JP2000143533A - Nanosphere - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce a nanosphere having high overall absorption of calcitonins, imparting durability and useful for the transpulmonary administration by coating a core part of a lactic acid-glycolic acid copolymer with a mucosa-adhesive polymer. SOLUTION: The objective nanosphere is produced by coating the core part of (A) a lactic acid-glycolic acid copolymer decomposable in vivo and containing calcitonins (e.g. elcatonin) known as a physiologically active peptide with (B) chitosan known as a tackifier polymer and preferably further with (C) a polyvinyl alcohol. Preferably, the copolymer of the component A contains lactic acid and glycol at a ratio of about 0.01:1 to 1:0.01 and has a molecular weight of about 2,000-200,000, the coating amounts of the components B and C are 0.005-5% (W/W) and 0.1-10% (W/W) based on the whole nanosphere, respectively, and the average particle diameter of the nanosphere is <=2,000 nm.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a nanosphere intended for use in pulmonary administration, and a lactic acid / glycolic acid copolymer which is a biodegradable polymer containing calcitonins which are physiologically active peptides. The present invention relates to a nanosphere in which the core portion of is coated with a chitosan polymer that is adherent to mucous membranes, and contains calcitonin which has a medically useful effect by improving the absorption of calcitonin and maintaining the absorption. The present invention relates to a technique for providing nanospheres for pulmonary administration.

[0002]

2. Description of the Related Art Advances in pharmaceutical research and genetic engineering have shown the usefulness of bioactive peptides such as calcitonins and insulin as pharmaceuticals, and the frequency of use in medical settings has increased. Are coming. However, considering oral administration, which is the most common dosage form, peptides are generally highly water-soluble and have a high molecular weight, so that absorption from the gastrointestinal tract is extremely low. Due to its susceptibility to degradation, oral bioavailability is very low.

[0003] For this reason, peptidic drugs such as calcitonins and insulin are currently used as injections in most clinical cases. However, injections are painful at the time of administration, and these peptide-based drugs generally require frequent administration due to their short blood half-life, and often require hospitalization or hospital visits. Lowering the patient's QOL (quality of life). For this reason, the route of administration of peptide pharmaceuticals from injections may be changed by transdermal preparations such as oral administration preparations, nasal administration preparations, suppositories, vaginal preparations, pulmonary administration preparations, eye drops, and iontophoresis. There have been many studies aimed at dosage formulations and the like.

[0004] Of these, transpulmonary administration preparations have recently attracted attention. In the alveoli, which is the site for drug absorption from the lung, one epithelial cell is in contact with the capillaries, and the thickness is 0.5 to 1
μm and a very large surface area. For this reason, the lung has great potential as an absorption site. A pulmonary absorption aerosol using a propellant as a calcitonin [Japanese Patent Laid-Open No. 60-161924] and a powdery composition for transpulmonary administration containing a water-soluble base containing calcitonin as an active ingredient. Product (Japanese Patent Application Laid-Open No. 6-100464), a preparation for transpulmonary administration containing a peptide drug and hyaluronic acid (Japanese Patent Application Laid-Open No. 9-309843), and various transpulmonary administrations using special inhalation equipment Formulations have been reported.

[0005] However, when inhaling air, the powder containing the drug is sent to the terminal alveoli, and when exhaling the air, the powder stays in the alveoli without returning with the exhaled air to release the drug for a long time. Sufficient technology has not yet been completed. On the other hand, in recent years, many researches on a control release formulation based on a biodegradable polymer have been made in a research area related to pharmaceutical formulation technology. Among them, microcapsules and microspheres have already been accumulated in considerable technology, and their use in injections and oral preparations has been attempted. These include leuprorelin acetate, which is a derivative of luteinizing hormone-releasing hormone (LH-RH), as a drug, and lactic acid.
The average particle size using a glycolic acid copolymer is 20 to 30.
Micrometer [Chem. Pharm. Bu
ll. , Vol. 36, 1095-1103, (198
8)] has already been used as a pharmaceutical.

Recently, research on nanocapsules and nanospheres has been advanced. Normal microcapsules and microspheres have a particle size of about 5-
In contrast to 500 micrometers, nanocapsules and nanospheres usually have a particle size of about 1000 nanometers or less, and the lower limit is about 50 nanometers, but the lower limit is not particularly limited.

[0007] There have already been numerous disclosures of methods for producing various microcapsules and microspheres (Japanese Patent Laid-Open No. 1-216918).
Regarding a method for producing nanocapsules, several techniques have already been disclosed (Japanese Patent Application Laid-Open No. 5-58882). In recent years, the use of chitin, a natural polymer, and chitosan, a deesterified product thereof, has been studied in a wide range of fields.In the field of medicine, effects on wound healing, Studies have been made on the use as a base.

[0008]

SUMMARY OF THE INVENTION The present invention relates to a nanosphere containing calcitonin for transpulmonary administration, wherein the total absorption rate of calcitonin (absorbed amount with respect to the dose) is obtained.
The purpose of the present invention is to provide a useful nanosphere for transpulmonary administration, which has a high degree of durability and has long-lasting properties.

[0009]

Means for Solving the Problems The present inventors have so far described
Research has been conducted on nanospheres using biodegradable polymers such as lactic acid / glycolic acid copolymer and polylactic acid as to their production method and usefulness as pharmaceuticals. On the other hand, various studies have been made on the use of chitosan, which is a natural polymer and has some interesting properties, in the field of pharmaceuticals.

As a result of these studies, the present inventors have already obtained
In the field of preparations for oral administration of bioactive peptides, nanospheres containing lactic acid / glycolic acid copolymer, which contains bioactive peptide elcatonin and biodegradable polymer, are used as the core, and this is attached to the gastrointestinal mucosa. It has been found that coating with chitosan, one of the conductive polymers, improves absorption from the gastrointestinal tract during oral administration, and that a sustained nanosphere for oral administration can be obtained. It has been reported that a suitable nanosphere for oral administration can be obtained by coating with polyvinyl alcohol together with the molecule. [Summary of the 14th Symposium on Formulation and Particle Design P.S. 121
126 (1997), Japanese Patent Application No. 9-283473, filed on October 16, 1997].

The present inventors have conducted further studies in the field of pulmonary administration, which is a field completely different from oral administration. As a result, the inventors found that nanospheres containing elcatonin, a kind of calcitonin coated with chitosan, were obtained in lungs. Was found to improve the overall rate of absorption of elcatonin (absorbed amount per dose) and show sustainability, and completed the present invention.

That is, the present invention relates to a nanosphere for pulmonary administration, wherein a core portion of a lactic acid / glycolic acid copolymer which is a biodegradable polymer containing a bioactive peptide calcitonin is applied to a mucoadhesive. Nanospheres coated with chitosan as a polymer, and chitosan and polyvinyl as mucoadhesive polymers with a core of lactic acid / glycolic acid copolymer as a biodegradable polymer containing calcitonins, which are physiologically active peptides The present invention provides nanospheres coated with alcohol.

The biodegradable polymer used in the present invention is a polymer having a property of decomposing and disappearing in a living body, a polymer having no physiological activity, and a lactic acid / glycolic acid copolymer. As the lactic acid / glycolic acid copolymer, about 0.01: 1 to 1: 0.
01, preferably 1: 5 to 10: 1, more preferably 1: 1 to 6: 1, with a molecular weight of about 2000 to 20.
0000, preferably 5000 to 150,000, and more preferably 10,000 to 100,000. These are produced, for example, by heating lactic acid and glycolic acid under weak reduced pressure using an ion-exchange resin as a catalyst and subjecting them to condensation polymerization. There are also suitable commercial products. In the present invention, these may be used alone,
Further, a mixture of several types may be used.

The mucosa-adhering polymer referred to in the present invention is a polymer having a property of adhering to mucous membranes of mammals including humans, and this adhesiveness can be easily determined by experiments using rat intestinal mucosa, for example. [Proceedings of the 14th Symposium on Formulation and Particle Design P. 121
126 (1997), Japanese Patent Application No. 9-283473, filed on October 16, 1997]. A polymer having such properties includes chitosan.

[0015] Chitosan is a natural polymer having excellent biocompatibility and biodegradability, and is also suitable from that aspect. Here, the chitosan does not mean chitin completely de-esterified, but chitin partially or entirely de-esterified. Chitosan used in the present invention includes those having a molecular weight of about 5,000 to 200,000, preferably 20,000 to 100,000, and
0000-70000 is more preferred. Further, the degree of deesterification is about 50 to 100%.
0-100% is preferable, and 80% -100% is more preferable.

[0016] Chitosan can be usually prepared by heating and hydrolyzing purified chitin as a raw material in a strong alkaline solution of 30 to 60%. There are also already suitable commercial products. The polyvinyl alcohol used in the present invention includes those having a degree of polymerization of about 100 to 5,000, preferably 100 to 3,000, and more preferably 200 to 2,000. The saponification degree is about 70 to 100, preferably 75 to 95, and more preferably 75 to 9
0. Such polyvinyl alcohol can be produced by partial or complete hydrolysis of polyvinyl acetate, but there are also suitable commercial products.

Further, the calcitonins used in the present invention include eel calcitonin, salmon calcitonin,
Natural calcitonin such as human calcitonin, porcine calcitonin and chicken calcitonin, and semi-synthetic calcitonin such as ASU1-7 eel calcitonin (elcatonin) and ASU1-7 chicken calcitonin are preferred, and elcatonin is preferred.

The size of the nanospheres used in the present invention is, as an average particle diameter, 2,000 nanometers or less, preferably 1,000 nanometers or less, more preferably 800 nanometers or less, and the lower limit of 50 nanometers. Meters. The coating amount of chitosan is 0.005 to 5% (W /
W). The coating amount of polyvinyl alcohol is 0.1 to 10% (W / W) of the total amount of nanospheres.
It is.

The nanospheres of the present invention are prepared by preparing a core part of a nanosphere of a lactic acid / glycolic acid copolymer containing calcitonins by a known method, and may be coated with chitosan to prepare the core part nanosphere. Sometimes it may be prepared by a method such that it is simultaneously coated with chitosan. In the nanosphere production process of the present invention, in the step of coating with chitosan, in order to suppress aggregation of the nanospheres during lyophilization, and to obtain nanospheres having excellent redispersibility after lyophilization, polyvinyl alcohol is optionally used. A polymer such as alcohol may be used in combination, and it is preferable to simultaneously use chitosan and polyvinyl alcohol for simultaneous coating.

As an example of the basic method for preparing the nanospheres of the present invention, the outlines of the “solvent diffusion method in oil” and the “solvent diffusion method in water” are shown below. You are not limited to just two approaches. In the “solvent diffusion in oil” method, first, a lipid such as fatty acid glyceride, a first organic solvent such as n-hexane, and a first emulsifier are mixed to prepare a dissolved external phase <A>. On the other hand, an internal phase <B> in which a lactic acid / glycolic acid copolymer, calcitonins, and a second emulsifier are dissolved in a second organic solvent is prepared. Internal phase <B> with stirring during external phase <A>
Slowly using a peristaltic pump or the like. Next, stirring is continued for several hours under heating and reduced pressure to generate nanospheres. About the obtained suspension of nanospheres, after adding a first organic solvent as necessary,
Centrifuge and remove the supernatant. If necessary, the first organic solvent is added to remove lipids on the particle surface, resuspended, centrifuged, and the operation of removing the supernatant is repeated.

The obtained pellets are dried and dispersed in an aqueous solution (aqueous solution, various buffer solutions) in which chitosan and, if necessary, polyvinyl alcohol are dissolved together. Then, after centrifugation and removal of the supernatant, the pellet is resuspended in water. By freeze-drying the obtained suspension, calcitonins are encapsulated, and chitosan is used.
Alternatively, nanospheres coated with chitosan and polyvinyl alcohol can be obtained.

The lipid used in the external phase <A> is a simple lipid having a property of hardly dissolving the lactic acid / glycolic acid copolymer and calcitonin, and examples thereof include soybean oil, sesame oil, castor oil, corn oil, and cottonseed oil. Such as vegetable oils and fatty acid glycerides, and two or more of these may be used as a mixture. Instead of these lipids, liquid paraffin or silicone oil may be used.

The first organic solvent is a solvent in which the lipid used is easily dissolved and the lactic acid / glycolic acid copolymer and calcitonin are hardly dissolved. For example, n-hexane,
and n-heptane. The outer phase <A> may use the above-mentioned lipid or the first organic solvent alone, but it is preferable to use a mixture of the lipid and the first organic solvent,
More preferably, a mixture of medium-chain fatty acid triglyceride and n-hexane is used.

The second organic solvent used in the internal phase <B> is easy to dissolve the lactic acid / glycolic acid copolymer and calcitonin, has a property of being soluble in the first organic solvent, and is lower than the first organic solvent. It is a boiling organic solvent. As a boiling point of the solvent, about 120 ℃ or less, preferably 100 ℃ or less,
More preferably, it is about 30 to 80 ° C. For example,
Acetone, methanol, ethanol, propanol, isopropanol, acetylacetone, tetrahydrofuran, dioxane, etc., and a mixed solvent of two or more of these may be used.

As the first and second emulsifiers, an appropriate one can be selected from those soluble in the respective solvents. The first emulsifier may not be required depending on the conditions. Examples of the emulsifier include anionic surfactants such as sodium stearate and sodium lauryl sulfate, and nonionic surfactants such as sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyglycerin fatty acid ester, and polyoxyethylene castor oil derivative. Agents, lecithin and the like. These emulsifiers may be used as a mixture of two or more kinds. Preferably, a nonionic surfactant is used for each.

The mixing ratio of the outer phase <A> and the inner phase <B> is from 01 to 100 parts by weight of the outer phase <A> per 1 part by weight of the inner phase <B>. Also, the inner phase of the lactic acid / glycolic acid copolymer <
The concentration in B> varies depending on the type and molecular weight of the polymer and the type of the second organic solvent, but is 0.1 to 20% (w /
w). The ratio of calcitonin to the lactic acid / glycolic acid copolymer varies depending on the type of calcitonin and the intended absorption amount and duration.
２０20% (w / w).

The concentration of chitosan in the aqueous solution varies depending on the molecular weight of chitosan.
% (W / w). The concentration of the polyvinyl alcohol in the aqueous solution is 0.3 to 3% (w / w). The concentration of the emulsifier when used is 0.05 to 10% (w
/ W). The stirring is usually performed under mild conditions such as a magnetic stirrer, but if necessary, a high-speed homogenizer may be used.

The prepared nanospheres can be isolated by freeze-drying as described above, ultracentrifugation, dialysis, or the like. In addition, the internal phase shown above <B
> Instead of the internal phase <B>
May be used to prepare nanospheres. That is, a lactic acid / glycolic acid copolymer and an emulsifier are dissolved in a volatile organic solvent such as chloroform, dichloromethane, dichloroethane, and trichloroethane, and calcitonins are dissolved in an aqueous solvent such as an aqueous solution or various buffer solutions. And a W / O emulsion may be prepared using a homogenizer or the like, and this may be used as the internal phase <B>.

In the "water solvent diffusion method", an aqueous solution (aqueous solution, various buffer solutions) in which chitosan and, if necessary, polyvinyl alcohol are dissolved together is prepared as an external phase <C>. Meanwhile, an internal phase <D> in which a lactic acid / glycolic acid copolymer and calcitonin are dissolved in an organic solvent is prepared. In addition, you may add a suitable emulsifier to the internal phase <D> as needed. While stirring the outer phase <C>, the inner phase <D
Is slowly dropped using a peristaltic pump or the like. If necessary, stirring is continued under heating and reduced pressure for several hours to generate nanospheres. The resulting nanosphere suspension was centrifuged, the supernatant was removed, and a washing operation was added if necessary.Then, the resulting pellet was resuspended in water and lyophilized to obtain calcitonin. Encapsulated and coated with chitosan or with chitosan and polyvinyl alcohol, nanospheres can be obtained.

The organic solvent used for the internal phase <D> is a volatile and water-miscible organic solvent that readily dissolves the lactic acid / glycolic acid copolymer and calcitonin. The solubility in water is about 80% or more, preferably 90% or more,
Those that are completely miscible are more preferred. The boiling point of the solvent is about 120 ° C. or less, preferably 100 ° C. or less, and about 30 ° C.
~ 80 ° C is more preferred. Examples include acetone, methanol, ethanol, propanol, isopropanol, acetylacetone, tetrahydrofuran, dioxane, and the like, or a mixed solvent of two or more of these.

The mixing ratio of the outer phase <C> and the inner phase <D> is 1 to 100 parts by weight of the outer phase <C> per 1 part by weight of the inner phase <D>. Also, the internal phase of the lactic acid / glycolic acid copolymer <D
The concentration in the medium varies depending on the type and molecular weight of the polymer and the type of the organic solvent, but is usually 0.01 to 50% (w /
w), preferably 0.1 to 20% (w / w). The ratio of calcitonin to the lactic acid / glycolic acid copolymer is 0.05 to 20% (w / w), depending on the type of the peptide, the target absorption amount and the duration. The concentration of chitosan in the external phase <C> varies depending on the molecular weight of chitosan, but is 0.2 to 0.6% (w
/ W). Further, the external phase of polyvinyl alcohol <C
The concentration in> is 1 to 3% (w / w).

Examples of the emulsifier which may be added to the internal phase <D> as needed include anionic surfactants such as sodium stearate and sodium lauryl sulfate, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, and the like. Examples include nonionic surfactants such as polyglycerin fatty acid esters and polyoxyethylene castor oil derivatives, and lecithin. These emulsifiers may be used as a mixture of two or more kinds. Preferably, a nonionic surfactant is used. The concentration when the emulsifier is used is preferably 0.05 to 10% (w / w).

The stirring is usually performed under mild conditions such as a magnetic stirrer, but if necessary, a high-speed homogenizer may be used. The prepared nanospheres can be isolated by freeze-drying as described above, ultracentrifugation, dialysis, or the like. The nanosphere of the present invention can be used as a transpulmonary administration type pharmaceutical containing the physiologically active peptide contained therein as a medicinal ingredient. At that time, when administered as a powder, the nanospheres of the present invention may be used alone, and may be administered using a suitable powder inhaler such as a spin spatula or a rotor spatula. May be added to prepare a powder for administration, which may be administered using a suitable powder inhaler.

Further, purified water, physiological saline, a pharmaceutically acceptable buffer, glycerin, ethylene glycol,
It may be dispersed and suspended in propylene glycol, polyethylene glycol, or an arbitrary mixed solution thereof, and administered using an inhaler such as a suitable nebulizer. In this case, a suitable pharmaceutically acceptable tonicity agent, pH
Additives such as adjusting agents and thickeners may be added. Further, using a gas for ejection, for example, Freon 11, Freon 12, etc.
Pulmonary administration may be performed by a conventional method.

The mechanism of improvement and / or sustained absorption of calcitonins from the lung in the pulmonary administration of nanospheres according to the present invention is presumed as follows. However, the present invention is not limited by the mechanism described below. When a drug is administered to the lungs by inhalation, large particles are difficult to reach the terminal alveoli and must reach a few microns or less to reach the alveoli. However, when the particle size is reduced, the probability that even if the particles enter the terminal alveoli together with inhalation, the particles will immediately exit with exhalation increases. In addition, part of what once stays in the alveoli is gradually excreted from the alveoli into the breath and the esophagus. Therefore, if the amount that goes out together with exhalation immediately after inhalation and the amount excreted thereafter can be reduced, a large part of the administered amount of the drug can be kept in the alveoli involved in absorption. Furthermore, if the particles can be positively attached to the alveoli inner surface, which is a place for drug absorption, and the drug can be continuously released from the particles, it is necessary to form a drug with good absorbability and a long-lasting preparation. Conceivable.

The nanospheres according to the present invention can be obtained by coating the nanospheres with chitosan, a mucoadhesive polymer, whereby the nanospheres adhere to the alveolar mucosa and gradually elute calcitonins therefrom continuously. It is considered to indicate an overall increase in the absorbed amount with respect to the dose and sustained absorption. Yet another factor is the presence of chitosan macromolecules near the eluted calcitonins, which inhibits the degradation of calcitonins by peptidase present in the lung mucosa, thereby increasing the amount of absorption, and Sustainability is also considered a possibility.

[0037]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described more specifically with reference to examples and reference examples, but the present invention is not limited to these. Further, the effects of the preparation of the present invention will be specifically described with reference to experimental examples. In the following examples, the lactic acid / glycolic acid copolymer is abbreviated as PLGA. .

[0038]

Example 1 (Preparation of PLGA (75:25) Nanospheres Encapsulated with Elcatonin Coated with Chitosan) Hexaglyceryl polyricinoleate (Hexaglyn)
Tri (caprylic / capric acid) glyceryl (Triester F) containing 2% of PR-15; Nikko Chemicals
810; Nikko Chemicals Co., Ltd.) (60 ml) was prepared, and 40 ml of n-hexane was mixed and dissolved to prepare an external phase.
Next, 100 mg of sorbitan monooleate (Span80: manufactured by Kishida Chemical Co., Ltd.) and PLGA (average molecular weight 2000
0, a lactic acid: glycolic acid polymerization ratio = 75: 25, 100 mg of Wako Pure Chemical Industries, Ltd.) and 2 mg of Elcatonin (manufactured by Asahi Kasei Kogyo Co., Ltd.) in a mixed solvent of 3 ml of acetone and 2 ml of methanol were stirred under an external phase solution. The solution was dropped at a rate of 2 ml / min using a peristaltic pump.

Then, stirring was continued at 35 ° C. under reduced pressure for 3 hours, 20 ml of n-hexane was added to the obtained suspension, and centrifugation (20,000 rpm, 4 ° C., 10 minutes) was performed. After the centrifugation operation, the supernatant was removed, n-hexane was added to resuspend, and the centrifugation operation was performed again. After the centrifugation operation, the supernatant was removed, and the precipitate was dried. This was washed with a 20% aqueous solution of 1% polyvinyl alcohol (PVA-403, manufactured by Kuraray).
l and 1% chitosan (average molecular weight 50,000, manufactured by Itakuno)
1 m solution (solvent: 0.05 M acetate buffer, pH 4.4)
were dispersed in the mixed solution. After the dispersion, a centrifugal operation was performed, and after removing the supernatant, the precipitate was resuspended in purified water. The resulting suspension is lyophilized to provide chitosan-coated elcatonin-encapsulated PLG for transpulmonary administration.
A (75:25) nanosphere powder 87.2 mg (elcatonin content 1.26 mg) was obtained.

[0040]

[Reference Example 1] (PLGA containing Elcatonin (75: 2
5) Preparation of nanospheres) The same procedure as in Example 1 was carried out except that chitosan was omitted, and
(75:25) 87.2 mg of nanosphere powder (elcatonin content 1.24 mg) was obtained.

[0041]

Reference Example 2 (Preparation of pyrene-encapsulated PLGA (75:25) microspheres (average particle diameter 12.5 μm)) 1 g of polyvinyl alcohol (PVA-403, manufactured by Kuraray) was used as a polymer and dissolved in 50 ml of distilled water. The resulting solution was used as an external phase, and the external phase was stirred at 400 rpm to obtain a PL.
A solution prepared by dissolving 200 mg of GA (average molecular weight: 20,000, lactic acid: glycolic acid polymerization ratio = 75: 25, manufactured by Wako Pure Chemical Industries, Ltd.) and 2 mg of pyrene (manufactured by Nacalai Tesque, Inc.) in 5 ml of acetone was used to obtain 2 ml / min
At a speed of. The obtained suspension was 26.8 × g, 1
After centrifugation for 0 min, a supernatant and a precipitate were obtained. The obtained precipitate was suspended in about 10 ml of purified water, and freeze-dried to obtain 69.7 mg of a pyrene-encapsulated PLGA (75:25) microsphere (average particle diameter: 12.5 μm) powder. The particle diameter was measured by resuspending pyrene-encapsulated PLGA (75:25) microsphere powder in purified water and using a laser diffraction light scattering method (wet method, LDSA-2400A, manufactured by Tohnichi Computer Co., Ltd.).

[0042]

REFERENCE EXAMPLE 3 (Preparation of pyrene-encapsulated PLGA (75:25) microspheres (average particle size 7.07 μm)) In Reference Example 2, the mixture was centrifuged at 26.8 × g for 10 minutes. 3 × g, centrifuged for 10 min,
A supernatant and a precipitate were obtained. The obtained precipitate was suspended in about 10 ml of purified water, and lyophilized to obtain pyrene-encapsulated PLGA.
(75:25) microspheres (average particle size 7.07
μm) 36.7 mg of a powder were obtained. The particle diameter was determined by re-suspending pyrene-encapsulated PLGA (75:25) microsphere powder in purified water and using a laser diffraction light scattering method (wet method, LD method).
(SA-2400A, manufactured by Tohnichi Computer Co., Ltd.).

[0043]

Reference Example 4 (Preparation of pyrene-encapsulated PLGA (75:25) nanospheres (average particle diameter: 660 nm)) In Reference Example 3, the mixture was centrifuged at 107.3 × g for 10 minutes, and the obtained supernatant was further subjected to 42931.2 ×. g, centrifuged for 10 min,
A supernatant and a precipitate were obtained. The obtained precipitate was suspended in about 10 ml of purified water, and freeze-dried to obtain pyrene-encapsulated P.
LGA (75:25) nanospheres (average particle size 660
nm) 81.0 mg of a powder were obtained. The particle diameter was determined by re-suspending pyrene-encapsulated PLGA (75:25) nanosphere powder in purified water and using a laser diffraction light scattering method (wet method, LDSA
-2400A, manufactured by Tonichi Computer Co., Ltd.).

[0044]

Reference Example 5 (Preparation of chitosan-coated pyrene-encapsulated PLGA (75:25) nanospheres) 1 g of polyvinyl alcohol (PVA-403, manufactured by Kuraray) and 0.2 g of chitosan (average molecular weight: 50,000, manufactured by Itakushi) A solution dissolved in 50 ml of 0.05 M acetate buffer (pH 4.4) was used as an external phase, and the external phase was stirred at 400 rpm.
PLGA (average molecular weight 20,000, lactic acid: glycolic acid polymerization ratio = 75: 25, manufactured by Wako Pure Chemical Industries, Ltd.) 200 mg,
2 mg of pyrene (manufactured by Nacalai Tesque) in 3 ml of acetone
A solution dissolved in a mixed solvent of ethanol and 2 ml of ethanol was dropped at a rate of 2 ml / min using a peristaltic pump. Then, stirring was continued at 35 ° C. under reduced pressure for 3 hours, and after the stirring, centrifugation operation (20,000 rpm, 4 ° C., 10 minutes) was performed. After the centrifugation operation, the supernatant was removed, purified water was added to resuspend, and the centrifugation operation was performed again. After the centrifugation operation, the supernatant was removed, and the precipitate was resuspended in purified water. The resulting suspension was lyophilized and chitosan-coated pyrene-encapsulated PLGA (75:25) nanosphere powder 164.6
mg was obtained.

[0045]

Reference Example 6 (Preparation of pyrene-encapsulated PLGA (75:25) nanospheres) Polyvinyl alcohol (PVA-40)
3, a solution prepared by dissolving 1 g of purified water in 50 ml of purified water was used as the external phase, and the external phase was stirred at 400 rpm to obtain PLG.
A (average molecular weight: 20,000, lactic acid: glycolic acid polymerization ratio = 75: 25, manufactured by Wako Pure Chemical Industries, Ltd.) 200 mg, pyrene (manufactured by Nacalai Tesque, Inc.) 2 mg in a mixed solvent of acetone 3 ml and ethanol 2 ml was used as a perimeter. The solution was dropped at a rate of 2 ml / min using a star pump. Then, stirring was continued at 35 ° C. under reduced pressure for 3 hours, and after the stirring, a centrifugal operation (20,000 rpm, 4 ° C., 10 minutes) was performed. After centrifugation, remove the supernatant, add purified water and resuspend,
The centrifugation operation was performed again. After centrifugation, the supernatant was removed and the precipitate was resuspended in purified water. The resulting suspension was freeze-dried to obtain 186.0 mg of pyrene-encapsulated PLGA (75:25) nanosphere powder.

[0046]

Test Example 1 (Measurement of Particle Size of Nanosphere) Example 1
The nanosphere powder prepared in Reference Example 1 was resuspended in purified water, and the particle diameter was measured by a dynamic light scattering method (LPA3100, manufactured by Otsuka Electronics Co., Ltd.). Further, the nanospheres prepared in Example 1 were observed with a scanning electron microscope.

The weight average particle diameter measured by the dynamic light scattering method was 658.4 nm in Example 1, and 522.
It was 9 nm. As shown above, the nanospheres prepared in Example 1 and Reference Example 1 had a weight average particle size of 500 nm to 700 nm. When observed with an electron microscope, the nanospheres of Example 1 were spherical and had a particle diameter of 1000 nm or less.

[0048]

Test Example 2 (Measurement of zeta potential of nanospheres) After suspending the nanospheres prepared in Example 1 and Reference Example 1 in distilled water, a solvent (0.1 M hydrochloric acid and 0.1 M potassium hydroxide aqueous solution) was suspended. Are mixed and adjusted at each pH) and the zeta potential on the surface of the nanosphere particles is measured with a zeta potentiometer (Zeta master, Malvern Incorporated).
instruments (manufactured by Agilent Technologies, Inc.).

As shown in FIG. 1, as compared with the nanosphere of Reference Example 1, the nanosphere of Example 1 showed a positive zeta potential in the acidic region. This indicates that the nanosphere surface of Example 1 was coated with chitosan having a positive charge in the acidic region.

[0050]

[Test Example 3] (Evaluation of the reach of nanospheres in the lung) The reach of the nanospheres in the lung was measured using a bath-type ultrasonic nebulizer (N
(E-U07, manufactured by OMRON) was connected to a cascade impactor (Andersen Nonverble Sampler, MODEL AN200, Tokyo Dylek) of an artificial lung model, and aspirated by a pump for evaluation.

The pyrene-encapsulated PLGA microspheres and the pyrene-encapsulated PLGA nanospheres prepared in Reference Examples 2, 3 and 4 were dispersed in 10 ml of purified water.
Spraying was performed for 20 minutes with a bath-type ultrasonic nebulizer (NE-U07, manufactured by OMRON) at a suction flow rate of 28.3 L / min. After spraying, nebulizer, Throat, St
age0 (> 11.0 μm), Stage1 (11.0 μm)
-7.0 μm), Stage 2 (7.0-4.7 μm)
m), Stage 3 (4.7-3.3 μm), Stage
e4 (3.3-2.1 μm), Stage5 (2.1-
1.1 μm), Stage 6 (1.1-0.65 μm)
And the amounts of pyrene-encapsulated PLGA microspheres and pyrene-encapsulated PLGA nanospheres collected on Stage 7 (0.65-0.43 μm) were measured with a fluorescence spectrometer (F-30).
10, manufactured by Hitachi, Ltd .: excitation wavelength 337 nm, fluorescence wavelength 395 nm).

Pyrene-filled PLG charged in a nebulizer
A Nebulizer, Throat and Sta for Amount of Microspheres and Pyrene-Encapsulated PLGA Nanospheres
The ratio (collection rate) of the amount collected in ge0 to ge7 was determined and is shown in Table 2. In addition, the ratio (spray efficiency) of the amount discharged from the nebulizer (total amount collected in the Throat and Stages 0 to 7) to the amount of the charged pyrene-encapsulated PLGA microspheres and pyrene-encapsulated PLGA nanospheres (spray efficiency), and reached the trachea, bronchi, and lungs The ratio (arrival efficiency) of the total amount collected to Stages 2 to 6 assumed to be deposited was calculated and shown in Table 1, and the in-lung reach was evaluated.

[0053]

[Table 1]

As shown in Table 1, Reference Examples 2 and 3
PGA of Reference Example 4 compared to PLGA microspheres of
LGA nanospheres dramatically increased spray and delivery efficiencies and significantly improved intrapulmonary reach. That is, nanospheres having an average particle diameter of 1 μm or less have an average particle diameter of 7 μm.
It was presumed that the microspheres having a diameter of not less than μm efficiently reached the lung during transpulmonary administration.

[0055]

[Test Example 4] (Evaluation of retentivity of pyrene-encapsulated PLGA nanospheres in lungs) Using a Hartley male guinea pig (6 weeks old, 3 animals each), 30 mg of the pyrene-encapsulated PLGA nanospheres prepared in Reference Examples 5 and 6 were used. A solution dissolved in 10 ml of physiological saline was sprayed for 20 minutes using a bath-type ultrasonic nebulizer, and the solution was inhaled from the trachea of a guinea pig. The administration device consists of a bath-type ultrasonic nebulizer, a chamber, and a respirator (MODEL683, H
ARVARD, 2.5 ml / st. × 80st. / Mi
n) and a Y-cannula connected to the trachea of a guinea pig anesthetized with pentobarbital. After administration, the trachea was sutured, and 1 hour and 4 hours later, the guinea pig was bled to death and the lung was removed. PLG encapsulating pyrene from the excised lung
A nanospheres were extracted and used for fluorescence spectroscopy (F-301).
0, manufactured by Hitachi, Ltd .: excitation wavelength 337 nm, fluorescence wavelength 3
95 nm), the amount of PLGA nanospheres remaining in the lung was measured.

The results are as shown in FIG. Reference Example 6
As compared with the administration group, the administration group in Reference Example 5 showed a high value immediately after administration, and the disappearance thereafter was also slow. That is, it was speculated that the nanospheres coated with chitosan adhered to the lung mucosa and stayed in the lung.

[0057]

[Test Example 5] (Evaluation of transpulmonary absorption of PLGA nanospheres encapsulating elcatonin) PLGA nanospheres encapsulating elcatonin prepared in Examples 1 and 2 using Hartley male guinea pigs (6 weeks old, 4 animals each) 2
A solution in which 0 mg was dissolved in 10 ml of physiological saline, and a solution in which elcatonin was dissolved in physiological saline as a control were 1
As in Test Example 4, a dose of 00 IU / kg was inhaled from the trachea of a guinea pig. After administration, blood was collected over time from the jugular vein of guinea pigs, and the calcium concentration in plasma was measured using Calcium C Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). The calcium reduction rate was calculated with the plasma calcium concentration before administration set as 100.

The results are as shown in FIG. As compared with the elcatonin solution administration group and the reference example 1 administration group, the calcium concentration of the administration group of Example 1 was reduced to 81% 8 hours after administration, indicating a strong plasma calcium lowering effect. In addition, the plasma calcium-lowering effect significantly continued 24 hours after administration. That is, it was speculated that the nanospheres coated with chitosan adhered to the lung mucosa and continuously released the drug. These results indicate that the chitosan-coated elcatonin-containing nanosphere preparation of the present invention has improved and long-lasting absorption of elcatonin from lung mucosa by pulmonary administration.

[0059]

According to the present invention, it is possible to provide a calcitonin-containing nanosphere having a high overall utilization rate (absorbed amount per dose) of calcitonin and a sustained absorption. A formulation for pulmonary administration can be provided. As a result, calcitonin, which could only be administered by injection or the like, can now be administered by pulmonary administration, leading to improvement in QOL of patients.

[Brief description of the drawings]

FIG. 1 shows the pH profile of the zeta potential of nanospheres.

FIG. 2 shows the retention of nanospheres in the lung.

FIG. 3 shows the profile of plasma calcium reduction after pulmonary administration of elcatonin-loaded nanospheres.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4C076 AA65 BB27 CC30 EE24H EE24M EE30H EE30M EE30N FF32 FF34 FF68 4C084 AA03 BA44 DB31 MA13 MA38 MA56 NA10

Claims (2)

[Claims] 1. A nanosphere intended to be used for pulmonary administration, wherein a core portion of a biodegradable polymer containing a physiologically active peptide, calcitonin, of a lactic acid / glycolic acid copolymer is treated with a mucous membrane. Nanospheres coated with chitosan, an adhesive polymer. 2. A nanosphere intended for use in pulmonary administration, wherein a core portion of a biodegradable polymer containing a bioactive peptide, calcitonin, of a lactic acid / glycolic acid copolymer is used as a mucosa. Nanospheres coated with the adhesive polymers chitosan and polyvinyl alcohol.