JP3941036B2 - Liposome composition for oral administration - Google Patents

Description

【００３５】
試験例２
ラクトフェリン溶液(ｐＨ６．６９のクエン酸緩衝液に３０ｍｇ/ｍｌの濃度で溶解)、ラクトフェリン封入リポソーム懸濁液及びフコイダンでコーティングしたラクトフェリン封入リポソーム懸濁液を用いて人工腸液による消化試験を行った。
【００３６】
ラクトフェリン溶液(ｐＨ６．６９のクエン酸緩衝液に３０ｍｇ/ｍｌの濃度で溶解)、ラクトフェリン封入リポソーム懸濁液及びフコイダンでコーティングしたラクトフェリン封入リポソーム懸濁液（実施例１で得られた溶液）の各０．６ｍｌをそれぞれＵＳＰ（米国薬局方）２４局に掲載されている人工腸液に添加し、３７℃で４時間撹拌する。４時間後、トリクロロ酢酸を添加し消化酵素を不活化し各サンプルの人工消化液を得る。得られた消化液中のラクトフェリン濃度をＥＬＩＳＡサンドイッチ法にて測定し、ラクトフェリンの残存率を算出した。その結果、ラクトフェリン溶液を消化した場合、その残存率は１５．９±０．２％であったが、ラクトフェリン封入リポソーム懸濁液ではその残存率は２６．１±０．２％、更にフコイダンで表面コーティングしたラクトフェリン封入リポソーム懸濁液においてはその残存率は３３．７±０．８％であった。ラクトフェリンをリポソームに封入し、更にそのリポソームの表面をフコイダンでコーティングすることによって、ラクトフェリンの人工腸液に対する安定性が高まることが明らかとなった。
【００３７】
【表２】

【００３８】
ラクトフェリンが腸管により吸収されることは知られているので、胃や腸で分解されないラクトフェリンの割合が高くなれば、腸管で吸収されるラクトフェリンの割合が高くなることが予想される。
【００３９】
【発明の効果】
生理活性のあるペプチド性化合物を経口投与したとき、胃酸や胃内、小腸内のタンパク分解酵素によって分解され生理活性が不活化されてしまうが、ペプチド性化合物を、硫酸基を含有する多糖類でコーティングしたリポソーム製剤とすることにより、ペプチド性化合物の消化管タンパク分解酵素に対する安定性を高めることができた。また、小腸粘膜へ吸着しやすいので消化管での滞留性の向上が期待でき、さらに上皮細胞からの吸収促進が期待できる。従って、ペプチド性化合物を経口投与した際の消化管からの吸収を高めることが期待できる。
【００４０】
本発明ではラクトフェリンをリポソーム製剤であるので、固形製剤のみに限られず、液体製剤などとすることも可能である。また、様々な形態の食品に配合することができ、例えば、飲料、ヨーグルトなど種々の形態としてラクトフェリンを摂取することも可能である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to providing a food composition containing liposomes and a pharmaceutical composition for oral administration.
[0002]
[Prior art]
When a peptidic compound is orally administered, it is degraded and easily inactivated by the action of gastric acid and proteolytic enzymes in the digestive tract. Peptide compounds are often water-soluble, and their molecular weight is large, so that membrane permeability from the gastrointestinal mucosa is poor, so that they are hardly absorbed from the gastrointestinal tract. At present, in order to increase the absorption of peptidic compounds, mainly a method using a protease inhibitor and a method for modifying a dosage form are being studied. Specific examples of the protease inhibitor include aprotinin and bacitracin. On the other hand, as a method for modifying the dosage form, an enteric-coated preparation or the like is used in order to prevent the peptide from being decomposed by gastric acid. However, intestinal solvents are limited to solid preparations and cannot be in various forms such as liquid preparations, and are particularly difficult to blend into foods.
In recent years, research on the use of microparticle preparations as drug carriers as one of the methods for modifying dosage forms has been vigorously conducted, and as one of them, studies on the use of liposomes as drug carriers for oral administration have been conducted in the past. Has been made. For example, it has been observed that the insulin bioavailability at the time of oral administration is increased by encapsulating insulin, which was normally invalidated by oral administration, in a modified liposome [(Takeuchi H et al. Pharam. Res. 13, 896, 1996), (Muramatsu K et al. Biol. Pharam. Bull. 19, 1055, 1996), (Iwanaga K et al. J. Pharm. Sci. 88, 248, 1999)]. It has also been reported that encapsulating erythropoietin in liposomes increases its bioavailability.
[(Maitani Y et al. J. Pharam. Sci. 85, 440, 1996)]. However, these peptide compound-encapsulated liposomes have drawbacks such as requiring a high dose compared to injection and the fact that absorption is likely to fluctuate, and have not yet been fully put into practical use. Is currently used as an injection in clinical practice.
[0003]
[Problems to be solved by the invention]
An object of the present invention is to provide an oral administration preparation having excellent stability in the digestive tract of a peptide compound.
[0004]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present invention stabilizes a peptide compound in the digestive tract by encapsulating a peptide compound in a liposome, which is a lipid membrane microcarrier, and coating the surface of the liposome with a sulfate group-containing polysaccharide. It is an oral liposome preparation that enhances the properties and can efficiently absorb peptide compounds from the digestive tract.
[0005]
That is, this invention provides the invention which concerns on each following item.
Item 1. A food composition comprising a liposome containing lecithin and sterol and encapsulating a physiologically active substance, the liposome being coated with a sulfate group-containing polysaccharide.
Item 2. The food composition according to Item 1, wherein the lecithin is at least one selected from the group consisting of egg yolk lecithin and soybean lecithin.
Item 3. The food composition according to Item 1 or 2, wherein the sterol is at least one selected from the group consisting of cholesterol and phytosterol.
Item 4: The food composition according to any one of Items 1 to 3, wherein the molar ratio of lecithin to sterol is 5: 5 to 9: 1.
Item 5. The food composition according to Item 4, wherein the molar ratio of lecithin to sterol is 6: 4 to 8: 2.
Item 6. The food composition according to any one of Items 1 to 5, wherein the sulfate group-containing polysaccharide is at least one selected from the group consisting of fucoidan, carrageenan and agar.
Item 7. The food composition according to Item 6, wherein the sulfate group-containing polysaccharide is fucoidan.
Item 8. The food composition according to any one of Items 1 to 7, wherein the physiologically active substance is a physiologically active peptide.
Item 9. The food composition according to Item 8, wherein the physiologically active peptide is at least one selected from the group consisting of lactoferrin, transferrin, interferon, interleukin, and lysozyme.
Item 10. The food composition according to Item 9, wherein the physiologically active peptide is lactoferrin.
Item 11. A pharmaceutical composition for oral administration comprising a liposome containing lecithin and sterol and encapsulating a physiologically active substance, the liposome being coated with a sulfate group-containing polysaccharide.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
[0007]
Liposomes are lipid vesicles having a bilayer formed by hydrating lipids mainly composed of phospholipids with a sufficient amount of water. Currently, liposomes are classified based on the number of lipid bilayers, and are classified into multilamellar liposomes (MLV) and single membrane liposomes. Single-membrane liposomes are further classified into SUV (small unilamella vesicle), LUV (large unilamella vesicle), and GUV (giant unilamella vesicle) according to the size. Any of these may be sufficient as the liposome of this invention.
[0008]
The size of the liposome of the present invention is preferably about 50 to 1000 nm, more preferably about 100 to 500 nm.
[0009]
Liposomes can incorporate water-soluble drugs into the inner water layer and fat-soluble drugs into the lipid bilayer, and can be applied as drug carriers for DDS preparations for the purpose of drug targeting, sustained release, and reduction of side effects. Has been tried. Liposomes are also known to have high safety because they are composed of components of biological membranes.
[0010]
In the present invention, examples of lecithin include egg yolk lecithin, soybean lecithin, rapeseed lecithin, corn lecithin, sunflower lecithin, peanut lecithin and the like.
In the present invention, these hydrogenated products can also be used. Among these, egg yolk lecithin and soybean lecithin are preferable as the lecithin of the present invention.
[0011]
As sterols, sterols derived from animals such as cholesterol, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol; sterols derived from plants such as stigmasterol, sitosterol, campesterol, brassicasterol (phytosterols); timosterol, ergosterol, etc. Sterols derived from these microorganisms. Among these, cholesterol or phytosterol is preferably used.
[0012]
The molar ratio of lecithin and sterol in the liposome is preferably about 5: 5 to 9: 1, and more preferably about 6: 4 to 8: 2.
[0013]
The physiologically active substance to be encapsulated in the liposome of the present invention is not limited as long as it is easily decomposed in the digestive tract and expected to be absorbed. Peptide compounds having physiological activity, compounds having amide bonds, glycosides Etc. are exemplified.
[0014]
As the peptidic compound having physiological activity, those having a molecular weight of about 1,000 to 200,000 can be preferably used. Specifically, physiologically active proteins such as lactoferrin, transferrin, interferon, interleukin, lysozyme; peptide hormones such as insulin, glucagon, calcitonin, luteinizing hormone, kallikrein, gastrin, secretin, growth hormone, erythropoietin; influenza vaccine, diphtheria Vaccines such as vaccines, tetanus vaccines, and pertussis vaccines. Among these, physiologically active proteins such as lactoferrin, transferrin, interferon, interleukin, and lysozyme are particularly preferable, and lactoferrin is more preferable.
[0015]
The blending ratio of the physiologically active substance is preferably about 20 to 1000 parts by weight and about 100 to 500 parts by weight with respect to 100 parts by weight of lecithin.
[0016]
Liposomes encapsulating physiologically active substances can be produced by conventional methods. For example, a predetermined amount of lecithin and sterol is solubilized with an appropriate organic solvent such as ethanol, the solvent is removed under reduced pressure, a membrane lipid is prepared, and an aqueous solution containing the physiologically active substance is added thereto. For example, it can be obtained by preparing a liposome suspension by stirring at about 1000 to 3000 rpm for about 2 to 5 minutes.
[0017]
If necessary, the obtained suspension may be subjected to an operation for removing the physiologically active substance in the liposome outer liquid, for example, an operation for filtering the resulting suspension and then dialyzing the obtained filtrate. Good.
[0018]
The surface of the obtained liposome is coated with a polysaccharide containing sulfate groups.
[0019]
Examples of sulfate group-containing polysaccharides include fucoidan, carrageenan, agar, and heparin. The sulfate group-containing polysaccharide of the present invention may be a sulfated polysaccharide not containing a sulfate group, such as chondroitin sulfate or dermatan sulfate.
[0020]
As the sulfate group-containing polysaccharide of the present invention, those having a molecular weight of about 5,000 to 300,000 are preferably used.
[0021]
Among these, fucoidan and carrageenan can be preferably used in the present invention, and fucoidan is particularly preferable.
[0022]
About 10-500 weight part is preferable with respect to 100 weight part of lecithin contained in a liposome, for example, and the usage-amount of a sulfate group containing polysaccharide is more preferable about 20-200 weight part.
[0023]
The coating can be performed, for example, by adding a sulfate group-containing polysaccharide to a suspension containing liposomes encapsulating a physiologically active substance and stirring at about 1000 to 3000 rpm for about 2 to 5 minutes. A plurality of liposomes may be contained in one coating film.
[0024]
The fact that the liposome is coated with the sulfate group-containing polysaccharide can be confirmed, for example, by changing the zeta potential of the liposome solution by adding and stirring the sulfate group-containing polysaccharide.
[0025]
The liposome suspension thus obtained can be used in liquid form such as beverages, solid foods such as tablets, granules, chewable tablets, etc., in a liquid state or lyophilized dry matter. It can also be used for semi-solid foods such as yogurt. In the case of a food form, it can be prepared by an ordinary method in combination with a food material, food additive, or the like according to the form of the food, if necessary. The food containing the liposome coated with the sulfate group-containing polysaccharide of the present invention is used for applications such as health food, functional food, food for specified health use, food for the sick according to the type of physiologically active substance encapsulated in the liposome. be able to. For example, when lactoferrin is enclosed, prevention and treatment of hepatitis C, maintenance of health of people who drink heavily, normalization of immune function, prevention of infection, prevention of allergies, improvement of rhinitis, improvement of atopic dermatitis, cancer Can be taken in anticipation of effects such as prevention of bowel, bowel regulation, improvement of bowel movements and feces.
[0026]
In addition, the liposome suspension is used as a liquid, or the lyophilized dry product is used as a solid preparation such as a tablet or granule, using appropriate excipients, diluents, and other additives as necessary. It can be used for orally administered drugs. There are various uses of pharmaceuticals depending on the type of encapsulated physiologically active substance. For example, when lactoferrin is encapsulated, immunomodulators, antibacterial agents, antiviral agents, anticancer agents, anti-inflammatory agents, antioxidants It can be used for applications such as an agent.
[0027]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples and test examples, but the present invention is not limited to these examples.
[0028]
Example 1
Preparation of preparation A liposome preparation encapsulating lactoferrin and coated with fucoidan was prepared by the thin film hydration method. Egg yolk lecithin [manufactured by NOF Corporation] and phytosterol [manufactured by Tama Seikagaku Co., Ltd.] are weighed so that the molar ratio is 7: 3 (egg yolk lecithin 63.0 mg, phytosterol 14.55 mg), ethanol 6 ml. In addition, it was dissolved. The solvent was distilled off from this solution with a rotary evaporator to form a thin film. 6 ml of a citrate buffer solution (pH 6.69) in which 180 mg of lactoferrin was dissolved was added to the thin film, and the mixture was stirred for about 3 minutes at 2000 rpm with a vortex mixer to obtain a liposome suspension. The suspension was filtered using a polycarbonate membrane having a pore size of 0.4 μm, and the obtained filtrate was dialyzed using a cellulose ester membrane (MWCO 300,000) to remove lactoferrin in the liposome outer solution, Lactoferrin-encapsulated liposomes were obtained. 36 mg of fucoidan [manufactured by Takara Shuzo Co., Ltd.] was added to 6 ml of the obtained suspension of lactoferrin liposomes, and stirred vigorously with a vortex mixer to obtain lactoferrin-encapsulated liposomes coated with the desired fucoidan.
[0029]
The fact that the liposome was coated with fucoidan was confirmed by the fact that the zeta potential of the liposome solution, which was near 0 before the addition of fucoidan, was shifted in the negative direction after the fucoidan was added and stirred.
[0030]
Example 2
A liposome preparation encapsulating insulin and coated with carrageenan was prepared by thin film hydration. Egg yolk lecithin [manufactured by NOF Corporation] and phytosterol [manufactured by Tama Seikagaku Co., Ltd.] are weighed so that the molar ratio is 7: 3 (egg yolk lecithin 63.0 mg, phytosterol 14.55 mg), ethanol 6 ml. In addition, it was dissolved. The solvent was distilled off from this solution with a rotary evaporator to form a thin film. To this thin film, 6 ml of an acetic acid buffer solution [0.01N HCl and an equal amount of acetic acid buffer solution (pH = 4.0)] in which 12.0 mg of insulin was dissolved was added, and vortex mixer was performed at 2000 rpm for about 3 minutes. The mixture was sufficiently stirred to obtain a liposome suspension. The suspension was filtered using a polycarbonate membrane having a pore size of 0.4 μm, and the resulting filtrate was dialyzed using a cellulose ester membrane (MWCO 10,000) to remove insulin in the liposome outer solution, Insulin-encapsulated liposomes were obtained. 30 mg of carrageenan [manufactured by Nitta Gelatin Co., Ltd.] was added to 6 ml of the obtained suspension of insulin liposomes, and the mixture was vigorously stirred with a vortex mixer to obtain insulin-encapsulated liposomes coated with the target carrageenan.
[0031]
It was confirmed that the liposome was coated with carrageenan because the zeta potential of the liposome solution, which was near 0 before the addition of carrageenan, shifted in the negative direction after the addition of carrageenan and stirring.
[0032]
Test example 1
Digestion test with artificial gastric juice of lactoferrin solution (dissolved in citrate buffer of pH 6.69 at a concentration of 30 mg / ml) and lactoferrin-encapsulated liposome suspension and lactoferrin-encapsulated liposome suspension coated with fucoidan.
[0033]
Lactoferrin solution (dissolved in citrate buffer pH 6.69 at a concentration of 30 mg / ml), lactoferrin-encapsulated liposome suspension and fucoidan-coated lactoferrin-encapsulated liposome suspension (suspension obtained in Example 1) Of each was added to a pH 2 aqueous HCl solution containing 0.025 mg of pepsin and stirred at 37 ° C. for 1 hour. After 1 hour, 0.75 μg of pepstatin was added to inactivate pepsin, and neutralized with a NaOH solution to obtain an artificial digest of each sample. The lactoferrin concentration in the obtained digestive juice was measured by ELISA sandwich method, and the residual rate of lactoferrin was calculated. As a result, when the lactoferrin solution was digested, the residual rate was 0.04 ± 0.01%, but in the lactoferrin-encapsulated liposome suspension, the residual rate was 1.98 ± 0.13%, and fucoidan In the surface-coated lactoferrin-encapsulated liposome suspension, the residual ratio was 39.55 ± 1.39%. It was revealed that lactoferrin was stabilized in the digestive tract by encapsulating lactoferrin in liposomes and coating the surface of the liposomes with fucoidan.
[0034]
[Table 1]

[0035]
Test example 2
A digestion test using an artificial intestinal fluid was performed using a lactoferrin solution (dissolved in a citrate buffer of pH 6.69 at a concentration of 30 mg / ml), a lactoferrin-encapsulated liposome suspension and a lactoferrin-encapsulated liposome suspension coated with fucoidan.
[0036]
Each of a lactoferrin solution (dissolved in a citrate buffer of pH 6.69 at a concentration of 30 mg / ml), a lactoferrin-encapsulated liposome suspension and a lactoferrin-encapsulated liposome suspension coated with fucoidan (the solution obtained in Example 1) Add 0.6 ml each to the artificial intestinal fluid listed in USP (US Pharmacopoeia) 24th, and stir at 37 ° C. for 4 hours. After 4 hours, trichloroacetic acid is added to inactivate the digestive enzyme to obtain an artificial digestion solution for each sample. The lactoferrin concentration in the obtained digestive juice was measured by ELISA sandwich method, and the residual rate of lactoferrin was calculated. As a result, when the lactoferrin solution was digested, the residual rate was 15.9 ± 0.2%. However, in the lactoferrin-encapsulated liposome suspension, the residual rate was 26.1 ± 0.2%, and fucoidan. In the surface-coated lactoferrin-encapsulated liposome suspension, the residual ratio was 33.7 ± 0.8%. It was revealed that the stability of lactoferrin to an artificial intestinal fluid is increased by encapsulating lactoferrin in the liposome and coating the surface of the liposome with fucoidan.
[0037]
[Table 2]

[0038]
Since it is known that lactoferrin is absorbed by the intestinal tract, it is expected that if the proportion of lactoferrin that is not degraded in the stomach or intestine increases, the proportion of lactoferrin absorbed in the intestinal tract increases.
[0039]
【The invention's effect】
When a physiologically active peptidic compound is administered orally, it is degraded by proteolytic enzymes in the stomach acid, stomach, and small intestine, and the physiological activity is inactivated, but the peptidic compound is a polysaccharide containing a sulfate group. By using a coated liposome preparation, the stability of the peptide compound to the digestive tract proteolytic enzyme could be improved. Moreover, since it is easily adsorbed to the small intestinal mucosa, it can be expected to improve the retention in the digestive tract, and further to promote absorption from epithelial cells. Therefore, it can be expected to enhance absorption from the digestive tract when a peptide compound is orally administered.
[0040]
In the present invention, since lactoferrin is a liposome preparation, it is not limited to a solid preparation but can be a liquid preparation. Moreover, it can mix | blend with various forms of foodstuffs, for example, can also ingest lactoferrin as various forms, such as a drink and a yoghurt.

Claims (9)

A food composition comprising a liposome containing lecithin and sterol and encapsulating a physiologically active substance, the liposome being coated with fucoidan .  The food composition according to claim 1, wherein the lecithin is at least one selected from the group consisting of egg yolk lecithin and soybean lecithin.  The food composition according to claim 1 or 2, wherein the sterol is at least one selected from the group consisting of cholesterol and phytosterol.  The food composition according to claim 1, wherein the molar ratio of lecithin to sterol is 5: 5 to 9: 1.  The food composition according to claim 4, wherein the molar ratio of lecithin to sterol is 6: 4 to 8: 2. The food composition according to any one of claims 1 to 5 , wherein the physiologically active substance is a physiologically active peptide. The food composition according to claim 6 , wherein the physiologically active peptide is at least one selected from the group consisting of lactoferrin, transferrin, interferon, interleukin, and lysozyme. The food composition according to claim 7 , wherein the physiologically active peptide is lactoferrin. A pharmaceutical composition for oral administration comprising a liposome containing lecithin and sterol and encapsulating a physiologically active substance, the liposome being coated with fucoidan .