HK1186381A - Agent for enhancing anti-tumor effect comprising oxaliplatin liposome preparation, and anti-tumor agent comprising the liposome preparation - Google Patents

Abstract

The object is to further enhance the anti-tumor effect when oxaliplatin is used in combination with a mixed preparation containing tegafur, gimeracil and oteracil potassium. By using oxaliplatin in the form of a liposome preparation, when the liposome preparation is used in combination with a mixed preparation containing tegafur, gimeracil and oteracil potassium, the anti-tumor effect can be significantly enhanced without increasing adverse side effects.

Description

Antitumor effect enhancer comprising oxaliplatin liposome preparation and antitumor agent comprising the liposome preparation

Technical Field

The present invention relates to an antitumor effect enhancer comprising an oxaliplatin (oxaliplatin) liposome preparation. The invention also relates to antitumor agents comprising the liposomal formulation.

Background

Many anticancer agents have been developed and used in the medical field so far. For example, tegafur (tegafur) is an anticancer agent which is activated in vivo and gradually releases an active form, i.e., 5-fluorouracil (hereinafter referred to as "5-FU"), thus alleviating toxicity or side effects exhibited by 5-FU. A combination comprising three agents of tegafur, gimeracil and oteracil potassium (trade name: TS-1, molar ratio of tegafur/gimeracil/oteracil potassium =1:0.4:1, manufactured by Dapeng Pharmaceutical industries, Ltd., hereinafter the combination is referred to as TS-1) has a stronger antitumor effect because gimeracil inhibits degradation of 5-FU. In this formulation, the therapeutic effect is improved because the oteracil potassium specifically inhibits the occurrence of gastrointestinal toxicity which may be accompanied by the enhanced antitumor effects achieved by both tegafur and gimeracil agents. Therefore, TS-1 contributes to the treatment of various malignant tumors (patent document 1).

However, therapeutic agents and methods of treatment with stronger therapeutic effects sufficient to prolong the survival of cancer patients are still considered necessary. Combination chemotherapy (combination therapy) of a plurality of agents whose antitumor effects and/or action mechanisms of side effects are different from each other attempts to improve the treatment effect and some combination therapies do contribute to improvement of cancer treatment (see, for example, patent documents 2,3 and 4). For example, oxaliplatin exhibits low antitumor effect when used alone and is therefore used in combination with other agents. Combination therapy using 5-fluorouracil and folinate (hereinafter folinate is referred to as LV) (FOLFOX) is commonly used worldwide (see, for example, non-patent documents 1,2 and 3). However, the use of FOLFOX is complicated, and the prolonged period of infusion of 5-fluorouracil reduces the quality of life of patients, because continuous intravenous infusion brings physical constraints, and in addition, there is a high medical cost. Therefore, the development of better combination therapies with oxaliplatin is ongoing worldwide. As an example, a combination therapy (XELOX) using oxaliplatin and capecitabine (capecitabine) (trade name: hiloda Xeloda) which is an oral fluorinated pyrimidine is reported to provide almost the same antitumor effect as FOLFOX (see, for example, non-patent document 4). As the new method has shown that TS-1 and oxaliplatin provide a significant antitumor effect, in this case, the method has superior therapeutic efficacy compared to the case where oxaliplatin is combined with capecitabine (patent document 5). However, a stronger therapeutic effect is still required.

As described above, oxaliplatin exhibits a low antitumor effect when used alone, one of the reasons being that accumulation of the agent in tumor tissues is low. When an antitumor agent is administered, it can rapidly disappear from the blood circulation or be distributed to healthy organs, and therefore, the antitumor agent cannot be effectively accumulated in tumor tissues. Therefore, many antitumor agents do not always exhibit sufficient antitumor activity, and they often have adverse effects (side effects) on normal tissues, causing serious toxicity. Enhancing the efficacy of antitumor agents is an important target of current cancer chemotherapy, and there is a high necessity to develop a Drug Delivery System (DDS) capable of effectively accumulating drugs in tumors.

Liposomes are closed vesicles containing phospholipids as the main component, which are derived from biological materials. Thus, when administered to a living body, the liposome exhibits low toxicity and low antigenicity. In addition, some reports indicate that encapsulation of a drug in liposomes enables control of the stability and biodistribution of the drug in blood, resulting in improvement in the delivery efficiency of its effective delivery to a target tissue (patent documents 6, 7, 8 and non-patent document 5). It is known that vesicles such as liposomes having a particle size of 100-200nm are efficiently accumulated in tumors because the neovasculature present in tumors shows relatively high permeability compared to the vasculature in healthy tissue (non-patent document 6).

Patent document 1: japanese patent No. 2614164

Patent document 2: japanese patent No. 2557303

Patent document 3: japanese unexamined patent publication No. 1996-

Patent document 4: japanese unexamined patent publication No. 2002-205945

Patent document 5: WO2005/120480

Patent document 6: WO95/24201

Patent document 7: japanese patent No. 3415131

Patent document 8: japanese unexamined patent publication No. 2006-248978

Non-patent document 1: journal of Clinical Oncology, Vol.22,23-30,2004

Non-patent document 2: journal of Clinical Oncology, Vol.21,2059-2069,2003

Non-patent document 3: journal of Clinical Oncology, Vol.18,2938-2947,2000

Non-patent document 4: journal of Clinical Oncology, Vol.22,2084-2091,2004

Non-patent document 5: journal of lipid Research, Vol.4,667-687,1994

Non-patent document 6: drug Delivery System, Vol.14,433-447,1999

Disclosure of the invention

Problems to be solved by the invention

The primary object of the present invention is to enhance the antitumor effect of a combination comprising tegafur, gimeracil and oteracil potassium without increasing toxicity. It is another object of the present invention to provide an antitumor agent and a kit which exhibit better antitumor effect than oxaliplatin in combination with an antitumor agent containing tegafur, gimeracil, oteracil potassium.

Means for solving the problems

In view of the above-mentioned prior art, the inventors have conducted extensive studies to develop a method for treating cancer that can significantly contribute to prolonging patient survival. As a result, l-OHP-containing liposomes which are liposome preparations stably encapsulating a platinum complex, namely cis-oxalato (1R, 2R-diaminocyclohexane) platinum (II) (common name: oxaliplatin, trade name: lexadine (Eloxatin) or eloplatin (Elplat), hereinafter referred to as l-OHP), and which have appropriately adjusted particle diameters were developed. The antitumor effect of the combination drug comprising tegafur, gimeracil, oteracil potassium can be significantly improved without increasing side effects by combining with l-OHP-containing liposomes. Based on these new findings, the present invention has been achieved.

Specifically, the present invention provides an antitumor effect potentiator, an antitumor agent kit, and the like, as described below.

An anti-tumor agent comprising, in combination, a liposomal formulation that encapsulates oxaliplatin within liposomes, and a combination comprising tegafur, gimeracil, and oteracil potassium.

The antitumor agent according to claim 1, wherein at least one of the lipid components constituting the liposome is a phospholipid.

The antitumor agent according to item 3, wherein the membrane surface of the liposome is modified with polyethylene glycol, a glycerol polymer or a cationic lipid.

The antitumor agent according to any one of claims 1 to 3, wherein oxaliplatin and tegafur are used in a ratio of 0.1 to 5 mol: 1 mol.

The antitumor agent according to claim 5, wherein the combination comprising tegafur, gimeracil and oteracil potassium is used in a ratio of 0.1 to 5 mol: 1 mol of gimeracil to tegafur, and in a ratio of 0.1 to 5 mol: 1 mol of oteracil potassium to tegafur.

The antitumor agent according to item 6, wherein the antitumor agent is composed as a kit comprising a liposome preparation containing oxaliplatin and a combination containing tegafur, gimeracil and oteracil potassium.

The anti-tumor agent according to item 7, wherein the liposome preparation is administered intravenously, intraperitoneally, intramuscularly or subcutaneously, and the combination comprising tegafur, gimeracil and oteracil potassium is administered orally.

An antitumor effect potentiator, which is a liposome preparation containing oxaliplatin in an effective amount in a liposome, for enhancing the antitumor activity of a therapeutically effective amount of a combination comprising tegafur, gimeracil and oteracil potassium.

The antitumor effect enhancer according to claim 9, wherein at least one of the lipid components constituting the liposome is a phospholipid.

The antitumor effect enhancer according to claim 9, wherein the membrane surface of the liposome is modified with polyethylene glycol, a glycerol polymer or a cationic lipid.

The antitumor effect enhancer according to any one of claims 8 to 10, wherein oxaliplatin and tegafur are used in a ratio of 0.1 to 5 mol: 1 mol.

The antitumor effect enhancer according to item 12, wherein in the combination comprising tegafur, gimeracil and oteracil potassium, the ratio of gimeracil to tegafur used is 0.1 to 5 mol: 1 mol, and the ratio of oteracil potassium to tegafur used is 0.1 to 5 mol: 1 mol.

The use of a liposome preparation obtained by encapsulating oxaliplatin in a liposome, and a combination drug containing tegafur, gimeracil, and oteracil potassium for producing an antitumor agent.

Use of a liposome preparation obtained by encapsulating oxaliplatin in a liposome, for producing an antitumor effect potentiator.

A method for treating cancer, characterized in that an effective amount of a liposome preparation encapsulating oxaliplatin, and a combination comprising tegafur, gimeracil and oteracil potassium are administered to a cancer patient.

Effects of the invention

The antitumor effect enhancer of the present invention can enhance the antitumor effect of a combination comprising three agents of tegafur, gimeracil and oteracil potassium without increasing toxicity, as compared with the case of using l-OHP in combination, which is known as an antitumor agent. Therefore, the antitumor agent and antitumor agent kit of the present invention achieve a remarkable effect of exhibiting a better antitumor effect without increasing the toxicity level, as compared with the combination therapy using a combination comprising three agents of tegafur, gimeracil and oteracil potassium in combination with l-OHP. The present invention is intended to be applied to therapeutic methods that further prolong the survival of cancer patients.

Brief description of the drawings

Fig. 1 shows the relative tumor volume increase in test example 1.

Fig. 2 shows the change in body weight of mice in test example 1.

Fig. 3 shows the relative tumor volume increase in test example 2.

Fig. 4 shows the change in body weight of mice in test example 2.

Figure 5 shows the survival of mice within days after cell inoculation in test example 3.

Figure 6 shows the survival of mice within days after cell inoculation in test example 4.

Fig. 7 shows the relative tumor volume increase in test example 5.

Fig. 8 shows the relative tumor volume increase in test example 6.

Fig. 9 shows the change in body weight of mice in test example 6.

Fig. 10 shows the survival of mice within days after cell inoculation in test example 7.

Best Mode for Carrying Out The Invention

(1)Antitumor effect potentiator

The present invention provides an antitumor effect potentiator for enhancing the antitumor activity of a therapeutically effective amount of a combination comprising tegafur, gimeracil, and oteracil potassium. An antitumor effect potentiator is prepared as a liposome preparation obtained by encapsulating oxaliplatin (l-OHP) in a therapeutically effective amount for improving an antitumor effect in a liposome composed of at least one lipid component.

(1-1) Liposome preparation

l-OHP

l-OHP is a known compound of platinum complex. l-OHP binds to DNA within cancer cells and then induces disruption of DNA function and DNA strand breaks, resulting in the agent becoming cytotoxic to cancer cells. The l-OHP can be produced according to a known method, for example, as disclosed in Japanese examined patent publication No. 1985-41077.

Liposomes

The liposome used in the liposome preparation of the present invention is a vesicle formed of a phospholipid dispersed in water, which is a main component of a cell membrane, and has an inner aqueous phase surrounded by a lipid membrane. Liposomes can be divided into three classes, i.e. multilamellar vesicles, depending on the particle size and number of lipid layers: MLV, large unilamellar vesicles: LUVs and small unilamellar vesicles: an SUV. Any type of liposome can be used in the present invention. The liposomes used in the present invention need to have a stable form before and after administration to the body. Examples of the liposome-forming phospholipid include hydrogenated purified egg yolk phosphatidylcholine (phase transition temperature of 50 ℃ C., hereinafter referred to as HEPC), hydrogenated purified soybean phosphatidylcholine (phase transition temperature of about 55 ℃ C., hereinafter referred to as HSPC), dipalmitoyl phosphatidylcholine (phase transition temperature of about 41 ℃ C., hereinafter referred to as DPPC), distearoylphosphatidylcholine (phase transition temperature of about 58 ℃ C., hereinafter referred to as DSPC) and 1-palmitoyl-2-oleoyl phosphatidylcholine (phase transition temperature of about-3 ℃ C.). Among these, HEPC, HSPC, DPPC and DSPC are preferred.

In the present specification, hydrogenated purified egg yolk phosphatidylcholine means that obtained by hydrogenation of egg yolk-derived phosphatidylcholine. An example of hydrogenated purified egg yolk phosphatidylcholine for use in the preferred embodiment comprises phosphatidylcholine as the major constituent, the acyl moiety of which is C from a saturated straight chain fatty acid_16-18An acyl group. In the present invention, hydrogenated purified egg yolk phosphatidylcholine is obtained by purifying hydrogenated egg yolk phosphatidylcholine; also, for example, those of hydrogenated egg yolk phosphatidylcholine having a purity of not less than 80% and preferably those having a purity of not less than 90% are usable.

In the present specification, hydrogenated soybean phosphatidylcholine means one obtained by hydrogenation of phosphatidylcholine derived from soybean. An example of hydrogenated soy phosphatidylcholine for use in the preferred embodiment comprises phosphatidylcholine as the major constituent, the acyl moiety of which is C from a saturated straight chain fatty acid_16-18An acyl group. In the present invention, hydrogenated purified soybean phosphatidylcholine is obtained by refining hydrogenated soybean phosphatidylcholine; and, for example, those of no purityHydrogenated soy phosphatidylcholine less than 80% and preferably those not less than 90% pure are useful.

These phospholipids may be used alone or in combination of two or more. By using phospholipids with different phase transition temperatures, the mobility of the liposomal bilayer lipid membrane can be altered. This allows selection of the most suitable phospholipid in view of encapsulation efficiency, stability in pharmaceutical formulations, in vivo kinetics after administration, and the like.

In addition to these phospholipids, the liposomes used in the present invention are preferably mixed with a stabilizing agent, such as cholesterol, which is reported to improve the stability of the liposomes or their derivatives. In addition, if necessary, stability in blood, tissue distribution, migration to tumor tissue, and the like of liposomes can be further improved by modifying the liposome membrane surface with a lipid containing an amino group, an amidino group, a guanidino group, or a similar basic functional group (hereinafter referred to as cationic lipid), or a peptide, lectin, antibody, saccharide, glycoprotein, glycolipid, or the like, from a ligand selected from polyethylene glycol, a glycerol polymer, or a similar hydrophilic high molecular material.

In the present invention, "polyethylene glycol" includes not only unsubstituted polyethylene glycol but also derivatives thereof in which a lipophilic (hydrophobic) side chain forms a covalent bond. Specific examples of lipophilic side chains include alkyl chains, phospholipids and cholesterol. Various derivatives of polyethylene glycol that are commonly used to improve the stability of liposomes can be used in the present invention. Similarly, the "glycerol polymer (polyglycerin)" of the present invention includes not only unsubstituted glycerol polymers but also derivatives thereof whose lipophilic (hydrophobic) side chains form covalent bonds. Specific examples of lipophilic side chains include alkyl chains, phospholipids and cholesterol. Various glycerol polymer derivatives commonly used to improve the stability of liposomes can be used in the present invention. In addition, glycerol, glucose, sodium chloride, etc. can also be added as isotonic agent. In addition, preservatives such as benzoic acid esters, chlorobutanol, benzyl alcohol, and propylene glycol.

Liposome formulations

The liposomal formulations of the invention can be produced by known methods. Examples of known methods for producing liposome preparations include a reverse evaporation method (Proc. Natl. Acad. Sci. USA, Vol.75,4194,1978, WO97/48398), a freeze-thaw method (Arch. biochem. Biophys, Vol.212,186,1981), a pH gradient method (biochem. Biophys. acta, Vol.816,294,1985, Japanese unexamined patent publication No. 1995-165560), and the like.

In these methods, when the reverse evaporation method is used, the liposome preparation of the present invention is produced according to the following procedure. For example, the lipid component is dissolved in chloroform, ether, ethanol or the like, and the resulting dissolved substance is placed in a pear-shaped bottle. The solvent was removed by evaporation under reduced pressure to form a lipid film. Subsequently, a mixture solution containing chloroform and diethyl ether was added to dissolve the film, and the chloroform/diethyl ether ratio was 1/2. An aqueous solution containing the active agent was added thereto, and the mixture was sonicated at 25 ℃ for 15 minutes to obtain an emulsion. The organic phase of the resulting emulsion was removed by evaporation in a rotary evaporator for one hour while vortexing to convert the w/o emulsion to an o/w emulsion. Liposomes are thereby formed and the agent is encapsulated in the liposomes. The above process allows for the encapsulation of the agent in the liposome.

The particle size of liposomes has been reported to strongly influence the biodistribution and tumor accumulation of their payloads (biol. pharm. bull., vol.17,935, 1994). In the present invention, particle sizing is preferably performed to obtain the desired, uniform size of liposomes having the agent contained therein. For example, the particle size of the liposome may be adjusted to have an average particle diameter of about 100-200nm by sonication using a biodissruptor (manufactured by Nippon Seiki co., Ltd., etc.), or high-pressure emulsification using a nanomizer (manufactured by Yoshida Kikai co., Ltd.), or the like. Alternatively, the solution containing liposomes may be sized using different polycarbonate membrane filters (0.4 μm, 0.2 μm, 0.1 μm and 0.08 μm) under nitrogen pressure, and the particle size of the liposomes may be adjusted to have an average particle diameter of about 100-300 nm.

In the present invention, the term "average Particle diameter" means an average Particle diameter measured by a light scattering method using a NICOMP370HPL submicron Particle analyzer (manufactured by Particle Sizing systems, inc.).

If necessary, the stability in blood, tissue distribution and tumor localization of liposomes can be further improved by modifying the liposome membrane surface with polyethylene glycol, glycerol polymers, cationic lipids or ligands such as peptides, lectins, antibodies, sugars, glycoproteins or glycolipids.

The liposome preparation of the present invention can also be produced by the method of patent document 7 (Japanese patent No. 3415131) or patent document 8 (Japanese unexamined patent publication No. 2006-248978), which disclose oxaliplatin (l-OHP) liposome preparations.

In a preferred embodiment, the liposome preparation of the present invention is produced using a solution as an l-OHP solution obtained by dissolving l-OHP into a 1-10% glucose solution in such a manner that the concentration of l-OHP becomes 1-20 mg/ml.

The l-OHP-containing liposome preparation thus obtained may be subjected to ultracentrifugation, gel filtration, ultrafiltration and dialysis, as necessary. These treatments may be suitably performed alone or in combination, thereby removing agents that are not encapsulated in the liposomes.

The l-OHP-containing liposome preparation obtained by the above method can be used as it is. However, in view of storage time, conditions, etc., the l-OHP-containing liposome preparation may be lyophilized after adding excipients such as mannitol, trehalose, lactose, glycine. Alternatively, the l-OHP-containing liposome preparation may be cryopreserved after adding a cryopreservative such as glycerol.

In a preferred embodiment, the l-OHP liposome formulation contains oxaliplatin in an amount of 1-50 μ g/mg lipid, and preferably 5-40 μ g/mg lipid.

The l-OHP-containing liposome preparation of the present invention has an average particle diameter of preferably 50 to 300nm and more preferably 80 to 200 nm.

The l-OHP-containing liposome preparation is usually suspended or diluted with a physiologically acceptable aqueous solution and then used as an injection preparation (intravenous, intraperitoneal, intramuscular or subcutaneous administration preparation); however, the l-OHP-containing liposome preparation can also be used as an oral preparation, nasal drops, inhalant, suppository, transdermal preparation, transmucosal preparation, etc. In this case, the l-OHP-containing liposome preparation is formed into a preparation composition using a suitable carrier according to a usual method. Carriers useful herein are those commonly used in conventional pharmaceutical formulations. Specific examples thereof include excipients, binders, disintegrants, lubricants, colorants, flavoring agents, surfactants, and the like.

Hereinafter, examples of combinations comprising tegafur, gimeracil and oteracil potassium, the antitumor effect of which can be potentiated by the antitumor effect potentiator of the present invention, are described.

(1-2) combination drug containing tegafur, gimeracil and oteracil potassium

Tegafur

Tegafur (common name, chemical name: 5-fluoro-1- (2-tetrahydrofuryl) -2,4- (1H,3H) -pyrimidinedione) is a known compound which is activated in vivo to release 5-FU, the active form exerting antitumor activity. Tegafur can be produced according to a known method, for example, the method disclosed in Japanese examined patent publication No. 1974-10510.

Gimeracil

Gimeracil (common name, chemical name: 2, 4-dihydroxy-5-chloropyridine) is also a known compound and does not exhibit any antitumor activity per se. However, it can inhibit the metabolic inactivation of 5-FU in vivo, resulting in an increase in the antitumor effect of 5-FU.

Potassium Oxazinate

Potassium oxonate (common name, chemical name: potassium 1,2,3, 4-tetrahydro-2, 4-dioxo-1, 3, 5-triazine-6-carboxylate) is also a known compound, which, although it does not exhibit any antitumor activity per se, mainly resides in the gastrointestinal tract, inhibits the activation of 5-FU in the gastrointestinal tract, thereby preventing gastrointestinal disorders caused by 5-FU.

With respect to a combination drug containing three ingredients, i.e., tegafur, gimeracil and oteracil potassium as active ingredients, the ratio of each active ingredient may be within the range described in connection with known pharmaceutical agents, such as disclosed in, for example, patent publication No. 2614164. The proportions are generally as follows: gimeracil is used in a ratio of about 0.1 to 5 moles and preferably about 0.2 to 1.5 moles, and Potassium Oxonate is used in a ratio of about 0.1 to 5 moles and preferably about 0.2 to 2 moles, per 1 mole of Tegafur. A particularly preferred ratio of the three components is tegafur: gimeracil: the molar ratio of oteracil potassium =1:0.4: 1.

combinations containing tegafur, gimeracil and oteracil potassium as active ingredients may be prepared in pharmaceutical form comprising two or more medicaments, wherein each medicament contains one or any combination of the active ingredients; or prepared in a pharmaceutical form comprising a single agent containing all of the active ingredients. In either case, the combination is prepared as a pharmaceutical composition according to conventional methods using a suitable pharmaceutical carrier. Carriers useful herein are those commonly used in conventional pharmaceutical formulations, such as excipients, binders, disintegrants, lubricants, colorants, flavoring agents, surfactants, and the like.

When a combination drug comprising two or more agents in a pharmaceutical form is used, each agent may be administered simultaneously, or one agent may be administered at any time before or after the administration of the other agent. Preferably, all agents are administered simultaneously, or one agent is administered within 4 hours, more preferably within 2 hours, before or after the administration of the other agent.

(1-3) method of administration

The above-mentioned antitumor effect enhancer (i.e., l-OHP-containing liposome preparation) may be administered separately or simultaneously with a combination comprising three components, i.e., tegafur, gimeracil and oteracil potassium, as active ingredients, which is prepared in a unit dosage form. More specifically, the antitumor effect enhancer of the present invention can be administered simultaneously with the antitumor agent or at any time before or after the administration of the antitumor agent, the antitumor agent containing three components, i.e., tegafur, gimeracil and oteracil potassium, as active ingredients. Preferably, the antitumor effect enhancer is administered simultaneously with the antitumor agent, or within 4 hours before or after the administration of the antitumor agent, preferably within 2 hours before or after the administration of the antitumor agent. When the administration is continued, the frequency of administration and the time interval between administrations of the antitumor agent and the antitumor effect enhancer should be appropriately selected.

When the liposome preparation containing l-OHP is administered simultaneously or separately with an antitumor preparation containing three active ingredients of tegafur, gimeracil and oteracil potassium, the antitumor effect enhancer is preferably administered in an amount such that the amount of l-OHP is about 0.1 to 5 moles, preferably about 0.1 to 3 moles, more preferably about 0.2 to 2 moles per 1 mole of tegafur.

(2) Antitumor agent

The present invention provides an antitumor agent comprising a combination comprising three active ingredients of tegafur, gimeracil and oteracil potassium, and an antitumor effect enhancer (liposome preparation containing l-OHP) in combination therewith. The antitumor agent may be a pharmaceutical preparation comprising a plurality of agents each containing the above-mentioned four components, i.e., one of the above-mentioned three components and l-OHP-containing liposome, or each agent containing any combination of these components; alternatively, the antineoplastic agent may be in the form of a drug comprising a single agent that includes all of the ingredients. More specifically, the antitumor agent of the present invention may be formulated into a single dosage form containing all of the above 4 components, or a multiple dosage form containing a medicament containing 1 to 3 components and one or more medicaments containing the remaining components. A particularly preferred example is a two-part formulation in which a medicament containing three components of tegafur, gimeracil and oteracil potassium as active ingredients and a medicament containing l-OHP encapsulated in a liposome preparation are presented as separate dosage forms.

Regarding the antitumor agent, the ratio of the constituent components is not limited whether it is composed of a single agent or a plurality of agents. Generally, Gimeracil is used in a ratio of about 0.1 to 5 moles and preferably about 0.2 to 1.5 moles per 1 mole of tegafur; potassium oxonate is used in a proportion of about 0.1 to 5 moles and preferably about 0.2 to 2 moles; and l-OHP is used in a ratio of about 0.1 to 5 moles, preferably about 0.1 to 3 moles, more preferably about 0.2 to 2 moles. In particular, the composition is preferably in a molar ratio of tegafur: gimeracil: potassium oxonate: l-OHP = about 1:0.4: 1: 0.1-5, more preferably about 1:0.4: 1: 0.1 to 3, particularly preferably about 1:0.4: 1: 0.2-2. When the antitumor agent is in two dosage forms in which a medicament containing three components of tegafur, gimeracil and oteracil potassium as active ingredients and l-OHP-containing liposomes are presented as the above-mentioned separate dosage forms, the antitumor agent preferably comprises a combination drug and a pharmaceutical preparation, the combination drug containing the components in a molar ratio of 1:0.4:1, comprising l-OHP in a ratio of about 0.1 to 5 moles, preferably about 0.1 to 3 moles, more preferably about 0.2 to 2 moles, per 1 mole of tegafur.

The active ingredient may be prepared as a pharmaceutical composition using suitable pharmaceutical carriers according to standard procedures. Carriers useful herein are those commonly used in conventional pharmaceutical formulations, such as excipients, binders, disintegrants, lubricants, colorants, flavoring agents, surfactants, and the like.

When an antitumor preparation having multiple dosage forms and containing two or more agents as described above is used, each agent may be administered simultaneously, or each agent may be administered at any time before or after the administration of the other agent. Preferably, all agents are administered simultaneously, or one agent is administered within 4 hours, more preferably within 2 hours, before or after the administration of the other agent.

(3) Reagent kit

The present invention provides a kit comprising a liposome preparation containing l-OHP and a combination drug containing tegafur, gimeracil and oteracil potassium. More specifically, the present invention provides a kit for the treatment of cancer in a mammal, said kit comprising:

(a) an antitumor composition comprising tegafur in a therapeutically effective amount, gimeracil in an amount effective for enhancing antitumor effect and oteracil potassium in an amount effective for inhibiting side effects, and

(b) a liposome preparation encapsulating an effective amount of l-OHP for enhancing antitumor effect.

The composition contained in the kit may be in any known pharmaceutical form. The composition is generally placed in any of the usual containers, depending on its pharmaceutical form.

The kit is for treating cancer in a mammal and comprises:

(i) a therapeutically effective amount of tegafur,

(ii) gimeracil in an amount effective to enhance the anti-tumor effect,

(iii) potassium oxonate in an amount effective to inhibit side reactions; these are all part of an anti-tumor composition, and

(iv) liposomes containing l-OHP in an amount effective to enhance the anti-tumor effect.

The kit contains at least 2 containers for these components, and tegafur and l-OHP are packaged in separate containers. The above-mentioned components (i) to (iv) are preferably in a pharmaceutical form prepared in combination with a pharmaceutically acceptable carrier. With regard to the above-mentioned kit, as long as the components (i) and (iv) are stored in separate containers, the components (ii) and (iii) may be independently stored in a container separate from the container in which the above-mentioned two components are stored, or the components (ii) and (iii) may be independently mixed with the component (i) or (iv) to be stored in the same container. Preferred kits are: the agents containing components (i) to (iii) are stored in one container and the agent containing component (iv) is stored in another container.

The usable unit dosage form of the antitumor agent of the present invention for treating mammals including humans suffering from malignant tumors is not limited and may be appropriately selected depending on the purpose of treatment. Specific examples are parenteral forms such as injections, suppositories, eye drops, ointments, aerosols, etc.; oral forms such as tablets, coated tablets, powders, granules, capsules, liquids, pills, suspensions, emulsions, and the like. These dosage forms of the antineoplastic agent can be produced according to methods well known in the art.

The amounts of tegafur, gimeracil, oteracil potassium and l-OHP as active ingredients of the antitumor agent of the present invention are varied depending on the dosage form, administration route, dosage arrangement and the like, and are not limited, and thus can be appropriately selected. It is generally preferred that the proportion of active ingredient is from about 1% to about 70% by weight of the pharmaceutical formulation.

The administration method of the pharmaceutical preparation of the present invention is not limited and may be decided according to its form, age, sex, condition of the patient and other factors; thus, enteral, oral, rectal, intraoral, intraarterial, intravenous, transdermal or the like may be used. For example, tablets, pills, solutions, suspensions, emulsions, granules, capsules, and the like are administered orally; the injection is administered intra-arterially or intravenously; the suppository is administered in the rectum; the ointment is applied to skin, oral mucosa, etc. With regard to the pharmaceutical preparation of the present invention, the agents which may contain tegafur, gimeracil and oteracil potassium are administered orally, while the l-OHP agent encapsulated in liposome is administered intravenously.

The dose of each active ingredient in the present invention may be appropriately selected depending on the application, the age and sex of the patient, the degree of disease, and other factors. The antitumor effect enhancer and the antitumor preparation of the present invention can be administered in 1 to 4 doses per day.

In oral administration, the amount of the pharmaceutical preparation of the present invention to be administered is preferably used using the following ranges as standards: the amount of tegafur is about 0.1-100 mg/kg/day, preferably about 0.2-40 mg/kg/day, more preferably about 0.5-20 mg/kg/day; gimeracil in an amount of about 0.02-30 mg/kg/day, preferably about 0.05-12 mg/kg/day, more preferably about 0.1-6 mg/kg/day; the amount of Potassium Oxonate is from about 0.1 to 100 mg/kg/day, preferably from about 0.2 to 40 mg/kg/day, more preferably from about 0.5 to 20 mg/kg/day; and l-OHP in an amount of about 0.08-200 mg/kg/day, preferably about 0.15-80 mg/kg/day, more preferably about 0.4-40 mg/kg/day.

When the pharmaceutical preparation is in an injectable form, it can be gradually administered to an adult human over a period of 5 minutes or more, usually in amounts equivalent to about 0.1 to 100 mg/kg/day of tegafur and about 0.08 to 200 mg/kg/day of l-OHP, and the pharmaceutical preparation may be diluted with an aqueous glucose solution as necessary. When the pharmaceutical preparation of the present invention is in the form of suppositories, it is administered to adults 1 or 2 times per day by inserting it into the rectum, at intervals of 6 to 12 hours, usually in amounts corresponding to about 0.1 to 100 mg/kg/day of tegafur and about 0.08 to 200 mg/kg/day of l-OHP.

The type of malignancy that can be treated by administration of the pharmaceutical preparation of the present invention is not limited as long as the active form, i.e., 5-FU, reacts therein; such as lung cancer, stomach cancer, colon cancer, rectal cancer, esophageal cancer, breast cancer, head and neck cancer, liver cancer, gallbladder/bile duct cancer, pancreatic cancer, uterine cancer, cervical cancer, ovarian cancer, kidney cancer, bladder cancer, prostate cancer, pharyngeal cancer, brain tumor, leukemia, melanoma, malignant lymphoma, etc. In particular, the pharmaceutical preparation of the present invention can be expected to have a significant effect on colon cancer, kidney cancer, stomach cancer, esophageal cancer, breast cancer, and head and neck cancer. In addition, significant effects can also be expected on typical drug-resistant tumors and tumors in which positive drug resistance is occurring.

Examples

Examples and test examples are given below to illustrate the present invention in more detail, but the scope of the present invention is not limited by these examples and test examples.

Example 1

DPPC (L- α -dipalmitoylphosphatidylcholine, Coatsome MC-6060, manufactured by NOF Co.), cholesterol (Special grade, manufactured by Wako Pure Chemical Industries, Ltd.), and mPEG₂₀₀₀DSPE (1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-n- [ methoxy (polyethylene glycol) -2000]Sunbright DSPE-020CN, manufactured by NOF corporation) was dissolved in chloroform to give concentrations of 20mM, 50mM and 5mM, respectively. Subsequently, 5ml of 20mM DPPC solution, 0.5ml of 50mM cholesterol solution and 1ml of 5mM mPEG₂₀₀₀The DSPE solution is placed in a pear-shaped bottle. While reducing the pressure using an evaporator, chloroform was removed to form a lipid film on the bottom of the pear-shaped bottle. The lipid film was dissolved with 6ml chloroform/diethyl ether (volume ratio 1: 2). l-OHP was dissolved in a 5% glucose solution so that the concentration of l-OHP became 8 mg/ml. The thus prepared l-OHP solution (2ml) was added to the lipid solution, followed by mixing. The resulting mixture was sonicated at 25 ℃ for 15 minutes to form a w/o emulsion, and the organic phase was removed using an evaporator under reduced pressure while vortexing; thus encapsulating the l-OHP in liposomes. Sonication of the particle size was performed using a biodissruptor (Nippon Seiki co., Ltd.) so that the average particle diameter of the liposomes containing therein the agent was slightly less than 200 nm. To remove the agent not encapsulated in the liposome, dialysis was performed at 4 ℃ for 2 hours using a dialysis cartridge (Slide-a-Lyzer dialysis cartridge, 10000MWCO, manufactured by seimer fisher Scientific Inc.) and a 5% glucose solution as an external solution while stirring with a magnetic stirrer. After changing the external solution, another dialysis was performed for 2 hours while stirringThus, a liposome preparation containing l-OHP was obtained. The liposome preparation containing l-OHP thus prepared had an average particle diameter of 185.2 ± 14.9nm (n = 5).

In the examples of the present invention, the average Particle diameter of the liposome preparation was measured by a light scattering method using a NICOMP370HPL submicron Particle analyzer (manufactured by Particle Sizing systems).

Example 2

HSPC (hydrogenated soybean phosphatidylcholine, Coatsome NC-21, manufactured by NOF corporation), cholesterol (Special grade, manufactured by Wako Pure Chemical Industries, Ltd.), and mPEG₂₀₀₀DSPE (1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-n- [ methoxy (polyethylene glycol) -2000]Sunbright DSPE-020CN, manufactured by NOF corporation) was dissolved in chloroform to give concentrations of 20mM, 50mM and 5mM, respectively. Subsequently, 5ml of 20mM HSPC solution, 1ml of 50mM cholesterol solution and 2ml of 5mM PEG₂₀₀₀The DSPE solution is placed in a pear-shaped bottle. While reducing the pressure using an evaporator, chloroform was removed to form a lipid film on the bottom of the pear-shaped bottle. The lipid film was dissolved with 6ml chloroform/diethyl ether (volume ratio 1: 2). l-OHP was dissolved in a 5% glucose solution so that the concentration of l-OHP became 8 mg/ml. The thus prepared l-OHP solution (2ml) was added to the lipid solution, followed by mixing. The resulting mixture was sonicated at 35 ℃ for 15 minutes to form a w/o emulsion, and the organic phase was removed using an evaporator under reduced pressure for about 1 hour while vortexing; whereby the l-OHP is encapsulated in liposomes. Thereafter, the particle size was controlled and the unencapsulated drug was removed in the same manner as in example 1, thereby obtaining a l-OHP liposome preparation. The liposome preparation containing l-OHP prepared in this way had an average particle diameter of 197.9 ± 22.0nm (n = 5).

Example 3

A liposome preparation containing l-OHP was prepared in the same manner as in example 1, except that PG-DSPE (Sunbright DSPE-PG10G, manufactured by NOF corporation) was used in place of mPEG₂₀₀₀-a DSPE. The liposome preparation containing l-OHP prepared in this way had an average particle diameter of 130.4 ± 48.3nm (n = 5).

Example 4

HSPC (hydrogenated soybean phosphatidylcholine, manufactured by NOF corporation), cholesterol (Special grade, manufactured by Wako Pure Chemical Industries, Ltd.), DC-6-14 (cationic lipid, O, O' -dimyristoyl-N- (. alpha. -trimethylacetamido) chlorodiethanolamine, manufactured by Sogo pharmaceutical Co., Ltd.), and mPEG2000-DSPE (1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000)]Sunbright DSPE-020CN, manufactured by NOF corporation) was dissolved in chloroform to give concentrations of 20mM, 50mM and 5mM, respectively. Subsequently, 5ml of 20mM HSPC chloroform solution, 1ml of 50mM cholesterol chloroform solution and 2ml of 5mM DC-6-14 and 5mM mPEG₂₀₀₀-the DSPE chloroform solution was placed in pear-shaped bottles. While reducing the pressure using an evaporator, chloroform was removed to form a lipid film on the bottom of the pear-shaped bottle. The lipid film was dissolved with 6ml chloroform/diethyl ether (volume ratio 1: 2). l-OHP was dissolved in a 5% glucose solution so that the concentration of l-OHP became 8 mg/ml. The thus prepared l-OHP solution (2ml) was added to the lipid solution, followed by mixing. The resulting mixture was sonicated at 35 ℃ for 15 minutes and the organic solvent was removed using an evaporator under reduced pressure for about 1 hour while vortexing; thus encapsulating the l-OHP in liposomes. Thereafter, the particle size was controlled and the unencapsulated drug was removed in the same manner as in example 1, thereby obtaining a l-OHP liposome preparation. The liposome preparation containing l-OHP thus prepared had an average particle diameter of 202.4 ± 14.7nm (n = 5).

Test example 1

Dosage form of pharmaceutical preparation

The l-OHP-containing liposome preparation obtained in example 1 (hereinafter referred to as DPPC liposome l-OHP) was used.

A preparation of l-OHP (hereinafter referred to as free l-OHP) was prepared by dissolving l-OHP in a 5% glucose solution.

TS-1 formulations (hereinafter referred to as TS-1) were prepared by dissolving the contents encapsulated in TS-1 capsules in water

Antitumor effect and side effects of pharmaceutical preparation

At 5% CO₂Highly metastatic lung cancer cells, Lewis Lung Carcinoma (LLC), were cultured in DMEM with 10% fetal bovine serum at 37 ℃. Containing 5X 10⁶0.1mL of LLC cells per cell/mL were injected subcutaneously into the backs of male C57BL/6 mice (5 weeks old, 20g in body weight) to obtain mice bearing solid tumors. It was confirmed that the tumor volume calculated by the following formula was more than 50mm 6 days from LLC cell infusion³。

DPPC liposome l-OHP or free l-OHP was administered intravenously at a dose of 4.2mg/kg l-OHP. TS-1 was administered orally at a dose of 6.9mg/kg of tegafur. DPPC liposome l-OHP and free l-OHP were administered on days 13 and 20 after cell inoculation, and TS-1 was administered daily from 6 days after cell inoculation until the end of the experiment.

From 6 days after cell inoculation, tumor volume was calculated according to the following formula. The antitumor effect was evaluated using the relative tumor volume ratio as an index.

Tumor volume =1/2 × a × b²

(a: major axis of tumor location, b: minor axis of tumor location)

Changes in mouse body weight were monitored as an indicator of side effects.

As is clear from the results shown in FIG. 1, the combination therapy of DPPC liposome l-OHP with TS-1 showed better antitumor effect than the combination of free l-OHP with TS-1. However, the results shown in FIG. 2 indicate that there was no significant difference in body weight change between TS-1 in combination with DPPC liposome l-OHP and TS-1 in combination with free l-OHP. No significant increase in toxicity was observed.

Test example 2

Antitumor effects and side effects were evaluated in the same manner as in test example 1, except that the l-OHP-containing liposome preparation obtained in example 2 (hereinafter referred to as HSPC liposome l-OHP) was used.

As is clear from the results shown in FIG. 3, the combination therapy of HSPC liposome l-OHP with TS-1 showed better antitumor effect than the combination of free l-OHP with TS-1. However, the results shown in FIG. 4 clearly show that there was no significant difference in body weight change between TS-1 in combination with HSPC liposome l-OHP and TS-1 in combination with free l-OHP. No significant increase in toxicity was observed.

Test example 3

A pharmaceutical preparation was prepared in the same manner as in test example 1. The resulting pharmaceutical formulation was administered to tumor-bearing mice, and the mice were monitored for days of survival.

It is clear from the results shown in FIG. 5 that when TS-1 was administered in combination with DPPC liposome l-OHP, the survival rate was higher than that in combination with free l-OHP.

Test example 4

A pharmaceutical preparation was prepared in the same manner as in test example 2. The resulting pharmaceutical formulation was administered to tumor-bearing mice, and the mice were monitored for days of survival.

It is clear from the results shown in FIG. 6 that when TS-1 is administered in combination with HSPC liposome l-OHP, the survival rate is higher than in combination with free l-OHP.

Test example 5

The antitumor effect was evaluated in the same manner as in test example 1 except that the l-OHP-containing liposome preparation obtained in example 3 (hereinafter referred to as PG10 liposome l-OHP) was used.

As is clear from the results shown in FIG. 7, PG10 liposome l-OHP in combination with TS-1 showed better antitumor effect than free l-OHP in combination with TS-1.

Test example 6

Dosage form of pharmaceutical preparation

The pharmaceutical preparation was formulated in the same manner as in test example 2.

Antitumor effect and side effects of pharmaceutical preparation

At 5% CO₂Mouse Colon cancer cells, Colon26, were cultured in DMEM with 10% fetal bovine serum at 37 ℃. Containing 2X 10⁷0.1mL of Colon26 cells per mL were injected subcutaneously into the back of male BALB/c mice (5 weeks old, 20g in body weight) to obtain mice bearing solid tumors. It was confirmed that 7 days after the Colon26 cell inoculation, the tumor volume calculated according to the following formula exceeded 50mm³。

HSPC liposome l-OHP or free l-OHP was administered intravenously at a dose of 4.2mg/kg in terms of l-OHP. TS-1 was administered orally at a dose of 6.9mg/kg of tegafur. HSPC liposome l-OHP and free l-OHP were administered on days 14 and 21 after cell inoculation, and TS-1 was administered daily from 7 days after cell inoculation until the end of the experiment.

Other antitumor effects and side effects were evaluated in the same manner as in test example 2.

It is clear from the results shown in FIG. 8 that the anti-tumor effect of the combination therapy of HSPC liposome l-OHP plus TS-1 is higher than that of the combination of free l-OHP plus TS-1. However, the results shown in FIG. 9 clearly indicate that there were no significant differences in body weight changes between TS-1 and HSPC liposome l-OHP and combination therapy of TS-1 and free l-OHP. That is, no significant increase in toxicity was observed.

Test example 7

The pharmaceutical preparation was formulated in the same manner as in test example 6. The resulting pharmaceutical formulation was administered to tumor-bearing mice, and the mice were monitored for days of survival.

It is clear from the results shown in figure 10 that the combination therapy with HSPC liposome l-OHP plus TS-1 shows a prolonged survival rate compared to the combination of free l-OHP plus TS-1.

The results shown above indicate that the antitumor effect and survival rate of l-OHP-containing liposomes in combination with TS-1 can be significantly increased by encapsulating l-OHP in liposomes, i.e., l-OHP-containing liposome preparations, while the toxicity level is almost the same as that of l-OHP and TS-1 administration. The increase in antitumor activity is probably due to the improved biodistribution and enhanced tumor localization of l-OHP as a result of its optimized lipidization (lipolysis).

The present invention discloses that the antitumor activity of a combination therapy of l-OHP plus a combination drug comprising tegafur, gimeracil and oteracil potassium can be potentiated by encapsulating l-OHP in a liposome preparation, and toxicity due to the lipidization of l-OHP has not been found. This suggests that combination therapy of l-OHP liposomal formulations with a combination containing tegafur, gimeracil and oteracil potassium is a promising cancer therapy.

Claims (15)

1. An antitumor agent comprising, in combination, a liposome preparation obtained by encapsulating oxaliplatin within liposomes, and a combination drug containing tegafur, gimeracil, and oteracil potassium.

2. The anti-tumor agent of claim 1, wherein at least one of the lipid components comprising the liposome is a phospholipid.

3. The anti-tumor agent of claim 2, wherein the membrane surface of the liposome is modified with polyethylene glycol, a glycerol polymer, or a cationic lipid.

4. An anti-tumour agent as claimed in any of claims 1 to 3 wherein oxaliplatin is used in a ratio to tegafur of 0.1-5 mole: 1 mole.

5. The antitumor agent according to claim 1, wherein the combination comprising tegafur, gimeracil and oteracil potassium is used in a ratio of 0.1-5 mol: 1 mol of gimeracil to tegafur, and in a ratio of 0.1-5 mol: 1 mol of oteracil potassium to tegafur.

6. The anti-neoplastic agent of claim 1, wherein the anti-neoplastic agent is comprised in a kit comprising a liposomal formulation comprising oxaliplatin and a combination comprising tegafur, gimeracil and oteracil potassium.

7. The anti-tumor agent according to claim 1, wherein the liposome preparation is administered intravenously, intraperitoneally, intramuscularly or subcutaneously and the combination comprising tegafur, gimeracil and oteracil potassium is administered orally.

8. An antitumor effect potentiator which is a liposome preparation containing an effective amount of oxaliplatin in liposomes and is for potentiating the antitumor activity of a therapeutically effective amount of a combination comprising tegafur, gimeracil, and oteracil potassium.

9. The antitumor effect enhancer as claimed in claim 8, wherein at least one of lipid components constituting said liposome is a phospholipid.

10. The antitumor effect enhancer as claimed in claim 9, wherein the membrane surface of the liposome is modified with polyethylene glycol, a glycerol polymer or a cationic lipid.

11. The antitumor effect enhancer as claimed in any one of claims 8 to 10, wherein oxaliplatin and tegafur are used in a ratio of 0.1-5 mol: 1 mol.

12. The antitumor effect enhancer as claimed in claim 8, wherein in the combination comprising tegafur, gimeracil and oteracil potassium, the ratio of gimeracil to tegafur used is 0.1-5 mol: 1 mol, and the ratio of oteracil potassium to tegafur used is 0.1-5 mol: 1 mol.

13. Use of a liposomal preparation obtained by encapsulating oxaliplatin within liposomes and a combination comprising tegafur, gimeracil and oteracil potassium for the production of an antitumor agent.

14. Use of a liposome preparation obtained by encapsulating oxaliplatin in a liposome for producing an antitumor effect potentiator.

15. A method for treating cancer characterized by administering to a cancer patient an effective dose of a liposome formulation containing oxaliplatin encapsulated within liposomes and a combination comprising tegafur, gimeracil and oteracil potassium, respectively.