JPH0859503A - Liposome preparation for administering pepide or protein medicine through mouth or mucosa - Google Patents

Abstract

PURPOSE: To obtain a liposome preparation containing a peptide or protein medicine and excellent in absorbability and persistence. CONSTITUTION: The liposome preparation for oral or mucosal administration contains one or more single layered liposomes and/or multilayered liposomes (inner-sealed liposomes) and, if necessary, an absorption-stimulating agent, preferably a protease-inhibiting agent, in a single layered liposome and/or a multilayered liposome (exodermis liposome), and contains a peptide or protein medicine or a vaccine in the inner-sealed liposomes. Examples of the peptide or protein medicine includes a calcitonin compound, an insulin compound, a growth hormone-releasing hormone, a corpus-forming hormone-releasing hormone and a parathyroid hormone, and the vaccine includes an influenza vaccine, diphtheria vaccine, a tetanus vaccine and a pertussis vaccine. The amount of the peptide or protein medicine or vaccine is preferably 2-10 times that used for their injection administration.

Description

Detailed Description of the Invention

[0001]

The present invention relates to unilamellar vesicles and / or multilamellar vesicles (envelope liposomes),
Oral administration comprising one or more unilamellar vesicles and / or multilamellar vesicles (encapsulated liposomes), and optionally an absorption enhancer, and the encapsulated liposomes containing a peptide / protein drug or vaccine Or, it relates to a liposome preparation for transmucosal administration.

[0002]

2. Description of the Related Art Currently known liposomes are multilamellar liposomes (MLV; mult) composed of several concentric membranes.
ilamellar vesicle), oligo-multilamellar liposome (OLV; oligolamellar) consisting of 2 to 4 concentric membranes
vesicles), MLV-stabilized liposomes (SPLV; stable plurilamellar vesicles), liposomes composed of several irregular membranes not concentric circles (MV
V; Multivesicular vesicle), small unilamellar vesicle (SUV), large 1
Single layer liposome (LUV; large unilamellar vesicl)
e) etc. It is possible to use lipids derived from biological membranes and it is safe for living organisms, that it has a structure similar to biological membranes and is considered to be natural even at interactions at the cell level, and it is possible to encapsulate polymers. For some reasons, liposomes are being actively studied as drug carriers. One use of liposomes as carriers is to study formulations for oral administration for drugs that are poorly absorbed from the digestive tract. These are mainly investigated by MLV liposomes and SUV liposomes, which are the easiest to prepare, and insulin, which was usually ineffective in oral administration (Kensuke Shirane et al., The Japanese Society of Pediatrics 91 (6) 1506 19
87), heparin (Masaharu Ueno et al., Pharmaceutical Science 47 (1) 56-60198)
It has been confirmed that 7) and others retain their activity and enter the blood by oral administration. Similarly, insulin
Encapsulation in V liposomes may improve the absorption rate of insulin by nasal absorption, which is one of the mucosal absorption.
Suggested by in vitro experiments (Y. Maitani et al.
Chem. Pharm. Bull. 40 (6) 1569-1572 1992).

[0003]

According to the report of Ueno et al. Mentioned above, a beagle dog is obtained by encapsulating heparin in a positively charged MLV liposome prepared from 90% phosphatidylcholine and 10% stearylamine. Has succeeded in oral absorption of heparin. In this report, since the comparison of the absorption rate with the injection of heparin is not described, it is not possible to make a precise reference to the absorption rate of heparin by oral administration, but from the results of our intravenous injection of heparin By analogy, the absorption rate of heparin is considered to be about several percent, and it is hard to say that efficient absorption was achieved.

Shirane et al., Supra, have insulin-encapsulated ML.
Although a decrease in blood glucose level has been confirmed by orally administering V liposomes to mice, its lowering effect does not reach that of subcutaneous injection.

Moreover, Yotsuyanagi et al. Conducted oral administration of liposomes encapsulating insulin to rats, but could not confirm absorption of insulin. (Ann.
Rept. Pharm. Nagoya City Univ. 31 1-17 1983) That is, MLV, SUV, which are general liposomes,
These methods using LUV also have drawbacks in that a high dose is required as compared with injection and that absorption is likely to fluctuate, so that they have not yet been completely put into practical use.

In addition, MVV liposome was announced by Kim et al. In 1983 as a novel liposome which is not MLV (Biochim Biophys Acta 728 (3) 339-348 1983), and it is an anticancer drug 5-FU (5-fluorouracil) eye. Administration to (Opht
almic Surg 19 (6) 408-413 1988) and intracerebroventricular administration of antiviral DDC (2 ', 3'-dideoxycytidine) (J In
fect.Dis. 162 (3) 750-752 1990) and the like have been reported to be used as carriers, but there has been no report to be used for oral administration or transmucosal administration of peptide / protein drugs or vaccines.

The object of the present invention is to administer a peptide / protein drug or vaccine for oral or transmucosal administration.
In the unilamellar liposome and / or multilamellar liposome,
It is intended to provide a liposome preparation containing one or more unilamellar vesicles and / or multilamellar vesicles containing a peptide / protein drug or a vaccine.

A further object of the present invention is to provide a peptide / protein drug in a unilamellar liposome and / or multilamellar liposome capable of efficiently absorbing the peptide / protein drug or vaccine by oral or transmucosal administration. Another object is to provide a liposome preparation containing one or more unilamellar liposomes and / or multilamellar liposomes containing a vaccine.

The object of the present invention is to provide peptides such as insulins, calcitonins, luteinizing hormone-releasing hormones (including agonists and antagonists).
One or more containing one or more of these peptide / protein drugs or vaccines in a single-membrane liposome and / or multilamellar liposome for efficiently absorbing a protein drug or a vaccine such as influenza vaccine by oral administration or transmucosal administration. It is intended to provide a liposome preparation in which single-layered liposomes and / or multilamellar liposomes are encapsulated.

Still further, an object of the present invention is to obtain a single-membrane liposome and / or a liposome capable of absorbing a peptide / protein drug or vaccine by oral or transmucosal administration in the presence of an absorption enhancer very efficiently. It is an object of the present invention to provide a liposome preparation in which one or more unilamellar vesicles containing a peptide / protein drug or a vaccine and / or multilamellar liposomes are encapsulated in the multilamellar liposome.

[0011]

Means for Solving the Problems The inventors of the present invention have earnestly studied a method of efficiently absorbing a peptide / protein drug and a vaccine by oral administration or transmucosal administration. Is similar to a conventional MVV liposome, but by changing the content of the MVV liposome from an oil phase to an aqueous phase, that is, one or more monolayer liposomes and / or multilamellar liposomes containing a drug, It was found that by using a liposome preparation encapsulated in large unilamellar vesicles and / or multilamellar vesicles, when a peptide / protein drug or vaccine is orally or transmucosally administered, it is more efficiently absorbed. Invented.

That is, the present invention provides one or more unilamellar vesicles and / or multilamellar vesicles (encapsulated liposomes) in a unilamellar vesicle and / or multilamellar vesicle (envelope liposome), and if necessary, The present invention relates to a liposome preparation for oral administration or transmucosal administration, which contains an absorption enhancer and contains a peptide / protein drug or a vaccine in an encapsulated liposome.

In the present invention, the peptide / protein drugs include calcitonin, insulins, glucagon, growth hormone, growth hormone releasing hormone, somatostatin, growth hormone releasing factor, enkephalins, luteinizing hormone releasing hormone, Insulin-like growth factors, calcitonin gene-related peptides, vasopressins, atrial natriuretic peptides, interferons, interleukins, erythropoietin, granulocyte colony-stimulating factor, macrophage formation-stimulating factor, parathyroid hormone, parathyroid hormone release Mention may be made of one or more peptide / protein drugs selected from the group consisting of hormones, oxytocin, prolactin and thyroid stimulating hormone releasing hormone.

Calcitonin is a peptide hormone that regulates calcium metabolism in the body, and has the functions of inhibiting bone resorption of calcium and reabsorbing calcium excreted from the kidney. As its kind, salmon calcitonin, eel calcitonin, human calcitonin, porcine calcitonin, chicken calcitonin, bovine calcitonin, sheep calcitonin, rat calcitonin are known as natural ones, and elcitonin etc. are also known as their synthetic derivatives. . In the present invention, any of these calcitonin and derivatives thereof can be used as the calcitonin.

Insulin is a hormone that is biosynthesized in B cells of the islets of Langerhans and secreted into the blood, and its blood glucose level is enhanced by the action of promoting the membrane penetration of sugars (glucose, galactose, etc.) and the action of utilizing sugar. Has the function of controlling Insulin of human, pig, cow, dog, rabbit, horse, sheep, rat, etc. is known as its kind. In the present invention, any of these insulins can be used as insulins.

Parathyroid hormone (PTH) is secreted by the parathyroid gland and has the function of maintaining the normal serum Ca and P concentrations. Bovine, human, porcine and rat are known and are peptides consisting of amino acid residue 84. In addition, as its derivative, N-terminal amino acid removed,
PTH (1-3 composed of 34 amino acid residues from the end
4) etc. are known. In the present invention, any of these parathyroid hormones and their derivatives can be used as the parathyroid hormones.

Growth hormone-releasing hormone (GHRH) is a peptide consisting of 43 to 44 amino acid residues that acts on the anterior pituitary of mammals to release growth hormone, and is of human, rat, sheep, bovine and porcine origin. Things are known. Further, as its derivative, GHRH (1-29) -NH2, etc., which is composed of 29 amino acid residues from the N-terminal without removing the C-terminal amino acid, is known. In the present invention, any of these growth hormone-releasing hormones and derivatives thereof can be used as the growth hormone-releasing hormones.

Luteinizing hormone-releasing hormone (LHR
H) is a peptide that acts on GTH cells in the anterior pituitary of vertebrates to release LH and FSH, and human, pig, chicken, salmon, and eel are known. As its derivative, [D-Ser (t-Bu) ⁶ , des-Gly ¹⁰ e
thylamide] LHRH, [D-Ala-3 (2-naphtyl) ⁶ ] LHRH, [D-Le
u ⁶ , des-Gly ¹⁰ ethylamide] LHRH, [D-Ser (t-Bu) ⁶ , aza-G
ly ¹⁰ ] LHRH and other super agonists and [N-Ac-D-Nal
(2) ¹ , D-pFPhe ² , D-Pal (3) ³ , Lys (Nic) ⁵ , D-Lys (Nic) ⁶ , Lys
Antagonists such as (iPr) ⁸ , D-Ala ¹⁰ ] LHRH are known. In the present invention, any of these luteinizing hormone-releasing hormones and derivatives thereof can be used as the luteinizing hormone-releasing hormones.

Among the peptide / protein drugs of the present invention,
Calcitonins, insulins, PTHs, GHR
Similarly, other than Hs and LHRHs also include conventionally known compounds derived from natural origin, chemically or genetically engineered, and their synthetic or partially amino acid substituted, inserted or deleted derivatives. .

In the present invention, the vaccine includes one or more peptide / protein drugs selected from the group consisting of influenza vaccine, diphtheria vaccine, tetanus vaccine and pertussis vaccine.

In the present invention, the amount of the peptide / protein drug or vaccine is a therapeutically effective amount, and is an amount specific to each peptide / protein drug or vaccine. Generally, the therapeutically effective amount is preferably the same amount to 20 times as much as the amount of each peptide / protein drug or vaccine used in injection administration, particularly preferably 2 times to 10 times. .

In the present invention, examples of the absorption enhancer include chelating agents such as EDTA, cyclodextrins such as dimethyl-β-cyclodextrin, saturated / unsaturated fatty acids such as sodium caprate, salts thereof, and saturated salts thereof. / Unsaturated fatty acid alcohols, alkyl esters thereof, proteolytic enzyme inhibitors and the like, which are conventionally known in oral preparations or transmucosal preparations, can be used. The absorption enhancer of the present invention is preferably present in the outer skin liposome together with the inner liposome, but may be further present in the inner liposome.

In the present invention, the proteolytic enzyme inhibitor may be any as long as it inhibits proteolysis by trypsin or chymotrypsin. That is, an inhibitor against serine protease is naturally included therein. It is also possible to use inhibitors for aminopeptidases. In particular, by combining a trypsin inhibitor, a chymotrypsin inhibitor, and further an aminopeptidase inhibitor, it may be possible to achieve more efficient oral administration by the liposome preparation of the present invention. Examples of these protease inhibitors are aprotinin, soybean trypsin inhibitor, potato type I / II inhibitor, pancreatic secretory trypsin inhibitor, α2 macroglobulin, leupeptin, chymostatin, elastatinal, benzamidine, aminocaproic acid, tranexamic acid, mesyl. Acid gabexate, camostat mesilate, nafamostat mesilate, DFP (diisopropyl fluorophosphate), PMS
F (phenylmethylsufonyl fluoride), TPCK (to
sylamino phenyletheyl chloromethyl ketone), α
1 antitrypsin, α1 antichymotrypsin and the like can be mentioned. Of these, aprotinin and α2 are preferable.
Examples include macroglobulin, chymostatin, gabexate mesylate, camostat mesylate, and nafamostat mesylate.

In the present invention, the amount of the protease inhibitor is influenced by the amount of the peptide / protein drug encapsulated in the liposome, and usually, the amount of the peptide / protein drug encapsulated in the liposome is used. The amount is preferably from the same amount to 100 times, and particularly preferably from 10 times to 50 times.

The liposome preparation of the present invention is, for example, (A)
It is produced by two steps, that is, the production of an internal liposome containing a drug, and (B) the production of an external liposome containing an internal liposome. Any known technique is adopted in the step (A). In the step (B), for example, a manufacturing method using a glass filter is adopted.

Examples of the manufacturing method of the step (A) include the following.

(1) The solvent of the phospholipid solution was removed by evaporation in a flask to form a thin film of phospholipid, and the membranous phospholipid was subjected to hydration using an aqueous solution containing a peptide / proteinaceous drug or vaccine. An MLV (or MLV and OLV; hereinafter the same) liposome containing a proteinaceous drug or vaccine is obtained. This MLV liposome corresponds to an example of the encapsulated liposome in the preparation of the present invention. Further, by shaking this MLV with an ultrasonic shaker, SUV liposomes can be obtained. This SUV liposome also corresponds to an example of the encapsulated liposome in the preparation of the present invention.

(2) In a flask, the solvent of the phospholipid solution is removed by evaporation to form a thin film of phospholipid, and the membranous phospholipid is subjected to hydration using an aqueous solution containing a peptide / proteinaceous drug or a vaccine. Obtain MLV liposomes containing a proteinaceous drug or vaccine. This MLV liposome corresponds to an example of the encapsulated liposome in the preparation of the present invention. Furthermore, when this MLV is put into a French press and extruded at 20,000 lbs / in ² , a non-uniform SUV containing the MLV is produced. A uniform SUV is produced by repeating the extrusion by the French press at least four times or more. This SUV liposome also corresponds to an example of the encapsulated liposome in the preparation of the present invention.

(3) An SUV liposome containing a peptide / protein drug or vaccine can be obtained by injecting an ethanol solution of phospholipid through a syringe into an aqueous solution in which the peptide / protein drug or vaccine is dissolved. . This SUV liposome also corresponds to an example of the encapsulated liposome in the preparation of the present invention.

(4) Diameter 29 mm, thickness 4 mm, hole diameter 10
Impregnate the phospholipid solution in a 16 μm glass filter and remove the solvent by circulating nitrogen at room temperature,
An aqueous solution containing a peptide / proteinaceous drug or vaccine is added and hydrated for 10 minutes at a temperature not lower than the phase transition temperature of the phospholipid used. Then, sonicate for 30 minutes, attach syringes to both ends of the glass filter, and rapidly pass the remaining buffer solution through the filter to prepare SUV liposomes containing a peptide / protein drug or vaccine. it can. This SUV liposome also corresponds to an example of the encapsulated liposome in the preparation of the present invention.

As the manufacturing method of the step (B), for example, the phospholipid solution is impregnated in a glass filter having a diameter of 30 mm, a thickness of 5 mm and a pore size of 40 to 100 μm, and the solvent is removed by circulating nitrogen at room temperature. , SU containing peptide / protein drug or vaccine prepared in step (A)
An aqueous suspension of V liposomes and / or MLV liposomes is added. The absorption enhancer added as necessary is
It is preferable to add it to this suspension aqueous solution. Next, the phospholipid used is hydrated for 10 minutes at a temperature equal to or higher than the phase transition temperature. After that, ultrasonic treatment is performed for 30 minutes, syringes are attached to both ends of the glass filter, and the remaining buffer solution is rapidly passed through the filter, whereby the unilamellar vesicles and / or multilamellar vesicles (outer shell) of the present invention are One or more unilamellar liposomes and /
Alternatively, it is possible to obtain a liposome preparation for oral or transmucosal administration, which comprises multilamellar liposomes (encapsulated liposomes) and, if necessary, an absorption enhancer and also contains a peptide / protein drug or vaccine in the encapsulated liposomes. it can.

In the thus obtained liposome preparation of the present invention, it is desirable that the peptide / protein drug or vaccine is contained only in the encapsulated liposome, but within the scope of the above-mentioned object of the present invention. It may be present in the sealed liposome membrane and / or in the aqueous phase of the outer liposome, or in the membrane.

The liposome preparation of the present invention is applied as a preparation for oral or transmucosal administration. Examples of such mucosa include oral mucosa, nasal mucosa, cavity mucosa, uterine mucosa, rectal mucosa, and the like. Among these, nasal mucosa and cavity mucosa can be mentioned as preferable mucosa.

As the phospholipid used in the production of the liposome of the present invention, those known per se can be used,
Examples include egg yolk lecithin, soybean lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, sphingomyelin, lysophosphatidylcholine, natural or synthetic phosphatidic acid. Phospholipids, hydrogenated phospholipids or glycerophosphoglyceroglycolipids. These phospholipids may be used alone or in combination of two or more.

In the liposome preparation of the present invention, in addition to these phospholipids, sterols such as cholesterol as a membrane stabilizer as an additive, tocopherol as an antioxidant, stearylamine, dicetyl phosphate as a charged substance, Gangliosides may be added.

The liposome preparation of the present invention comprises a combination of the above phospholipids and, if necessary, a combination with other additives. Among these combinations, soybean lecithin and phosphatidylserine are selected. Preferred examples include liposome preparations composed of phospholipids selected from the group consisting of soybean lecithin, hydrogenated soybean lecithin, and liposome preparations composed of phospholipids selected from the group consisting of phosphatidylserine and charged substances such as stearylamine. You can

Further, in these liposome preparations of the present invention, the inner-encapsulated liposome is preferably a single-membrane and / or multilamellar liposome, and 1 to several single-membrane liposomes are preferable, and the outer-liposomes are one-membrane and / or Among the multilamellar liposomes, the unilamellar liposome can be mentioned as a preferable one. In the inner and outer liposomes of such a preferred example, as a preferable combination with a phospholipid, an additive, a peptide / protein drug or a vaccine, an absorption enhancer, etc., the above-mentioned preferred examples of phospholipid to absorption enhancer, etc. A combination can be mentioned.

The drug encapsulated in the liposome preparation of the present invention is in the form of an aqueous solution. This aqueous solution contains isotonic substances such as phosphoric acid, acetic acid, citric acid and the like which constitute a buffer solution, sodium chloride, glucose and glycerin. Agent, tocopherol,
Those to which an antioxidant such as glutathione, an antiseptic such as chlorbutanol and paraben, a stabilizer such as albumin and the like are added may be used.

The liposome preparation of the present invention is usually administered by the following administration methods. That is, when used as a preparation for oral administration, there is a method in which the obtained liposome solution is used as it is, or when it is used as a concentrated liposome, it is dispersed in a dispersion liquid before use. In addition, if possible, there is a method such as freeze-drying and use as it is or by redispersion before use.

When used as a preparation for transmucosal administration, there is a method in which the obtained liposome solution is put in a spray container or a syringe and sprayed or dropped on the mucosal site.
In addition, when freeze-drying is possible, there is a method in which it is dissolved at the time of use, put in a spray container or syringe, and sprayed or dropped on the mucosal site. In addition, the freeze-dried powder is filled into the capsule as it is, set in a dedicated spray device, the needle is pierced, thereby opening microscopic holes in the top and bottom of the capsule, and then air is sent with a rubber ball etc. to the mucous membrane site. There is a method to make it gush out.

It is of great medical value to provide a liposome preparation capable of efficiently orally or transmucosally administering a peptide / protein drug or vaccine according to the present invention.

[0042]

The present invention will be described in detail below with reference to examples.
This is provided to illustrate the invention,
It does not limit the invention.

Examples 1 to 6 Liposomes having the compositions shown in Table 1 below were prepared using a glass filter.

(1) That is, the lipids shown in Table 1 were dissolved in chloroform according to the composition, and the diameter was 29 mm, the thickness was 4 mm, and the pore size was 1
After impregnating a glass filter of 0 to 16 μm, nitrogen was passed at room temperature to completely distill off the solvent. A phosphate buffer solution having a pH of 7.4 in which insulin or salmon calcitonin was dissolved was added thereto, hydrated for 10 minutes at the phase transition temperature of hydrogenated soybean lecithin or higher, and then sonicated for 30 minutes.
Next, attach syringes to both ends of the glass filter and p
A H7.4 phosphate buffer solution (PBS) was rapidly passed through the filter to prepare an encapsulated liposome for encapsulating the drug.

(2) A lipid having the same composition as that of (1) was prepared with a diameter of 30.
After impregnating a glass filter having a size of 5 mm, a thickness of 5 mm and a pore size of 40 to 100 μm, nitrogen was passed at room temperature to completely distill off the solvent. In addition to this, the encapsulated liposomes prepared in (1), pH
7.4 phosphate buffer or pH 7.4 phosphate buffer in which aprotinin and / or chymostatin is dissolved is added, hydrated for 10 minutes at a temperature above the phase transition temperature of hydrogenated soybean lecithin, and then sonicated for 30 minutes. Processed. Then, syringes were attached to both ends of the glass filter, and PBS was rapidly passed through the filter to prepare a liposome preparation used in the present invention. It was confirmed that the thus-obtained liposome preparation contains 1 to several SUVs as the internal liposomes in the LUVs as the outer liposomes.

[0046]

[Table 1]

[Comparative Examples 1 to 6] SUV liposomes having the compositions shown in Table 2 below were prepared using a glass filter.
That is, the following lipids were dissolved in chloroform according to the composition and impregnated in a glass filter having a diameter of 29 mm, a thickness of 4 mm and a pore size of 10 to 16 μm, and then nitrogen was passed at room temperature to completely distill off the solvent. A phosphate buffer solution of pH 7.4 in which insulin or salmon calcitonin was dissolved was added thereto, hydrated for 10 minutes at the phase transition temperature of hydrogenated soybean lecithin or higher, and then sonicated for 30 minutes. Then, attach syringes to both ends of the glass filter, and use phosphate buffer (PB
S) is rapidly passed through the filter, and the target SU
V liposome formulations (Control Examples 1 to 6) were prepared.

[0048]

[Table 2]

[Embodiments 7 to 9] Embodiment 1 as Embodiment 7
The insulin-encapsulated liposomes prepared in 1 above were dispersed in PBS, and male rats (Wistar strain, body weight: 180 to 200 g) fasted for 16 hours were forcibly orally administered with a sonde so that the insulin amount was about 20 IU / kg. The liposome prepared in Example 2 as Example 8 and the liposome prepared in Example 3 as Example 9 were similarly administered. Blood was collected from the jugular vein after a certain period of time, and the glucose concentration in the serum was measured by the glucose oxidase method using a glucose B-test kit (Wako Pure Chemical Industries, Ltd.).

At the same time, as a comparison, Comparative Examples 7 to 10 were carried out. The liposome prepared in Control Example 1 was used as Control Example 7, and the liposome prepared in Control Example 2 was used as Control Example 8.
Further, as Control Example 9, the liposomes prepared in Control Example 3 were orally administered to rats under the same conditions as in Examples 1 to 3.
In addition, Control Example 10 is an example of a rat when not administered.

The obtained results are shown in FIG.

From FIG. 1, the liposome preparation of the present invention (preparation of Examples 7, 8 and 9) was compared with the conventional liposome preparation (preparation of Control Examples 7, 8 and 9) and blood glucose after oral administration. It can be seen that the value is significantly reduced. That is, it was shown that the present invention efficiently absorbs encapsulated insulin in the body as compared with the conventional technique.

[Embodiment 10] Embodiment 6 as Embodiment 10
The salmon calcitonin-encapsulated liposomes prepared in step 1 were dispersed in PBS so that the amount of calcitonin was about 500 IU / kg.
Male rats fasted for a long time (Wistar strain, weight: 180-200
g) was orally administered by a sonde. Blood was collected from the jugular vein after a certain period of time, and the calcitonin concentration in plasma was measured by RI.
It was measured by Method A.

At the same time, as a comparison, Comparative Example 11 and Comparative Example 12 were carried out. The liposome preparation prepared in Control Example 4 as Control Example 11 and the liposome preparation prepared in Control Example 6 as Control Example 12 were orally administered to rats under the same conditions as in Example 10.

The results obtained are shown in Table 3.

[0056]

[Table 3]

From Table 3, the salmon calcitonin absorption amount of the salmon calcitonin-containing liposome preparation for oral administration of the present invention (Example 10) was about 4.3 times that of Control Example 11,
3.8 times that of Control Example 12 (AUC; pg · h / ml)
The results show that the absorption rate and durability are excellent.

[Embodiment 11] Embodiment 4 as Embodiment 11
The salmon calcitonin-encapsulated liposome preparation prepared in
Female rats (Wistar strain, weight: 18) were dispersed in S and fasted for 16 hours so that the amount of calcitonin was about 200 IU / kg.
0-200 g) was administered to the vaginal mucosa. Blood was collected from the jugular vein after a certain period of time, and the calcitonin concentration in plasma was measured by the RIA method.

At the same time, as a comparison, Comparative Example 13 was carried out. As Control Example 13, the liposome preparation prepared in Control Example 4 was administered to the vaginal mucosa of rats under the same conditions as in Example 11.

The results obtained are shown in Table 4.

[0061]

[Table 4]

From Table 4, it can be seen that the salmon calcitonin-containing lysosome preparation for transmucosal administration (Example 11) of the present invention is superior in absorption rate and sustainability to the control example.

[Embodiment 12] Embodiment 5 as Embodiment 12
The salmon calcitonin-encapsulated liposome preparation prepared in
Male rabbits (Japanese White strain, dispersed in S and fasted for 16 hours so that the amount of calcitonin was about 200 IU / kg)
Body weight: 2.5-3.5 kg) was sprayed into the nasal cavity. Blood was collected from the ear vein after a certain period of time, and the calcitonin concentration in plasma was measured by R
It was measured by the IA method.

At the same time, as a comparison, Comparative Examples 14 and 15 were carried out. As Control Example 14, the liposome preparation prepared in Control Example 5 was intranasally administered to a rabbit under the same conditions as in Example 12.

Further, as a control example 15 (aqueous solution), an acetic acid buffer solution of salmon calcitonin was added in an amount of about 20 calcitonin.
It was intranasally administered to a rabbit under the same conditions as in Example 12 so that the dose would be 0 IU / kg.

The results obtained are shown in Table 5.

[0067]

[Table 5]

From Table 5, it can be seen that the salmon calcitonin-containing liposomal preparation for transmucosal administration (Example 12) of the present invention is superior in absorption rate and sustainability to the control example.

Examples 13 to 18 Liposomes having the compositions shown in Table 6 below were prepared using a glass filter.

(1) That is, the lipids shown in Table 6 were dissolved in chloroform according to the composition, and the diameter was 30 mm and the thickness was 5 m.
m and a glass filter having a pore size of 40 to 100 μm were impregnated, and then nitrogen was passed at room temperature to completely distill off the solvent. To this, a phosphate buffer solution having a pH of 7.4 in which the peptide / proteinaceous drug shown in Table 6 was dissolved was added, and hydrated for 10 minutes at a temperature above the phase transition temperature of hydrogenated soybean lecithin, and then 30
Sonicated for minutes. Then, a syringe equipped with both ends of a glass filter was attached, and a phosphate buffer of pH 7.4 was rapidly passed through the filter to prepare liposomes encapsulating the peptide / proteinaceous drug shown in Table 6.

(2) A lipid having the same composition as in (1) was prepared with a diameter of 3
After impregnating a glass filter having a hole diameter of 40 to 100 μm with a thickness of 0 mm and a thickness of 5 mm, nitrogen was passed at room temperature to completely distill off the solvent. To this, the encapsulated liposomes prepared in (1) and a phosphate buffer of pH 7.4 or a phosphate buffer of pH 7.4 in which gabexate mesylate is dissolved are added, and the phase transition temperature of hydrogenated soybean lecithin or higher is exceeded. In 1
It was hydrated for 0 minutes and then sonicated for 30 minutes. Next, attach syringes to both ends of the glass filter and adjust the pH to 7.
The phosphate buffer solution of No. 4 was rapidly passed through the filter to prepare a liposome preparation corresponding to the present invention.

[0072]

[Table 6]

[Brief description of drawings]

FIG. 1 shows the measurement results of serum glucose concentration when an insulin-encapsulated liposome preparation (Examples 7 to 9, Control Examples 7 to 10) was orally administered to rats. Figure 1 (A)
Shows the results of administration of the liposome preparations of Control Examples 7 to 10, the white circles show Control Example 10, the black circles show Control Example 7, the white horns show Control Example 8, and the black horns show Control Example 9. Further, FIG. 1 (B) shows the results of administration of the liposome preparations of Examples 7 to 9 and Control Example 10. The white circles are Control Example 10, the black circles are Example 7, the white horns are Example 8, and the black horns are Examples. 9 is shown.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication location A61K 9/127 V 38/00 38/28 38/26 38/23 38/11 38/27 38 / 04 38/21 39/00 G 47/20 E 47/42 E A61K 37/24 37/26 37/28 37/30 37/34 37/36 37/43 37/66 H

Claims (6)

[Claims] 1. One or more unilamellar vesicles and / or multilamellar vesicles (encapsulated liposomes) and, if necessary, an absorption enhancer in a unilamellar vesicle and / or multilamellar vesicle (outer coat liposome). A liposome preparation for oral administration or transmucosal administration, which comprises a peptide / protein drug or a vaccine in an encapsulated liposome. 2. The peptide / protein drug is calcitonin, insulin, glucagon, growth hormone, growth hormone releasing hormone, somatostatin, growth hormone releasing factor, enkephalins, luteinizing hormone releasing hormone, insulin-like growth. Factors, calcitonin gene-related peptides, vasopressins, atrial natriuretic peptides, interferons, interleukins, erythropoietin, granulocyte colony-stimulating factor, macrophage-stimulating factor, parathyroid hormone, parathyroid hormone-releasing hormone, oxytocin The liposome preparation according to claim 1, which is one or more kinds of peptide / protein drugs selected from the group consisting of, prolactin, and thyroid-stimulating hormone-releasing hormone. 3. The peptide / protein drug is one or more peptide / protein drugs selected from the group consisting of calcitonins, insulins, growth hormone releasing hormones, luteinizing hormone releasing hormones, and parathyroid hormones. The liposome preparation according to claim 1, which is 4. The vaccine is influenza vaccine,
The liposome preparation according to claim 1, which is a diphtheria vaccine, a tetanus vaccine or a pertussis vaccine. 5. The liposome preparation according to claim 1, wherein the absorption enhancer is a protease inhibitor. 6. The protease inhibitor is one or more protease inhibitors selected from the group consisting of aprotinin, gabexate mesylate, camostat mesylate, nafamostat mesylate, soybean trypsin inhibitor, α2 macroglobulin, chymostatin. The liposome preparation according to claim 5, which is an agent.