with entrapped topotecan liposomes had a growth of 09 mm3 per day for a dose of 2 mg / kg, -1 9 mm3 per day for a dose of 5 mg / kg and -08 mm3 per day for a dose of 8 mg / kg. The negative growth rate indicates a regression of the size of the tumor below the initial tumor volume. The size of the tumors treated according to the size of the tumors with trol (% T / C) was examined for all treatment groups and summarized in Table 6 The National Cancer Institute (National Center Institute) defined significant antitumor activity as% T / C less than 42 Table 7 % T / C defined as the average tumor volume on the indicated day on the average tumor volume of control animals treated with saline 3 Live administration of CKD-602 Example 5 describes another study carried out in support of the invention using the topoisomerase inhibitor CKD-602 The drug was remotely loaded into the liposomes against a gradient of ammonia-sulfate with dextran as an agent of entrapment The lipid composition of liposome was identical to that used for the study using topotecan-HSPC and mPEG-DSPE in a ratio of 95/5 mol Figure 6 is a graphical representation showing the plasma concentration of CKD-602 in function time after administration to rats at a dose of 1 mg / kg The entrapped form of drug liposomes (solid circles) had a calculated half-life of 98 hours and an AUC of 274 μg / mL / hr. the drug had a calculated half-life of 02 hours and an AUC of 037 μg / mL / hr. The therapeutic efficacy of the CKD-602 formulation was assessed using a HT-20 colon cancer xenograft in the ear of the patients. atones Seventy-two mice were inoculated with tumor cells HT-29 and were boosted nine days later in six treatment groups. The animals in each group were treated with one of the following saline formulations., MPE-camptothecin trapped liposomes 4 mg / kg, CKD-602 free 20 mg / kg, CKD-602 entrapped liposomes in drug doses of 1 mg / kg, 2 mg / kg or 4 mg / kg All treatments are administered as intravenous bolus injections determined weekly for 3 treatments, specifically on days 11, 18, and 25 The tumor size in each animal was measured twice weekly during the study to assess therapeutic efficacy. The body weight of each animal was monitored twice. times weekly to evaluate the toxicity of the formulations The results are shown in Tables 8 and 9 and in Figures 7A-7B Table 8 Complete remission defined as removal of the tumor mass until the end of the experiment Partial remission defined as a tumor volume less than 50% of the peak tumor volume for an individual animal Non-sensitive was defined as a tumor volume equal to or greater than the initial tumor volume As can be seen in Table 8 and Figure 7B, the animals treated with saline experienced continuous tumor growth, at a rate of 15.45 mm3 per day for the duration of the study. The animals treated with the trapped MPE-camptothecin liposomes (positive control animals) had a tumor growth rate of -0.63 mm3 per day for the duration of the study. The animals treated with free CKD602, not entrapped, had a tumor growth of 15.21 mm3 per day. The animals treated with liposomal CKD602 had a tumor growth of -221 mm3 per day for the animals treated with a dose of 1 mg / kg, -096 mm3 per day for a dose of 2 mg / kg and -237 mm3 per day for a 4 mg / kg dose The negative growth rate indicates regression of tumor size below the initial tumor volume. The size of treated tumors as a function of the size of the control tumors (% T / C) was examined for all treatment groups and is summarized in Table 9 The National Cancer Institute (National Cancer Institute) defines significant antitumor activity as less than 42 Table 9 % T / C defined as the average tumor volume on the indicated day on the average tumor volume of control animals treated with saline IV EXAMPLES The following examples illustrate the methods of preparation, characterization and use of the composition of the present invention. The examples are not intended to limit the scope of the invention in any way.

Materials The Topoisomerase Inhibitor tpfluoroacetate (7- (4-meth? L-piperazine-metien) -10,11-et? Lend? Ox? -20 (S) -camptotec? Na (G 1147211) (MPE-camptothecin), was provided by Glaxo Research Institute, Research Tpangle Park, NC CKD602 (7- (2- (N-? Soprop? Lam? No) et? L) - (20S) -camptotec? N) was provided by Chong Kun Dang Corporation, Seoul, Korea Topotecan (Hycamtin®) was purchased commercially Materials for preparation of liposomes and other reagents came from commercially available sources Methods Animal studies: Naked homozygous mice were obtained from Tacome Farms (Germantown, NY) and allowed to acclimate for 7 days before the start of the experiment. The animals were housed in appropriate boxes with sterile rodent feed ad libitum and acidified water and a dark light cycle 12 12 Animals were housed in treatment groups before tumor inoculation based on body weight. Aleatopization was confirmed based on tumor size immediately before treatment start. Tumors: Tumors were inoculated by placement By fragments trochanter derived from rapid growth tumors in donor animals The HT-29 human colon cancer cell line was used to initiate subcutaneous xenograft tumors Cultured cells were tppsmized, rinsed, counted and resuspended in 50 m illions of cells per m L of normal growth media Tumors they were inoculated by injecting 0 1 μl (5 μm of cells) into the back of the neck. The tumors were allowed to grow to an average size of 1 00 mm3 before the start of the treatment. Monitoring: Daily well-being was observed. All animals were tested by experiments. The animals were weighed before the tumor inoculation and weekly thereafter. The tumors were measured twice a week throughout the experiment, beginning 5-10 days after the tumor inoculation. poorly observed that it had 1 5% or greater in weight losses from the initial weight and any observed animal that had more than 4, 000 m m3 of tumoral volume was excluded from the study EXAMPLE 1 Preparation of posomesomes with Entranded Topoisomerase Inhibitor The liposomes were prepared and loaded with a selected topoisomerase inhibitor as explained below Preparation of Liposome The hydrogenated soy phosphatidylcholine (HSPC), cholesterol (Col) and mPEG-DSPE (in a proportion of 56438353 mol / mol) of lipids were dissolved in ethanol at 65 ° C in a 250 mL round bottom flask. lipids were stirred continuously for at least 30 minutes at 65 ° C. The total concentration of lipids in ethanol solution was 37 g of total lipid per 10 mL of ethanol. The dissolved lipid solution was transferred to another 250 mL round bottom flask. containing 100 mL of 250 mM ammonia sulfate solution equilibrated at 65 ° C. The hydration mixture of ethanol lipid sulfate ammonia was mixed continuously for at least one hour while maintaining the temperature using a water bath at 65 ° C for form hydrophobic oligolamellar ethanol hiposomes Liposomes or golamelars were reduced in size using a Lipex thermobarble extruder to pass the hydration mixture through poly membranes arbonate with known pore size dimensions The mixture was passed 5 times through a membrane with a pore diameter of 020 μm, followed by 10 passes through a membrane with a pro-diameter of 010 μm. The extruded posomes contained ammonia sulfate within the aqueous (s) compartments (s) of the liposomes, as well as in the medium of the aqueous exterior bulk phase in which they are suspended. The dimensioned liposomes were stored in the refrigerator until the diafiltration prior to the remote loading procedure. 1000 mg of a selected topoisomerase inhibitor, MPE-cam ptotecma, CKD-602 or topotecan, was dissolved in 40 mL of 10% sucrose solution to deliver a concentration of 2 5 mg / m L After dissolution, the solution was passed through a 0 20 μm filter to extract insoluble particles B Remotely Loading Liposomes Ammonia sulfate and ethanol were extracted from the exterior bulk aqueous phase immediately before remote loading by diafiltration of hollow fiber tangential flow with a nominal molecular weight cutoff cartridge of 1 00KDa. constant feed volume, and at least seven exchange volumes were used resulting in the liposomes suspended in an exterior aqueous phase comprised of 10% sucrose After diafiltration, the liposomes were mixed with a selected drug solution. in a proportion (solution of drug hposomes) of 1 4 (mol / mol) and heated rapidly to 65 ° C using a preheated heating jacket with water equilibrium. The temperature of the mixture was maintained at 65 ° C for 40 to 60 minutes, after which the mixture was quickly cooled in an ice-water bath After remote loading, a sample of the liposomes was taken to verify the presence of crystals, to determine the percentage of encapsulation and to measure the diameter of the medium particle The non-encapsulated drug was extracted from the mass phase medium by the diafiltration of hollow fiber tangential flow using a weight-cutting cartridge Molecular molecular weight of 1 00KDa At least eight exchange volumes were used, resulting in a liposomal encapsulated drug suspended in an exterior aqueous phase comprised of 1 0% sucrose 1 0 millimolar H istid ma pH 6 5 The final posomal preparation sterile filtered using a 0 22 μm cellulose acetate syringe filter and stored refrigerated and protected from light until use C Characterization of Liposomes The percentage of encapsulation was determined using size exclusion chromatography to compare the percentage of drug in the null volume (encapsulated liposomal) with the total drug (null volume plus volume included) The drug concentration in the fractions The column diameter was determined by absorbance. The mean particle diameter was determined using quasi-stratified laser light scattering (QELS). The total lipid concentration was analyzed in the post-plug filtration step in order to determine the ratio of drug to lipid. prepared and characterized the liposomes loaded with M PE-cam ptotecma, topotecan and 7- (2- (N-? soprop? lam? no) et? l) - (20S) -cam ptothecin (CKD-602) The results are shown in the table below Example 2 In Vivo Efficacy of Trapped Liposome MPE-Camptothecin Liposomes containing entrapped MPE-camptothecin were prepared as described in Example 1. The enteric drug of liposomes and the free drug were given in 5% dextrose in water as required for the desired concentrations. The nude mice were inoculated with the human colon cancer cell line HT-29 as described above in the methods section. Seventy mice were randomized to one of seven treatment groups as follows: free drug at 24 mg / kg, 15 mg / kg or 6 mg / kg; liposome entrapped drug at 24 mg / kg, 15 mg / kg or 6 mg / kg; saline . Treatment was started when the average tumor volume was approximately 75 mm3 on day 1 0 after inoculation. All treatments were administered as intravenous bolus injections given weekly for 3 treatments, specifically on days 1 0, 1 6 and 23 Tumor size during and after each experiment was used as the primary assessment of therapeutic efficacy Body weight was evaluated to assess toxicity All animals bearing tumors after cessation of treatment were observed, up to That the euthanasia was applied to them based on the aforementioned criterion. The experiments concluded when a majority of the control tumors reached the maximum allowed volume (4, 000 mm3). The tumor size in each animal was measured repeatedly in d At various points in time, therefore, these measurements were considered as correlated information. Because the sizes of After the examination of the data, a logarithmic transformation seemed reasonable. Let Y denote the measurement of original tumor, be Z = log (Y + 1) After transforming the data, repeated measurement analyzes were made for the transformed Z data. The SAS PROC MIXED procedure was used. The logarithmic growth rate was calculated for each treatment group and was used to compare the data. r the different treatment groups The statistical significance was declared at the level of 0 05, but due to multiple comparisons, the adjustment to type I error and a P-value of < 00033 indicated a statistically significant difference in any designated comparison The results were summarized in Tables 1 and 2 and in Figures 2A-2B EXAMPLE 3 Dose Discovery Study for Liposome Trapped MPE-Camptothecin Liposomes containing trapped MPE-camptothecin were prepared as described in Example 1 The posomes trapped drug and the free drug were diluted in dextrose in 5% water as described. required to achieve the desired concentrations Naked mice were inoculated with the HT-29 human colon cancer cell line as described above in the methods section Seventy mice were randomized to one of seven treatment groups as explained below trapped drug of liposomes at 01 mg / kg, 05 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg or 20 mg / kg, and saline Treatment was initiated when the average tumor volume was approximately 75 mm3 in on day 9 after tumor injection All treatments were administered as intravenous bolus injections administered weekly for 3 treatments, specifically on days 9, 16 and 23. Tumor year was evaluated and analyzed as described in Example 2, and the results are shown in Tables 3 and 4 and in Figures 3A-3B. Example 4 In vivo efficacy of entrapped liposomal topotecan A. Preparation of liposomes Liposomes containing topotecan were prepared as explained below. Lipid distearoylphosphatidylcholine (DSPC) and (N- (carbonyl-methoxypolyethylene glycol 2000) -1 were combined, 2-distearyoli-sn-glycero-3-phosphoethanolamine) (mPEG-DSPE) in a molar ratio of 95: 5 and dissolved in ethanol at 70 ° C using continuous stirring. The concentration of lipids in the ethanol solution was 8.9 grams per 10 mL of ethanol. Ammonia salt of dextran sulfate was prepared by ion exchange chromatography using sodium salt of dextran sulfate as the raw material. A 100 mg / mL solution of ammonia salt of dextran sulfate was prepared by dissolving ammonia salt of dextran sulfate in water and adjusting the pH of the solution to 5 using ammonia hydroxide. 100 mL of post-hydration mixing solution was heated to 70 degrees and the size was reduced using a Lipex thermobarrel extruder through a series of polycarbonate membranes to a particle size close to 100 nm particle diameter medium. Typically, the sequence involved 5 passes through a membrane 0.2 μm pore diameter, followed by 10 passes through a membrane of 0.1 μm pore diameter.

The non-entrapped dextran sulfate polymer and the remaining ethanol were extracted from the bulk aqueous exterior phase immediately before the active drug loading step with eight volume exchanges using 350 mM sodium chloride solution, followed by eight volume exchanges using a 10% sucrose solution. The diafiltration cartridge employed had a specified nominal molecular weight cutoff of 100,000 Daltons. A topotecan solution was prepared at a concentration of 2.5 mg / mL in 10% sucrose. The drug solution and the diafiltered liposomes were combined in a volume ratio of 4: 1, and the temperature of the resulting mixture was raised to 70 ° C and maintained with constant agitation for one hour. The loading of the active drug was terminated by rapidly cooling the post-charge mixture using an ice water bath. The trapped drug was extracted by diafiltration using a cartridge having a nominal molecular weight cutoff of 100,000 Daltons. Typically, 8-10 volume exchanges were used using 10 mM 10% sucrose of Histidine pH 6.5 as the exchange buffer. The drug concentration was adjusted to the final value when evaluating the power with a uv-vis absorbance measurement and diluting according to the above. The step of the final process involved sterile-grade filtration using a 0.22 μm filter before filling bottles.

B Characterization of Liposomes The percentage of encapsulation was determined using size exclusion chrom atography to determine the percentage of drug in the null volume ("posom al drug") to the total amount recovered in both the included and null fragments. Drug concentration was monitored using uv-vis absorbance spectrophotometry. The mean particle diameter was determined using quasi-stratified laser light scattering. The total lipids were determined using phosphorus assay. The results are summarized in the table below C Pharmacokinetics m alive and efficacy Seventy-two mice were inoculated with HT-29 cancer cells as described previously in the methods section Nine days after tumor inoculation, the animals were treated weekly with one of the following treatments: ientos B Famacokinetics m alive and efficacy Seventy-two mice were inoculated with HT-29 cancer cells as described in the method section. Eleven days after tumor inoculation, the animals were treated with one of the following intravenous treatments, saline, MPE-camptothecin entrapped posomes 4 mg / kg, CKD602 free 20 mg / kg, CKD602 enterm pada of hposomes in drug doses of 1 mg / kg, 2 mg / kg or 4 mg / kg All treatments are adm administered intravenous bolus injections determined weekly for 3 treatments, specifically on days 1, 1, 18, and 25 The tumor size in each animal was measured twice weekly during the study to assess therapeutic efficacy It was monitored twice per week the body weight of each animal to evaluate the toxicity of the formulations The results are shown in Tables 8 and 9 and in Figures 7A-7B Even though the invention has been described with respect to intravenous saline, MPE-camptotecma entrapped liposomes 4 mg / kg, topotecan free 25 mg / kg, topotecan entrapped liposomes in drug doses of 2 mg / kg, 5 mg / kg or 8 mg / kg All treatments were administered as intravenous bolus injections administered weekly for 3 treatments, specifically days 9, 16 and 23 Tumor size was evaluated and analyzed as described in Example 2, and the results are shown in Tables 6 and 7 and in Figures 5A -5B EXAMPLE 5 In Vivo Efficiency of CKD-602 Liposome Trapping A Preparation and Characterization of Hposomes Liposomes containing CKD-602 were prepared as described in Example 4, except using a drug solution of CKD-602. The liposomes were characterized as is described in Example 4 and the results are summarized below the particular embodiments, it will be apparent to those skilled in the art that various changes and modifications may be made without being isolated from the invention.

Claims (8)

1. REVIVAL NAME IS 1. A composition for treating a tumor in a patient, characterized in that it comprises liposomes composed of a vesicle-forming lipid and between about 1-20 mole percent of a vesicle-forming lipid derivative with a hydrophilic polymer, said liposomes being formed under conditions which distribute the polymer on both sides of the bilayer membranes of the liposomes; and entrapped in the liposomes, a topoisomerase inhibitor in a concentration of at least about 0.1 μmol of drug per μmol of lipid, wherein said liposomes have a sufficient inner / outer ion gradient to maintain the topoisomerase inhibitor within the liposomes in the specified concentration prior to administration in vivo, and wherein said liposomal entrapped topoisomerase inhibitor has a longer bloodstream duration than the topoisomerase inhibitor in free form.
2. 2. The composition according to claim 1, characterized in that the topoisomerase inhibitor is a topoisomerase I inhibitor selected from the group consisting of camptothecin and camptothecin derivatives.
3. 3. The composition according to claim 2, characterized in that the camptothecin derivative is selected from the group consisting of 9-aminocamptothecin, 7-ethylcamptothecin, 1-hydroxycamptothecin, 9-nitrocamptothecin, 1.01-methylenedioxycamptothecin, 9-amino-1,1,1-methylenedioxycamptothecin, 9-chloro, 1,1,1-methylenedioxy camptothecin, 9-chloro, 10,1-methylenedioxycamptothecin, irinotecan, topotecan, (7- (4-methylpiperazinomethylene) -1 0, 1 1-ethylenedioxy-20 (S) -camptothecin, 7- (4-methylpiperazinomethylene) -1,0,1-methylenedioxy-20 (S) -camptothecin and 7- (2-N-isopropylamino) ethyl) - ( 20S) -camptothecin.
4. 4. The composition according to claim 1, characterized in that the topoisomerase inhibitor is a topoisomerase I / II inhibitor selected from the group consisting of 6 - [[2- (dimethylamino) -ethyl] amino] -3- dihydrochloride. hydroxy-7H-indene [2, 1 -c] quinolin-7-on, azotoxin and 3-methoxy-1 1 H -pyrido [3 ', 4'-4,5] pyrrolo [3,2-c] quinolin- 1, 4-dione.
5. The composition according to claim 1, characterized in that the hydrophilic polymer is polyethylene glycol having a molecular weight of between 500-5,000 daltons.
6. 6. The composition according to any of the preceding claims, characterized in that the liposomes include a vesicle-forming lipid having a phase transition temperature greater than 37 ° C. 7. The composition according to claim 6, characterized in that the vesicle formation lipid is selected from the group consisting of hydrogenated soybean polyphosphatidylcholine, distearoylphosphatidylcholine and sphingomyelin. The composition according to claim 6, characterized in that the liposomes are composed of 20-94 mole percent of hydrogenated soy phosphatidylcholine and 1-20 mole percent of distearoylphosphatidylethanolamine derived with polyethylene glycol and 5-60 percent by weight. mol of cholesterol. The composition according to claim 6, characterized in that the liposomes are composed of 30-65 mole percent of hydrogenated soy phosphatidylcholine, 5-20 mole percent of distearoylphosphatidylethanolamine derived with polyethylene glycol and 30-50 mole percent of cholesterol. The composition according to claim 6, characterized in that the liposomes are composed of 20-94 mole percent of distearoylphosphatidylcholine and 1-20 mole percent of distearoylphosphatidylethanolamine derived with polyethylene glycol. eleven . The composition according to any of claims 1-10, characterized in that the liposomes include a polyanionic polymer within the liposomes, said polymer capable of forming a complex with said topoisomerase inhibitor. The composition according to claim 1, characterized in that said polyanionic polymer is selected from dextran sulfate, chondroitin sulfate A, polyvinyl sulfuric acid and polyphosphoric acid. 13. A composition for the administration of a topoisomerase inhibitor, characterized in that it comprises liposomes composed of vesicle formation lipids and having an effective interior / exterior ion gradient to maintain the drug within the liposomes.; and entrapped in the liposomes, the topoisomerase inhibitor in a concentration of at least about 0.20 μmol of drug per μmol of lipid. said encapsulated topoisomerase inhibitor of liposomes has a longer bloodstream duration than the topoisomerase inhibitor in free form. 14. The composition according to claim 1 3, characterized in that the topoisomerase inhibitor is a topoisomerase I inhibitor selected from MPE-camptothecin, topotecan and (7- (2-N-isopropylamino) ethyl) - (20S) - camptothecin. The composition according to claim 1 3, characterized in that the topoisomerase inhibitor is a topoisomerase I / II inhibitor selected from the group consisting of 6 - [[2- (dimethylamino) -ethyl] amino] - dihydrochloride 3-hydroxy-7H-indene [2, 1-c] quinolin-7-on and 3-methoxy-1 1 H -pyrido [3 ', 4'-4,5] pyrrolo [3,2-c] quinoline- 1, 4-d iona. The composition according to claim 13, characterized in that the liposomes further include a polyanionic polymer within the liposomes, said polymer capable of forming a complex with a topoisomerase inhibitor.
7. 7. A composition according to any of the preceding claims for use as a medicament for treating a tumor in a patient. 1
8. 8. The use of a composition according to any of claims 1-16, for the manufacture of a medicament for treating a tumor in a patient.