Oral pharmaceutical composition containing pemetrexed and production method thereof

Abstract

The present invention relates to an oral pharmaceutical composition containing pemetrexed as a water-soluble anticancer drug and a method of manufacturing the same, and more particularly to an oral pharmaceutical composition, in which an ion-binding complex of pemetrexed and a bile acid derivative as an oral absorption enhancer is formed, mixed with a pharmaceutical additive, and then provided in the form of a capsule or a tablet through pressing or included in the internal water phase of a w/o/w (water-in-oil-in-water) multiple nanoemulsion, thus increasing the oral bioavailability of pemetrexed as the water-soluble anticancer drug, whereby pemetrexed, which is currently administered only in the form of an injection formulation, can be manufactured into a formulation capable of being orally administered, ultimately alleviating inconvenience and problems with use of injection formulations, improving patient compliance and contributing to a reduction in medical expenses.

Claims

1. An oral pharmaceutical composition, comprising an ion-binding complex comprising pemetrexed, a positively charged bile acid derivative and poloxamer, wherein the bile acid derivative is N.sup.α-deoxycholyl-L-lysyl-methylester (DCK).

2. The oral pharmaceutical composition of claim 1, wherein the DCK is used in an amount of 0.5 to 5 moles based on 1 mole of pemetrexed.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the blood drug concentration over time after intravenous injection of 5 mg/kg pemetrexed into rats;

(2) FIG. 2 shows the blood drug concentration over time after oral administration of 50 mg/kg pemetrexed or a capsule or tablet composition including a PMX/DCK ion-binding complex of Example 3, corresponding to 50 mg/kg pemetrexed, into rats;

(3) FIG. 3 shows the blood drug concentration over time after intravenous injection of 10 mg/kg pemetrexed into rats; and

(4) FIG. 4 shows the blood drug concentration over time after oral administration of 20 mg/kg pemetrexed or a nanoemulsion composition including a PMX/DCK ion-binding complex of Example 5, corresponding to 20 mg/kg pemetrexed, into rats.

MODE FOR INVENTION

(5) Hereinafter, a detailed description will be given of an oral pharmaceutical composition containing pemetrexed according to the present invention and a method of manufacturing the same.

(6) According to the present invention, the method of manufacturing an oral pharmaceutical granular composition containing pemetrexed comprising (a) dissolving pemetrexed in a solution including a dispersant, (b) forming an ion-binding complex by adding a positively charged bile acid derivative to the pemetrexed solution, (c) drying the ion-binding complex solution, (d) preparing granules by mixing the dried product with a binder and a diluent as pharmaceutical additives, and (e) adding the granules with a lubricant and filling the granules in a capsule or pressing the granules into a tablet.

(7) In addition, the method of manufacturing an oral pharmaceutical emulsion composition containing pemetrexed according to the present invention comprising (a) forming an ion-binding complex by adding a positively charged bile acid derivative to pemetrexed and a dispersant in a solution, (b) adding a primary surfactant and a primary auxiliary surfactant to the ion-binding complex solution, (c) preparing a water-in-oil (w/o) primary nanoemulsion by dispersing the mixture obtained in step (b) in a primary oil phase, and (d) preparing a water-in-oil-in-water (w/o/w) secondary nanoemulsion by adding the w/o primary nanoemulsion with a mixture of a secondary surfactant and a secondary auxiliary surfactant.

(8) Here, pemetrexed is a hydrophilic and negatively charged active material, and the pemetrexed aqueous solution is added with a bile acid derivative, which is a positively charged oral absorption enhancer, thus forming an ion-binding complex.

(9) The bile acid derivative is an amphiphilic material having a positive charge in an aqueous solution, and is preferably N.sup.α-deoxycholyl-L-lysyl-methylester (DCK) resulting from chemical bonding of L-lysine and deoxycholic acid. Moreover, it is preferably included in an amount of 0.5 to 5 moles, and more preferably 1 to 2 moles, based on 1 mole of pemetrexed.

(10) The DCK functions as an oral absorption enhancer, and is positively charged in an aqueous solution to form an ion-binding complex with the negatively charged hydrophilic active ingredient, thus increasing the lipophilicity of the hydrophilic drug molecule to thereby enhance the distribution thereof into the oil phase. Deoxycholic acid is an amphiphilic molecule composed of a hydrophobic α portion and a hydrophilic β portion, and is capable of increasing the membrane permeability of the drug by increasing the flexibility of the intestinal cell membrane and the solubility of the fat-soluble drug in the membrane by vertically binding to the membrane surface of the cell lipid bilayer and disturbing the acyl chain of the lipid matrix. Moreover, deoxycholic acid, which is a bile acid derivative coupled with the drug, may be recognized by a bile acid reabsorption transporter present on the surface of the intestinal membrane, which forms a high concentration gradient of the drug on the surface of the intestinal mucosa, thus accelerating the diffusion of the drug to the intestinal membrane. Therefore, the absorption enhancement effect due to the selective interaction of the ion-binding complex of the drug and the absorption enhancer with the bile acid reabsorption transporter may minimize the decrease in the action of the absorption enhancer diluted by the fluid present in the gastrointestinal tract after administration, making it possible to increase the gastrointestinal absorption rate of the hydrophilic drug even upon minimal use thereof, unlike conventional absorption enhancers that are simply added through physical mixing.

(11) In order to prepare the ion-binding complex formed in an aqueous solution phase into a solid powder phase, it may be additionally dried through hot-air drying, freeze-drying, spray drying, or the like. Moreover, a dispersant may be further added in the formation of the ion-binding complex in order to realize rapid redispersion in the aqueous solution of the ion-binding complex in a dry powder form.

(12) The dispersant may be selected from among monosaccharides, polysaccharides, dietary fiber, gums, surfactants and proteins, and is preferably selected from among poloxamer, polyvinylpyrrolidone, caprylocaproyl macrogol-8 glyceride (Labrasol™), Cremophor™, glycerol monocaprylocaprate (Capmul™ MCM), lauroyl macrogol-32 glyceride (Gelucire™ 44/14), Solutrol®, polysorbate (Tween), sorbitan monolaurate (Span), mannitol, sucrose, lactose, glucose, trehalose, glycerol, fructose, maltose, dextran, cyclodextrin, glycine, alanine, and lysine, and is more preferably selected from among poloxamer, polyvinylpyrrolidone, caprylocaproyl macrogol-8 glyceride (Labrasol™), cyclodextrin, lactose, and mannitol.

(13) The dispersant is preferably included in an amount of 0.1 to 100 parts by weight, and more preferably 0.1 to 60 parts by weight, based on 1 part by weight of pemetrexed.

(14) The w/o primary nanoemulsion may further include, in the internal water phase thereof, a hydrophilic active ingredient that may exhibit an anticancer synergistic effect in combination with the ion-binding complex, and the hydrophilic active ingredient may be selected from among 5-fluorouracil (5-FU) and leucovorin. By incorporating the hydrophilic active ingredient into the nanoemulsion formulation, it is possible to improve oral bioavailability by enhancing the gastrointestinal absorption rate of the drug.

(15) The primary oil phase used in the preparation of the w/o primary nanoemulsion is preferably at least one selected from the group consisting of silicone oil, ester oil, hydrocarbon oil, propylene glycol monocaprylate (Capryol® 90), propylene glycol dicaprylocaprate (Labrafac PG), oleoyl macrogol-6 glyceride (Labrafil® M1944 CS), lauroyl macrogol-6 glyceride (Labrafil® M2130 CS), linoleoyl macrogol-6 glyceride (Labrafil® M2125 CS), heavy-chain triglyceride (Labrafac), oleic acid, stearic acid, glyceryl behenate (Compritol® 888), glycerol monostearate, and castor oil.

(16) The amount of the primary oil phase in the w/o/w secondary nanoemulsion is preferably 0.1 to 40 wt %, and more preferably 1 to 20 wt %, based on the total weight of the composition. If the amount of the oil phase is less than 1 wt % or exceeds 20 wt %, the particles of the emulsion may be enlarged, thus deteriorating emulsion stability. Here, the amount of the oil phase means the total amount of the oil phase relative to the total weight of the w/o/w secondary nanoemulsion including the secondary external water phase, but not including an emulsion stabilizer.

(17) The w/o/w secondary nanoemulsion in step (d) may further include, in the primary oil phase thereof, a fat-soluble active ingredient capable of improving the pharmacological therapeutic efficacy of the ion-binding complex, and the fat-soluble active ingredient may be a poorly soluble anticancer agent such as paclitaxel, docetaxel or doxorubicin, curcumin, quercetin, a natural extract containing curcumin or quercetin as an active ingredient, or mixtures thereof. The fat-soluble active ingredient is included in the oil phase of the nanoemulsion formulation, thus solubilizing the active ingredient to thereby increase the gastrointestinal absorption rate thereof, ultimately improving oral bioavailability and pharmacological activity.

(18) The primary and secondary surfactants allow the internal water phase in which the ion-binding complex is dispersed to be dispersed in the oil phase and also allow the w/o primary nanoemulsion to be dispersed well in the external water phase of the w/o/w secondary nanoemulsion. It is preferred that each of the primary and secondary surfactants independently be at least one selected from the group consisting of poloxamer, caprylocaproyl macrogol-8 glyceride (Labrasol™), Cremophor™, glycerol monocaprylocaprate (Capmul MCM), lauroyl macrogol-32 glyceride (Gelucire™ 44/14), Solutrol®, polysorbate (Tween), and sorbitan monolaurate (Span).

(19) The primary and secondary auxiliary surfactants serve to reduce the surface energy so that the internal water phase in which the ion-binding complex is dispersed may be dispersed in the oil phase by the surfactant, and also allow the w/o primary nanoemulsion to be dispersed well in the external water phase of the w/o/w secondary nanoemulsion by the surfactant. It is preferred that each of the primary and secondary auxiliary surfactants independently be at least one selected from the group consisting of diethylene glycol monoethyl ether (Transcutol HP), polysorbate, polyethylene glycol, butylene glycol, propylene glycol, ethanol, and isopropanol.

(20) The amount of the mixture of the primary surfactant and the primary auxiliary surfactant and the mixture of the secondary surfactant and the secondary auxiliary surfactant is preferably 0.1 to 40 wt %, and more preferably 1 to 20 wt %, based on the total weight of the composition. If the amount thereof is less than 1 wt % or exceeds 20 wt %, the stability of the emulsion may decrease. Here, the amount of the mixture of the primary surfactant and the primary auxiliary surfactant and the mixture of the secondary surfactant and the secondary auxiliary surfactant means the total amount of the primary and secondary surfactants and the primary and secondary auxiliary surfactants relative to the total weight of the w/o/w secondary nanoemulsion including the secondary external water phase.

(21) Moreover, the primary auxiliary surfactant and the secondary auxiliary surfactant are mixed with the primary surfactant and the secondary surfactant, respectively, and are each independently mixed at a weight ratio of preferably 1:0.1 to 1:10, and more preferably 1:0.5 to 1:2, relative to the primary surfactant and the secondary surfactant, respectively.

(22) In addition, the present invention pertains to an oral pharmaceutical composition containing pemetrexed manufactured by the above method.

(23) As a drug transporter of the ion-binding complex of pemetrexed and bile acid derivative according to the present invention, a w/o/w multiple nanoemulsion form may be used. The oil phase in this system protects the ion-binding complex in the gastrointestinal tract, minimizes the dilution effect of the absorption enhancer, and enables high dispersibility of the amphipathic ion-binding complex in the gastrointestinal tract after administration. Moreover, the multiple nanoemulsion is known to enhance the absorption of the drug by modifying the structure and fluidity of the intestinal membrane in the gastrointestinal tract. In particular, when 5-fluorouracil, which is a hydrophilic drug that cannot be ionically complexed with a bile acid derivative, is also loaded in the internal water phase, the intestinal membrane permeability and oral bioavailability of the drug may be improved by the multiple nanoemulsion.

(24) Moreover, Labrasol™ and Cremophor™ EL, which are surfactants used for multiple nanoemulsion, may be used to disperse the primary internal water phase in the oil phase or to disperse the w/o primary nanoemulsion in the external water phase of the w/o/w secondary nanoemulsion. Labrasol™ is known to interact with F-actin (filamentous actin) and ZO-1 (zonula occludens-1) to disassemble tight junctions between small intestinal epithelial cells, and in the case of Cremophor™ EL, it was confirmed that it is possible to promote absorption by increasing the fluidity of the cell membrane and loosening tight junctions. Therefore, the membrane permeability of the hydrophilic active ingredient, 5-FU, may be increased through the intercellular path by the application of a surfactant that induces a decrease in binding between small intestinal epithelial cells. Moreover, Cremophor™ EL may bind to the hydrophobic region of P-gp, which results in a change in the shape of P-gp, thus reducing the drug's release into the intestinal tract, thereby increasing the limited gastrointestinal absorption rate of the P-gp-mediated drug, like 5-FU.

(25) A better understanding of the present invention will be given through the following examples. However, these examples are set forth to illustrate the present invention, and are not to be construed as limiting the scope of the present invention.

Example 1: Preparation of Bile Acid Derivative

(26) As an oral absorption enhancer, a bile acid derivative was prepared by chemically binding positively charged lysine to deoxycholic acid.

(27) 2.6 g of deoxycholic acid was dissolved in a mixture of 6.4 ml of ethyl chloroformate, 7.4 ml of N-methylmorpholine and 800 ml of tetrahydrofuran (THF), added with a solution of 2.7% (w/v) H-Lys(Boc)-OMeHCl in N-methylmorpholine, and then refluxed for 2 hr. After stirring at room temperature overnight, the resulting precipitate was filtered and dried by evaporating the solvent. The precipitate thus obtained was passed through a column using a mixture of chloroform and methanol to separate Lys(Boc)DOCA. The Lys(Boc)DOCA thus obtained was dissolved again in a mixture of ethyl chloride and methanol in an ice bath, the solvent was thoroughly removed, and the resulting residue was dissolved in purified water and then washed with chloroform. The aqueous solution layer was recovered and then lyophilized, thereby obtaining a deoxycholic acid-lysine derivative (DCK) in a powder form.

Example 2: Preparation of PMX/DCK Ion-Binding Complex

(28) An ion-binding complex was formed by dissolving pemetrexed (PMX) in purified water and slowly adding a DCK aqueous solution with stirring so that the molar ratio of pemetrexed to DCK was 1:1. The mixed solution thus obtained was centrifuged and then lyophilized, thereby preparing a PMX/DCK ion-binding complex in a powder form.

Example 3: Preparation of Tablet and Capsule Including PMX/DCK Ion-Binding Complex

(29) 500 mg of poloxamer and 375 mg of Labrasol™ were dissolved in 10 ml of purified water and then 50 mg of pemetrexed was added thereto and dissolved therein. Separately, 66.825 mg of DCK was dissolved in 10 ml of purified water, after which the DCK aqueous solution was slowly added to the pemetrexed solution with stirring so that the molar ratio of pemetrexed to DCK was 1:1, thus forming an ion-binding complex. The mixed solution thus prepared was lyophilized to afford a dispersant-added PMX/DCK ion-binding complex in a powder form. Here, poloxamer and Labrasol™ were used as dispersants. 198.364 mg of the dispersant-added PMX/DCK ion-binding complex was added with 30 mg of copovidone, 30 mg of microcrystalline cellulose and 38.636 mg of lactose, mixed, added with 3 mg of magnesium stearate, further mixed, and then pressed into an appropriate form to yield a tablet, or filled into a capsule.

Example 4: Preparation of PMX/DCK Ion-Binding Complex

(30) Pemetrexed was dissolved in purified water, added with a hydroxypropyl beta-cyclodextrin (HP-beta CD) aqueous solution at a molar ratio of 1:1 with pemetrexed, and stirred overnight. Thereafter, poloxamer 188 was added at the same weight ratio as pemetrexed and stirred, after which a DCK aqueous solution was slowly added thereto with stirring so that the molar ratio of pemetrexed to DCK was 1:1, thus forming a PMX/DCK ion-binding complex. The mixed solution thus obtained was lyophilized to afford a dispersant-added PMX/DCK ion-binding complex in a powder form. Here, hydroxypropyl beta-cyclodextrin and poloxamer 188 were used as dispersants.

Example 5: Preparation of w/o/w Nanoemulsion Including PMX/DCK Ion-Binding Complex

(31) 108.82 mg of the lyophilized powder mixture of the PMX/DCK ion-binding complex and the dispersants (hydroxypropyl beta-cyclodextrin and poloxamer) was redispersed in purified water, and was then added with 100 mg of a 1:2 primary-surfactant/primary-auxiliary-surfactant mixture (Labrasol™ Transcutol HP=1:2 (w/w)). As a primary oil phase, 57.2 mg of Capryol® 90 was added and mixed, thus preparing a primary w/o nanoemulsion. The primary w/o nanoemulsion was added with 600 mg of a 1:2 secondary-surfactant/secondary-auxiliary-surfactant mixture (Cremophor™ EL:Transcutol HP=1:1 (w/w)), mixed, and dispersed in 1200 mg of purified water, thereby preparing a w/o/w secondary nanoemulsion.

Example 6: Preparation of w/o/w Nanoemulsion Including PMX/DCK Ion-Binding Complex and Quercetin

(32) The mixture of 66.02 mg of the PMX/DCK ion-binding complex (corresponding to 10 mg of pemetrexed) and the dispersants (hydroxypropyl beta-cyclodextrin and poloxamer) was redispersed in 38 mg of purified water and then added with 112 mg of a 1:1 primary-surfactant/primary-auxiliary-surfactant mixture (Labrasol™:Tween 80=1:1, w/w). As a primary oil phase, 50 mg of Labrafil® M1944 was added and mixed, thereby preparing a primary w/o nanoemulsion.

(33) The primary w/o nanoemulsion was added with 20 mg of quercetin and 630 mg of a secondary-surfactant/secondary-auxiliary-surfactant mixture (Tween 80:Cremophor™ EL:PEG 400=1:2:2, w/w/w), mixed, and then dispersed in 800 mg of purified water, thereby preparing a w/o/w secondary nanoemulsion.

Example 7: Measurement of Artificial Intestinal Membrane Permeability of Oral Composition Including PMX/DCK Ion-Binding Complex

(34) The intestinal membrane permeability of pemetrexed (PMX), the mixture of hydroxypropyl beta-cyclodextrin and pemetrexed (HP-beta CD/PMX), the mixture of hydroxypropyl beta-cyclodextrin and PMX/DCK ion-binding complex (HP-beta CD/PMX/DCK), the mixture of hydroxypropyl beta-cyclodextrin, PMX/DCK ion-binding complex and poloxamer (HP-beta CD/PMX/DCK/P188) prepared in Example 4, and the w/o/w nanoemulsion including the mixture of hydroxypropyl beta-cyclodextrin, PMX/DCK ion-binding complex and poloxamer (HP-beta CD/PMX/DCK/P188) prepared in Example 5 was evaluated using an artificial intestinal membrane (PAMPA; BD Biosciences, San Jose, Calif., USA). The results thereof are shown in Table 1 below.

(35) Each sample was diluted with phosphate-buffered saline (PBS, pH 6.8) so that the concentration of pemetrexed was 200 μg/ml, and was then added in an amount of 200 μl to the donor compartment of each well of a PAMPA plate, each acceptor compartment thereof was added with 300 μl of phosphate-buffered saline (PBS, pH 6.8), and the donor compartment and the acceptor compartment were linked and then allowed to stand at room temperature for 5 hr, after which the sample was recovered from the donor compartment and the acceptor compartment of each well of the PAMPA plate and the concentration of the permeated drug was measured as follows.

(36) The concentrations of PMX and PMX/DCK were analyzed by introducing 20 μl of the sample into an HPLC system with a C18 column (4.6×250 mm, 5 μm, 100 □) and allowing a mixture of purified water (pH 3.2, adjusted with phosphoric acid) and acetonitrile at 80:20 (v/v) as a mobile phase to flow at a flow rate of 1.0 ml/min. The detection of PMX and PMX/DCK was performed at 254 nm.

(37) The effective permeability (P.sub.e) of each drug was calculated using the following equation.
P.sub.e=−ln[1−C.sub.A(t)/C.sub.equilibrium]/[A×(1/V.sub.D+1/V.sub.A)×t]

(38) (P.sub.e: permeability (cm/s); A: effective filter area (f×0.3 cm.sup.2; f: pores on surface of filter (f=0.76)); V.sub.D: volume of donor compartment (0.2 ml); V.sub.A: volume of acceptor compartment (0.3 ml); t: permeation time (sec); C.sub.A(t): drug concentration in acceptor compartment at time t; C.sub.equilibrium: [C.sub.D(t)×V.sub.D+C.sub.A(t)×V.sub.A]/(V.sub.D+V.sub.A); C.sub.D(t): residual drug concentration of donor compartment at time t)

(39) TABLE-US-00001 TABLE 1 Permeability Sample (×10.sup.−6, cm/s) PMX 3.73 ± 1.15 HP-beta CD/PMX 5.78 ± 1.37 HP-beta CD/PMX/DCK 14.0 ± 2.08 HP-beta CD/PMX/DCK/P188 19.6 ± 1.27 (Example 4) w/o/w nanoemulsion including 21.0 ± 3.27 HP-beta CD/PMX/DCK/P188 (Example 5)

(40) As shown in Table 1, the artificial intestinal membrane permeability of pemetrexed was increased 3.75 times after formation of the ion-binding complex with DCK, and when the ion-binding complex was included in the w/o/w multiple nanoemulsion formulation, the artificial intestinal membrane permeability of pemetrexed was increased 5.63 times compared to pemetrexed alone.

Example 8: Measurement of Artificial Intestinal Membrane Permeability of w/o/w Nanoemulsion Including PMX/DCK Ion-Binding Complex and Quercetin

(41) The intestinal membrane permeability of pemetrexed (PMX), quercetin and the w/o/w secondary nanoemulsions including the mixture of hydroxypropyl beta-cyclodextrin, PMX/DCK ion-binding complex and poloxamer (HP-beta CD/PMX/DCK/P188) prepared in Example 5 and quercetin was evaluated using an artificial intestinal membrane (PAMPA; BD Biosciences, San Jose, Calif., USA). The results thereof are shown in Table 2 below.

(42) Each sample was diluted with phosphate-buffered saline (PBS, pH 6.8) so that the concentration of pemetrexed was 200 μg/ml and the concentration of quercetin was 400 μg/ml, and was then added in an amount of 200 μl to the donor compartment of each well of a PAMPA plate, each acceptor compartment thereof was added with 300 μl of phosphate-buffered saline (PBS, pH 6.8), and the donor compartment and the acceptor compartment were linked and then allowed to stand at room temperature for 5 hr, after which the sample was recovered from the donor compartment and the acceptor compartment of each well of the PAMPA plate, and the concentration of the permeated drug was measured as follows.

(43) The concentrations of PMX and PMX/DCK were analyzed by introducing 20 μl of the sample into an HPLC system with a C18 column (4.6×250 mm, 5 μm, 100 □) and allowing a mixture of purified water (pH 3.2, adjusted with phosphoric acid) and acetonitrile at 80:20 (v/v) as a mobile phase to flow at a flow rate of 1.0 ml/min. The detection of PMX and PMX/DCK was performed at 254 nm.

(44) The concentration of quercetin was analyzed at 35° C. by introducing 20 μl of the sample into an HPLC system with a C18 column (4.6×150 mm, 5 μm, 100 □) and allowing a mixture of purified water (pH 2.6, adjusted with 2% acetic acid) and acetonitrile at 40:40 (v/v) as a mobile phase to flow at a flow rate of 1.0 ml/min. The UV detection was performed at 370 nm.

(45) The effective permeability (P.sub.e) of each drug was calculated in the same manner as in Example 7.

(46) TABLE-US-00002 TABLE 2 Permeability Sample (×10.sup.−6, cm/s) PMX 2.52 ± 0.60 w/o/w nanoemulsion including HP-beta 35.5 ± 5.89 CD/PMX/DCK/P188 (Example 6) Quercetin 4.51 ± 5.10 w/o/w nanoemulsion including quercetin 29.3 ± 1.88 (Example 6)

(47) As shown in Table 2, the artificial intestinal membrane permeability of pemetrexed from the w/o/w multiple nanoemulsion including the PMX/DCK ion-binding complex of Example 6 was increased 14.1 times compared to pemetrexed alone, and when quercetin was included in the w/o/w multiple nanoemulsion of Example 6, the artificial intestinal membrane permeability thereof was increased 6.50 times compared to quercetin alone due to the solubilization of quercetin.

Example 9: Measurement of Intestinal Cell Membrane Permeability of w/o/w Nanoemulsion Including PMX/DCK Ion-Binding Complex

(48) The intestinal cell membrane permeability of pemetrexed (PMX), the tablet or capsule composition including the PMX/DCK ion-binding complex prepared in Example 3, the mixture of hydroxypropyl beta-cyclodextrin and pemetrexed (HP-beta CD/PMX), the mixture of hydroxypropyl beta-cyclodextrin and PMX/DCK ion-binding complex (HP-beta CD/PMX/DCK), the mixture of hydroxypropyl beta-cyclodextrin, PMX/DCK ion-binding complex and poloxamer (HP-beta CD/PMX/DCK/P188) prepared in Example 4, and the w/o/w nanoemulsion including the mixture of hydroxypropyl beta-cyclodextrin, PMX/DCK ion-binding complex and poloxamer (HP-beta CD/PMX/DCK/P188) prepared in Example 5 was evaluated using a Caco-2 cell monolayer membrane. The results thereof are shown in Table 3 below.

(49) Caco-2 cells were treated at 3×10.sup.5 cells in each well of a 12-well Transwell (pore size: 0.4 μm, surface area: 1.12 cm.sup.2; Corning, N.Y., USA). Dulbecco's modified Eagles medium (DMEM; Lonza, Basel, Switzerland) containing 10% fetal bovine serum (FBS; Gibco™, Thermo Fisher Scientific) and 1% penicillin/streptomycin (Gibco™) was replaced every 48 hr for 21 days to 29 days, and a Caco-2 cell membrane having a transepithelial electrical resistance (TEER) of 350 Ωcm.sup.2 or more was used for in-vitro intestinal cell membrane permeability tests.

(50) Specifically, the culture medium was removed, culture was performed at 37° C. for 20 min with 0.5 ml of a Hanks' balanced salt solution (HESS), HBSS was removed, and each sample was diluted with HBSS so that the concentration of pemetrexed was 200 μg/ml, after which each sample thus diluted was added in an amount of 0.5 ml to the apical compartment of the Transwell, and the basolateral compartment of the Transwell was added with 1.5 ml of HBSS, followed by culturing at 37° C. After sample treatment, 100 μl of the sample was collected from the basolateral compartment of each well at 0.5, 1, 2, 3, 4 and 5 hr and then filtered with a membrane filter, after which the concentrations of PMX and the PMX/DCK ion-binding complex that permeated the intestinal cell membrane were measured using the same HPLC system as in Example 7. Moreover, the apparent permeation coefficient (P.sub.app) of PMX and the PMX/DCK ion-binding complex was calculated using the following equation.
P.sub.app=dQ/dt×1/(A×C.sub.0)

(51) (dQ/dt: weight of drug that permeated basolateral compartment per unit time (μmol/s), C.sub.0: initial concentration of PMX or PMX/DCK ion-binding complex in apical compartment (μmol/l), A: permeation area (cm.sup.2)).

(52) TABLE-US-00003 TABLE 3 Permeability Sample (×10.sup.−6, cm/s) PMX 0.50 ± 0.09 Tablet and capsule composition including 21.3 ± 0.67 PMX/DCK (Example 3) HP-beta CD/PMX 0.76 ± 0.19 HP-beta CD/PMX/DCK 1.15 ± 0.10 HP-beta CD/PMX/DCK/P188 (Example 4) 4.23 ± 0.46 w/o/w nanoemulsion including HP-beta 4.89 ± 0.13 CD/PMX/DCK/P188 (Example 5)

(53) As shown in Table 3, the Caco-2 intestinal cell membrane permeability of pemetrexed when prepared into a tablet or capsule composition after formation of the ion-binding complex with DCK was increased 42.6 times compared to pemetrexed alone. The Caco-2 intestinal cell membrane permeability after formation of the ion-binding complex of pemetrexed and DCK (HP-beta CD/PMX/DCK) was increased 2.30 times compared to pemetrexed alone, and when the ion-binding complex was included in the w/o/w multiple nanoemulsion formulation of Example 5, the Caco-2 intestinal cell membrane permeability thereof was increased 9.78 times compared to pemetrexed alone.

Example 10: Measurement of In-Vivo Bioavailability of Oral Pharmaceutical Composition Including PMX/DCK Ion-Binding Complex

(54) Each of pemetrexed and a tablet or capsule composition including the PMX/DCK ion-binding complex prepared in Example 3 was dissolved in distilled water and then orally administered in an amount of 500 μl into rats. Separately, in order to evaluate oral bioavailability, a solution of pemetrexed in distilled water was injected in an amount of 200 μl into rats through the tail vein thereof. Blood samples (200 μl) were collected at scheduled times after individual drug administrations and immediately mixed with 50 μl of a sodium citrate (3.8%) solution. The collected blood samples were centrifuged at 2,500×g and 4° C. for 15 min, pemetrexed in plasma was extracted using a solid-phase extraction (SPE) cartridge, and the concentration of pemetrexed was measured through liquid chromatography/mass spectrometry (LC/MS).

(55) FIGS. 1 and 2 show venous plasma pemetrexed concentration-time profiles after intravenous injection (5 mg/kg) and oral administration (50 mg/kg) of pemetrexed into rats or after oral administration of the tablet or capsule composition including the PMX/DCK ion-binding complex of Example 3 (corresponding to 50 mg/kg pemetrexed) into rats. Each value represents mean±standard deviation (n=4 in each group). As shown in FIG. 2, after oral administration of 50 mg/kg pemetrexed, C.sub.max was 451.78±221.38 ng/ml, AUC.sub.last was 1,393.3±395.12 ng.Math.h/ml, and oral bioavailability compared to intravenous injection was evaluated to be 1.49%±0.42%. On the other hand, as shown in Table 4, when the tablet or capsule composition including the PMX/DCK ion-binding complex of Example 3, corresponding to 50 mg/kg pemetrexed, was orally administered, C.sub.max was increased 6.15 times (2,779.8±1,051.3 ng/ml), AUC.sub.last was increased 3.94 times (5,490.2±1,013.4 ng.Math.h/ml) and oral bioavailability was increased 3.94 times (5.86%±1.08%), compared to when pemetrexed was orally administered alone. Therefore, the intestinal membrane permeability and oral absorption rate of pemetrexed in rats were significantly increased by the formation of the ion-binding complex with the bile acid derivative.

(56) TABLE-US-00004 TABLE 4 Tablet and capsule composition including PMX/DCK Sample Pemetrexed Pemetrexed (Example 3) Administration Intravenous Oral Oral route injection Pemetrexed   5 50 50 dose (mg/kg) T.sub.max (h) — 0.83 ± 0.58 0.67 ± 0.29 T.sub.1/2 (h) 0.35 ± 0.08 6.06 ± 1.83 4.10 ± 1.40 C.sub.max (ng/ml) 15,318 ± 6100.3 451.78 ± 221.38 2,779.8 ± 1,051.3 AUC.sub.last 9,377.5 ± 4,169.4 1,393.3 ± 395.12  5,490.2 ± 1,013.4 (ng .Math. h/ml) AUC.sub.inf 9,414.5 ± 4,223.6 2,559.0 ± 755.48  6,988.4 ± 1,625.2 (ng .Math. h/ml) Bioavailability 100 1.49 ± 0.42 5.86 ± 1.08 (%) {in Table 4, T.sub.max: time to reach maximum plasma drug concentration; T.sub.1/2: plasma drug concentration half-life; C.sub.max: maximum plasma drug concentration; AUC.sub.last: area under plasma drug concentration-time curve up to final plasma drug concentration measurement time; AUC.sub.inf: area under plasma drug concentration-time curve up to infinite time; bioavailability: (AUC.sub.last,oral administration/oral dose)/(AUC.sub.last,intravenous .sub.injection/intravenous injection dose) × 100}

Example 11: Measurement of In-Vivo Bioavailability of Oral Nanoemulsion Including PMX/DCK Ion-Binding Complex

(57) Each of pemetrexed and an oral nanoemulsion composition including the PMX/DCK ion-binding complex prepared in Example 5 was dissolved in distilled water and then orally administered in an amount of 500 μl into rats. Separately, in order to evaluate oral bioavailability, a solution of pemetrexed in distilled water was injected in an amount of 200 μl into rats through the tail vein thereof. Blood samples (200 μl) were collected at scheduled times after drug administration and immediately mixed with 50 μl of a sodium citrate (3.8%) solution. The collected blood samples were centrifuged at 2,500×g and 4° C. for 15 min, pemetrexed in plasma was extracted using a solid-phase extraction (SPE) cartridge, and the concentration of pemetrexed was measured through liquid chromatography/mass spectrometry (LC/MS).

(58) FIGS. 3 and 4 show venous plasma pemetrexed concentration-time profiles after intravenous injection (10 mg/kg) and oral administration (20 mg/kg) of pemetrexed into rats or after oral administration of the nanoemulsion composition including the PMX/DCK ion-binding complex of Example 5 (corresponding to 20 mg/kg pemetrexed) into rats. Each value represents mean±standard deviation (n=4 in each group). As shown in FIG. 4, after oral administration of 20 mg/kg pemetrexed, C.sub.max was 1,487±332 ng/ml, AUC.sub.last was 7,546±938 ng.Math.h/ml, and oral bioavailability compared to intravenous injection was 12.0%±1.50%. On the other hand, as shown in Table 5, when the nanoemulsion composition including the PMX/DCK ion-binding complex of Example 5, corresponding to 20 mg/kg pemetrexed, was orally administered, C.sub.max was increased 3.23 times (4,802±579 ng/ml), AUC.sub.last was increased 2.23 times (16,810±1,868 ng.Math.h/ml) and oral bioavailability was increased 2.23 times (26.8%±2.98%) compared to when pemetrexed was orally administered alone. Therefore, the intestinal membrane permeability and oral absorption rate of pemetrexed in rats were significantly increased by the formation of the ion-binding complex with the bile acid derivative and by the nanoemulsion composition.

(59) TABLE-US-00005 TABLE 5 Nanoemulsion composition including PMX/DCK Sample Pemetrexed Pemetrexed (Example 5) Administration Intravenous Oral Oral route injection Pemetrexed  10 20 20 dose (mg/kg) T.sub.max (h) — 2.75 ± 3.50 0.90 ± 0.22 T.sub.1/2 (h) 0.57 ± 0.07 7.55 ± 0.94 7.74 ± 2.74 C.sub.max 32,220 ± 13,900 1,487 ± 332   4,802 ± 579   (ng/ml) AUC.sub.last 31,340 ± 5,342  7,546 ± 938   16,810 ± 1,868  (ng .Math. h/ml) AUC.sub.inf 29,390 ± 4,844  14,300 ± 2,272  30,870 ± 2,831  (ng .Math. h/ml) Bioavailability 100 12.0 ± 1.50 26.8 ± 3.00 (%) {in Table 5: T: time to reach maximum plasma drug concentration; T.sub.1/2: plasma drug concentration half-life; C.sub.max: maximum plasma drug concentration; AUC.sub.last: area under plasma drug concentration-time curve up to final plasma drug concentration measurement time; AUC.sub.inf: area under plasma drug concentration-time curve up to infinite time; bioavailability: (AUC.sub.last,oral administration/oral dose)/AUC.sub.last,intravenous .sub.injection/intravenous injection dose) × 100}

(60) Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications are possible without departing from the technical idea of the present invention. Therefore, the embodiments disclosed in the present specification should be understood to be non-limiting and illustrative in every way, and are not to be construed as limiting the spirit or scope of the present invention. The scope of the present invention should be interpreted by the appended claims, and it will be understood that all technologies within the scope equivalent thereto belong to the scope of the present invention.