WO1993019771A1

WO1993019771A1 - Pernasal preparation containing granulocyte colony-stimulating factor

Info

Publication number: WO1993019771A1
Application number: PCT/JP1993/000368
Authority: WO
Inventors: Yoshiteru Watanabe; Mitsuo Matsumoto; Kazutoshi Maruyama; Hideaki Nomura; Satoshi Akamisaka
Original assignee: Kirin Brewery Company, Limited
Priority date: 1992-03-27
Filing date: 1993-03-26
Publication date: 1993-10-14

Abstract

A pernasal preparation containing a granulocyte colony-stimulating factor (G-CSF), a physiologically active protein, as an active ingredient.

Description

Description Nasal formulation containing granulocyte colony-stimulating factor

[Technical field]

The present invention relates to a preparation for nasal administration containing, as an active ingredient, granulocyte colony stimulating factor (G-CSF) which is a physiologically active protein.

[Background technology]

G—CSF is one of the hematopoietic factors and has been shown to be present in the culture of the human bladder cancer cell line 5637 (ATCC HT8-9). Natl Acad. Sci. U. S. A. 82, 1526-1530, (1985)). In addition, the DNA sequence encoding this gene has been determined (Japanese Patent Publication No. 63-500636), and G-CSF can be produced by genetic recombination.

G—CSF is effective in the treatment of normal hematopoietic disorders, chemotherapy, or the treatment of hematopoietic disorders by radiation therapy, bone marrow transplantation, and the like (前述, et al., Supra).

G-CSF-containing preparations include preparations prepared by adding a surfactant to G-CSF for the purpose of inactivating the preparation during storage and preventing and stabilizing adsorption to the container wall (Japanese Patent No. 63-146826), and a formulation containing saccharides (JP-A 63-146827). Formulations containing molecules (JP-A-63-148688) and formulations containing any one of amino acids, sulfur-containing reducing agents, and antioxidants (JP-A-1414) No. 6 829) or a protein-added preparation (Japanese Patent Application Laid-Open No. 63-143). However, all of these are in the form of a propellant.

In addition, in order to prevent inactivation of G-CSF during administration and to increase the sustainability of the drug effect, a sustained-release preparation containing G-CSF in a base composed of a biocompatible polymer in vivo ( Japanese Patent Application Laid-Open No. 63-91 1325) has been filed. In addition, for oral administration, an oral enteric preparation comprising G—CSF, a surfactant, a fatty acid, and an enteric base and a power has been reported.

In general, proteins having physiological activity are easily degraded by stomach acid or enzymes in the digestive tract or in the wall of the digestive tract, and it is extremely difficult to absorb them from the digestive tract due to their large molecular weight / complex molecular structure. So far, its application has been limited to intravenous, subcutaneous or intramuscular injection.

However, administration of such an injection causes pain to the patient, and involves problems such as tissue damage at the administration site and difficulty in ingestion by the patient.

In addition to the above administration methods, the administration method is relatively simple and painless, Nasal preparations that can be ingested by the patient themselves have been considered, but those that are currently in practical use as nasal preparations are oximetazoline hydrochloride and hydrochloride for nasal mucosal hyperemia and depressed blood. Most are intended for local effects, such as sodium cromoglycate, flunisolide, and betamethasone propionate for tetrahydrozolin and allergic rhinitis, and are expected to have systemic effects These include desmopressin acetate for central diabetes insipidus, pselelin acetate for endometriosis, and salmon calcitonin for osteoporosis. Moreover, the former, which is intended for local action, is a low molecular weight organic compound having a molecular weight of about 200 to 700, and the latter, which is intended for systemic action, is a comparison of molecular weights of 1,200 to 3,400. Low molecular weight type. In the case of physiologically active proteins with a molecular weight of 100,000 or more, intranasal administration has a low absorption rate compared to injection and does not have a sufficient pharmacological effect, so that it has been commercialized to date. do not do.

Therefore, an object of the present invention is to provide an intranasal G-CSF preparation as a non-injectable preparation which can be put to practical use of G-CSF having a molecular weight of about 190,000 to 200,000. It is to be.

[Disclosure of the Invention]

In view of the above situation, the present inventors have effectively applied G-CSF to the nasal mucosa. As a result of investigations aimed at the development of a drug that can be absorbed from the skin, surprisingly, the fact that G-CSF, a high molecular weight protein with a molecular weight of about 20,000, can be absorbed by the nasal mucosa alone, and that an absorption enhancer must be added As a result, the present inventors have found that they are more effectively absorbed from the nasal mucosa, and reached the present invention.

The G—CSF in the present invention can be produced from a transformed host cell obtained by transformation by gene recombination and isolated and purified. Examples of host cells include Escherichia coli, mammalian cells (C-127, CHO cells, etc.). The detailed production method of these G—CSFs is disclosed in, for example, Japanese Patent Publication No. Sho 63-3-5003636. In the nasal administration preparation of the present invention, a G-CSF derivative as disclosed in Japanese Patent Application Laid-Open No. Sho-sho can also be used.

The preparation for nasal administration according to the present invention comprises G-CSF or G-CSF and saccharides, surfactants, organic acids, lipids, amino acids, alcohols, glycols and vitamins as absorption enhancers. Contains one or more selected from the group consisting of:

For example, saccharides include aldose, amino sugar, cyclodextrin, sugar alcohol, sialic acid, dalconic acid, glucuron Acids and the like. Examples of the surfactants include cationic surfactants such as benzalcodium chloride and benzozetnium chloride, and anionic surfactants such as sodium sodium polysodium sulfate and sodium octyl sulfosuccinate. Activator, lauromacrogol, polyoxyl stearate 40, polyoxyethylene hydrogenated castor oil 10, 50, 60, glyceryl monostearate, polysorbate 20, 40, 60 , 65, 80, non-ionic surfactants such as sucrose fatty acid esters, cholic acid, glycocholic acid, dehydrocholic acid, deoxycholic acid, taurocholic acid, and salts thereof, savones, etc. Can be exemplified.

Organic acids include salicylic acid, glycyrrhetinic acid, glycyrrhizic acid, hyaluronic acid, tartaric acid, lingic acid, lactic acid, fumaric acid, citric acid, and salts thereof. Oleic acid, linoleic acid, linolenic acid, arachidonic acid, and salts thereof, lecithin, and acylglycerol. Alcohols include benzyl alcohol and ethanol; glycols include propylene glycol, polyethylene glycol, and petylene glycol; and vitamins include ascorbic acid, thiamin, and pyridoxal. In the present invention, as the local retention improving substance, Glucose, methylcellulose, ethynoresenorelose, hydroxyshethylcellulose, hydroxypropylcellulose, hydroxypropyl pillmethylcellulose, carboxypropylcellulose, sodium carboxymethylcellulose, and proteolytic enzymes Addition of inhibitors as aprotinin, soybean trypsin inhibitor, pacitracin, nafamostat mesilate, gabexat mesylate, potash mesylate, albumin, globulin, and gelatin In addition, the absorption can be increased.

The preparation for nasal administration according to the present invention can be obtained by dissolving it in an O liquid diluent such as a buffer solution to prepare a nasal solution, or by diluting it into a powdery carrier to obtain a nasal powder.

In the preparation for nasal administration according to the present invention, 0.1 ljtig to: L0000 / igZkg, preferably ljag to 500iig can be usually administered to an adult. The amount of the absorption enhancer is usually 0.1 to 50% based on the preparation. Dimethyl: In the case of S-cyclodextrin, the amount is preferably about 2 to 1000 ing per ml of nasal solution or about 2 to 1000 mg per lg of nasal powder.

Further, in the present invention, a preservative, a stabilizer and an excipient are used as necessary. As preservatives, acrylonitrile and cetylpyridinyl chloride And benzalkonium chloride, benzethonium chloride, etc. Stabilizers: polysorbate 40, 60, 65, 80, polyoxyl stearate 40, polyoxyethylene hydrogenated castor oil 10, 50, 60, sucrose fatty acid Ester, methylcellulose, carboxymethylcellulose and the like are included. Excipients include starches, sugars, minerals, organics, celluloses, synthetic and semi-synthetic polymers, amino acids, and the like. Starches include corn starch, wheat starch, potato starch and the like. Sugars include lactose, sucrose, sucrose, fructose, D-sorbitol, D-mannitol, inositol, and sucrose. Minerals include magnesium stearate, calcium phosphate, calcium hydrogen phosphate, magnesium carbonate, sodium chloride, calcium sulfate, and the like. Examples of the organic acids include succinic acid, tartaric acid, citric acid, fumaric acid, lingic acid, gluconic acid, and glucuronic acid. Cellulose includes microcrystalline cellulose, methylcellulose, ethylcellulose, hydroxyethyl cellulose, hydroxypropylcellulose, carboxypropylcellulose, and carboxymethylcellulose sodium. You. Synthetic and semi-synthetic polymers include polyvinyl alcohol Water, carboxyvinyl polymer, polyethylene glycol, polyvinylpyrrolidone, and sodium polyacrylate. The amino acids, L one-arginine, D, L- Mechio two emissions, L- phenylene Ruaranin, _c which etc. L- glutamicum phosphate

[Brief description of drawings]

FIG. 1 is a graph showing the results of Experimental Example 1. FIG. 2 is a graph showing the results of Experimental Example 2. FIG. 3 is a graph showing the results of Experimental Example 2. FIG. 4 is a graph showing the results of Experimental Example 3 _c . FIG. 5 is a graph showing the results of Experimental Example 3 FIG. 6 is a graph showing the results of Experimental Example 4. FIG. 7 is a graph showing the results of Experimental Example 5. FIG. 8 is a graph showing the results of Experimental Example 6. FIG. 9 is a daraf showing the results of Experimental Example 7. FIG. 10 to FIG. 12 are graphs showing the results of Experimental Example 8.

[Best Mode for Carrying Out the Invention]

Hereinafter, the present invention will be described specifically with reference to Examples and Experimental Examples.

w / V%

Granulocyte colony stimulating factor 0.02

Adjust to a total volume of 100 with 16.7 mM phosphate buffer. Example 2

V%

Granulocyte colony stimulating factor 0.06

6 Make the total volume 0 with 7 mM acetate buffer Example 3

VJ / V%

Granulocyte colony stimulating factor 0, 2

6 Make the total volume 0 with 7 mM 齚 acid buffer.Example 4

f / V%

Granulocyte colony-stimulating factor 0,02 Dimethyl-cyclodextrin 20

6 Make the total volume 100 with 7 mM 齚 acid buffer.Example 5

f / V% Granulocyte colony stimulating factor 0.06 dimethyl-cyclodextrin 20

6 Make the total volume 0 with 7 mM acetate buffer Example 6

ντ / V%

Granulocyte colony stimulating factor 0.2 dimethyl mono-; 8-cyclodextrin 20

Example 6 Granulocyte colony-stimulating factor 0, 37 75

Adjust the total volume to 0 with 10 mM MS acid buffer Example 8

w / V% granulocyte colony stimulating factor 0.3 7 5 Benzalkonium chloride

Adjust the total volume to 0 0 with 0 m Mg ^ acid buffer Example 9

w V%

Granulocyte colony stimulating factor 0.375 sodium peryl sulfate

Adjust the total volume to 0 with 0 mM M acid buffer.Example 10

V%

Granulocyte colony stimulating factor 0.3 7 5 Polysorbate 80

The total volume is adjusted to 0 with a 0 mM phosphate buffer. Example Granulocyte colony stimulating factor 0.375 sodium sodium deoxycholate

Adjust the total volume to 0 with 0 mM acetate buffer. Granulocyte colony stimulating factor 0.3 7 5 Salicylic acid

Adjust the total volume to 0 with 0 mM acetate buffer.Example 13

/ V%

Granulocyte colony stimulating factor 0.3 7 5 Tartaric acid 1

Adjust the total volume to 0 with 1 OmM acid buffer.Example 14

/ V% granulocyte colony stimulating factor 0.3 7 5 lecithin

Adjust the total volume to 0 with 0 mM drunk buffer. Granulocyte colony stimulating factor 0.3 7 5 sodium carboxylate

Example 1 6 Decrease the total volume to 0 with 0 mM acetate buffer.

Granulocyte colony stimulating factor 0.3 7 5 Resin

Adjust the total volume to 0 with a 0 mM M acid buffer.

w / V%

Granulocyte colony stimulating factor 0.3 7 5 Glycine

Adjust the total volume to 0 with 0 mM acetate buffer. Example 18

wZ V%

Granulocyte colony stimulating factor 0.375 benzyl alcohol

Example 19 The total volume was adjusted to 00 with 0 mM 緩衝 acid buffer.

. V%

Granulocyte colony stimulating factor 0.375 Propylene glycol

Adjust the total amount to 00 with 0 mM 齚 acid buffer.Example 20

τ / V% granulocyte colony stimulating factor 0.375 ascorbic acid

Adjust the total volume to 00 with 0 mM MS acid buffer. Example 2

w / V%

Granulocyte colony stimulating factor 0.3 7 5 aprotinin

Adjust the total volume to 100 with 0 mM acetate buffer.Example 22

/ V%

Granulocyte colony stimulating factor 0.3 3 5 Basitracin

Adjust the total volume to 0 with 0 mM acetate buffer.Example 23

w / V%

Granulocyte colony stimulating factor 0.3 7 5 Nafamostat mesilate

Adjust the total volume to 0 with 0 mM 齚 acid buffer Example 24 Granulocyte colony stimulating factor 0.3 7 5 Hydroxypropyl methylcellulose 30

Adjust to a total volume of 0 with 0 mM phosphate buffer Example 25 Granulocyte colony stimulating factor .5 Magnesium stearate

Adjust the total amount to 0 with mannitol Example 2 6

w / V% granulocyte colony stimulating factor 5 magnesium stearate

Adjust the total amount to 100 with hydroxypropylcellulose Example 2 7

w / γ%

Granulocyte colony stimulating factor 0.2

a Cyclodextrin 20

6 Make the total volume 0 with 7 mM acetate buffer.Example 28

ί / V%

Granulocyte colony stimulating factor 0, 2

One cyclodextrin 2 0

Adjust the total volume to 0 with 6 7 mM acid buffer.Example 2 9

V%

Granulocyte colony stimulating factor 0.3 7 5 sodium collate

Adjust the total volume to 0 with 0 mM 齚 acid buffer Example 30 Granulocyte colony stimulating factor 0, 3 7 5

; 3—cyclodextrin 20

Adjust the total volume to 0 with 0 mM drunk acid buffer Example 3

w / v% granulocyte colony stimulating factor 0.337 5 dimethyl-jS—cyclodextrin 20

Adjust the total volume to 0 with 0 mM acetate buffer.Example 32

f / V% granulocyte colony stimulating factor 0.2

Bring the total volume to 0 with O mM sulphate buffer Example 3 3

f /%

Granulocyte colony-stimulating factor 0.2 dimethyl-cyclodextrin 20

Adjust the total volume to 0 with 0 mM acetate buffer.Example 3 4

f / V%

Granulocyte colony stimulating factor 0.2

-Cyclodextrin 20

Experiment example: Adjust the total volume to 0 with 0 mM sulphate buffer.

The drug of the present invention prepared in Example 1 was used as a G-CSF in male rabbits (Japanese white breed, weighing 2.9 to 4.1 kg) which had been fasted with water only overnight before administration as l-Ojt Nasal administration of 501 / kg was carried out as a / gZkg preparation. At the same time, a preparation prepared in the same manner as in Example 1 without containing G-CSF as a control was intranasally administered, and g was intravenously administered as G-CSF as a control. Blood was collected from the ear vein at regular intervals after administration. Blood collected The white blood cell count was counted using a Coulter force counter ZM. FIG. 1 shows the white blood cell count at each blood collection.

As a result, in the preparation of the present invention of Example 1 in which the dose was 10 kg as G—CSF, the leukocyte count decreased once and then increased over 0.5 to 2 hours after the administration, and then increased after administration. Peaked at hour. At around 96 hours thereafter, leukocyte increase was again observed. The temporary decrease in the white blood cell count in the early stage is thought to be due to the behavior of the white blood cells attaching to the blood vessel wall and migrating to peripheral tissues due to the action of G-CSF, and apparently the white blood cell count was reduced in the blood. You. The increase in the number of leukocytes is biphasic, but the initial peak is considered to be the release of leukocytes matured by G-CSF into the blood from the bone marrow, etc., and the later peak is G-CSF. It is probable that as a result of acting on bone marrow stem cells, mature leukocytes increased and appeared in the blood. These series of phenomena have been observed even after intravenous administration and have been characterized as the pharmacological effects of G-CSF. At the same dose (10 g Z kg), the preparation of the present invention showed a remarkable leukocyte count increasing effect, although it did not reach the effect of intravenous administration. In contrast, no pharmacological effect was observed for the formulation without G-CSF (control). Experimental example 2

The preparation of the present invention prepared in Example 2 was used as a G—CSF in a male rabbit (Japanese white breed, weighing 2.9 to 4.1 kg) which had been fasted with water only overnight before administration, and was given as G—CSF at 30 // g. / kg, and 501 / kg as a preparation were administered intranasally. As a control, 10 ^ gZkg as G—CSF was intravenously administered. Blood was sampled from the ear vein at regular intervals after administration. The number of leukocytes in the collected blood was counted using Kohl-Koun-Yuichi-ZM. The G-CSF concentration in the serum was also measured using the ELISA method (Enzyme-Linked Immunosorbent Assay). The white blood cell count at each blood collection is shown in FIG. 2, and the serum G—CSG concentration is shown in FIG.

As a result, in the preparation of the present invention of Example 2 in which the dose was 30 as ugZkg as G—CSF, the leukocyte count decreased once and then increased in 0.5 to 2 hours after transdermal administration. From time h to 72 h, it decreased after maintaining a nearly constant high white blood cell count. Compared to the intravenous administration of G-CSF at 10 βg / kg, the effect was almost the same as that of the whole leukocyte count, although it did not reach the leukocyte peak. On the other hand, in the change of blood G-CSF concentration after administration, the case of intranasal administration of the preparation of the present invention of Example 2 was lower than the case of intravenous administration of G-CSF at 10 gg, although the concentration was lower at each time. It was confirmed that G-CSF was absorbed into the body and translocated into the blood by nasal administration.

Experiment 3

The preparation of the present invention prepared in Examples 3 and 6 using male rabbits (Japanese white breed, weighing 2.9 to 4.1 kg) which had been fasted with water only overnight before administration was used as G-CSF. Nasal administration of 50 / gZkg and 50 I / kg as a preparation was performed. At the same time, a preparation prepared in the same manner as in Example 6 without containing G—CSF as a control was assaulted. Blood was collected from the ear vein at regular intervals after administration. The number of leukocytes in the collected blood was counted using a Coulter-Counter ZM. The G-CSF concentration in the serum was measured using the ELISA method (Enzyme-Linked Immunosorbent Assay). The white blood cell count at each blood sampling is shown in FIG. 4, and the serum G—CSG concentration is shown in FIG.

As a result, the dosage of 100 / i gZkg was set as G-CSF. In the preparations of the present invention of Examples 3 and 6, the white blood cell count decreased once and then increased over 0.5 to 2 hours after nasal administration. The peak reached 24 hours after the administration. Thereafter, white blood cells increased again around 120 hours in Example 3 and around 72 hours in Example 6. Examples containing dimethyl monocyclodextrin The case of administering the preparation of the present invention in No. 6 showed clearly higher leukocytes almost in most cases than the case of administering the preparation of the present invention nasally which does not contain the preparation. In addition, in the blood G-CSF concentration change after administration, the preparation of the present invention of Example 6 containing dimethyl-cyclodextrin was administered nasally to the preparation of the present invention of Example 3 containing no dimethyl-cyclodextrin. The blood G-CSF concentration was clearly higher from 0.5 to 0.6 hours after administration than in the case of the administration.

Experiment 4

The preparation of the present invention prepared in Examples 3 and 27 using male rabbits (Japanese white breed, weighing 2.9 to 4.1 kg) fasted with water only overnight before administration was designated as G-CSF. Nasal administration of 100 / z gZkg and 50 ^ 1 Zkg as a preparation was performed. Blood was collected from the ear vein at regular intervals after administration. The leukocytes in the collected blood were counted using Coulter Counter ZM. Fig. 6 shows the white blood cell count at each blood collection.

As a result, compared to the control Example 3 containing only granulocyte colony stimulating factor, Example 27 containing α-cyclodextrin showed a significantly higher white blood cell count almost at most of the time after administration. Experimental example 5 shown

Male rabbits fed water only overnight prior to administration and fasted (Japanese white species, The preparation of the present invention prepared in Example 3 and Example 28 using a body weight of 2.9 to 4.1 kg) was subjected to nasal administration of 100 gZkg of G-CSF and 50 g / kg of Zkg as a preparation. Was administered. Blood was collected from the ear vein at regular intervals after administration. The leukocytes in the collected blood were counted using a Coulter Counter ZM. Fig. 7 shows the white blood cell count at each blood collection.

As a result, as compared to the control Example 3 containing only granulocyte colony stimulating factor, Example 28 containing one cyclodextrin showed a clearly higher white blood cell count almost at most time after administration. . Experiment 6

In order to examine the effect of the buffer concentration on the absorption of G-CSF by the recruited mucosa, the formulation of the present invention of Example 32 was prepared using a phosphate buffer at a concentration of 10 mM.

The preparations of the present invention prepared in Examples 32 and 3 using male rabbits (Japanese white breed, weighing 2.9 to 4.1 kg) fasted with water only overnight before administration were used as G-CSF. OOii gZkg, and 50 l Zkg as a preparation were administered differently. Blood was collected from the ear vein at regular intervals after administration. The leukocytes in the collected blood were counted using a Coulter Power Counter ZM. Fig. 8 shows the white blood cell count at each blood collection. As a result, as compared with the case of administering the preparation of Example 3 containing only granulocytes prepared using T-167 mM 齚 -acid buffer, the Example prepared using 10 mM acetate buffer was used. In the case of the 32 preparation, the increase in white blood cell count was reduced almost at the time after administration.

Experimental example 7

A book prepared in Examples 32, 33 and 34 using male rabbits (Japanese white breed, weighing 2.9 to 4.1 kg) that had been fasted with water only overnight before administration. The preparation of the present invention was intranasally administered with 100 μg / kg as G-CSF and g as the preparation. Blood was collected from the ear vein at regular intervals after administration. Leukocytes in the collected blood were counted using a Coulter Counter ZM. Fig. 9 shows the white blood cell count at each blood collection.

As a result, as compared to the control Example 32 containing only granulocyte colony stimulating factor, the Example 33 containing dimethyl-cyclodextrin and the Example containing α-cyclodextrin were compared. 34 showed a clearly high leukocyte count most of the time after administration.

Experiment 8

The preparations of the present invention prepared in Examples 7, 13, 17, 17, 20, 23, 29, 30, 31 using male SD rats were treated with G-CSF. Then, 50 ii g / kg and g as a preparation were intranasally administered through the nostrils into the nasal cavity and administered via microcrops. At this time, the rat had undergone surgery in advance according to the method of Hirai et al. (After anesthesia with intraperitoneal administration of pentobarbital and phenobarbital, the neck was incised and polyethylene was inserted into the trachea. Insert a tube to ensure breathing, and insert another polyethylene tube with a plug at the end of the esophagus to block the posterior nares and prevent the solution from flowing out of the nasal passages). After administration of the preparation, blood was collected from the jugular vein at regular intervals and serum was obtained by centrifugation. G-CSF concentration in serum was measured by ELISA. Fig. 10, Fig. 11 and Fig. 12 show the time course of serum G-CSF concentration.

As a result, as compared with the control Example 7 containing no absorption enhancer, Examples 13, 20, 23, 29 (FIG. 10), Examples 17, 21 (FIG. 11) (Figures) and Examples 30 and 31 (Fig. 12) showed high serum G-CSF concentrations, indicating that absorption was increased.

[The invention's effect]

Based on the above, it was confirmed that G-CSF alone was absorbed from the nasal mucosa and exhibited efficacy, and that the addition of an absorption enhancer was more effective when absorbed from the violent mucosa and exerted efficacy. The absorption of G-CSF through the nasal mucosa was dependent on the concentration of the buffer, but the effect of the absorption enhancer was observed in all cases.

These can lead to a decrease in the dose, facilitate accurate control of the dose, and enhance the practicality of a nasal formulation.

Claims

The scope of the claims

1. Nasal preparation containing granulocyte colony-stimulating factor as an active ingredient.

2. The intranasal preparation according to claim 1, further comprising an absorption enhancer.

3. The preparation for nasal administration according to claim 2, wherein the absorption enhancer is dimethyl-β-cyclodextrin.

4. The drug delivery preparation according to claim 2, wherein the absorption enhancer is α-cyclodextrin.

5. The drug delivery preparation according to claim 2, wherein the absorption enhancer is -cyclodextrin.

6. The transdermal formulation according to claim 2, wherein the absorption enhancer is tartaric acid.

7. The method of claim 2, wherein the absorption enhancer is glycine.

8. The preparation for nasal administration according to claim 2, wherein the absorption enhancer is ascorbic acid.

9. The preparation for nasal administration according to claim 2, wherein the absorption enhancer is aprotinin.

10. Claims wherein the absorption enhancer is nafamostat mesilate 3. The nasal administration preparation according to item 2.

1 1. The preparation for nasal administration according to claim 2, wherein the absorption enhancer is sodium cholate.