JP2000086501A - Liposome formulation of human calcitonin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a liposome formulation having excellent preserving stability and high enclosing percentage of human calcitonin by dispersing liposome containing human calcitonin in an aqueous solution having a specific pH. SOLUTION: This formulation is obtained by dispersing liposome preferably containing 5-35 mol% of lipid (e.g. phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, phosphatidic acid, cardiolipin, dicetylphosphoric acid or the like) containing human calcitonin and having a negative charge and 40-60 mol% of cholesterol in an aqueous solution having pH 5-10. In the formulation, an amount of the lipid constituting liposome is preferably used as 10-100 wt. times of human calcitonin. The objective formulation is usable as an injection formulation for subcutaneous administration or intramuscular administration, parenteral formulation for intrarectal administration, rhinenchysis administration, etc., or for peroral administration, etc. A human calcitonin concentration in the formulation is preferably 0.01-10 mg /mL.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a liposome preparation which improves the storage stability of human calcitonin and has a high human calcitonin encapsulation rate.

[0002]

2. Description of the Related Art Calcitonin is a hormone having a function of suppressing bone resorption by osteoclasts, and is used as a therapeutic agent for osteoporosis (Shiraki Masataka, Hormone and Clinical, 38
Vol., Pp. 547-554, 1990). This hormone is a polypeptide consisting of 32 amino acid residues, and is derived from various species such as salmon, eel, pig, and human. Pods differ in physicochemical properties (European Journal of Biochemistry: Eur. J. Bioche
m., 221: 1117-1125, 1994).

[0003] Among them, human-derived calcitonin is expected to have small side effects when administered to humans, but has the property of extremely low stability in aqueous solution as compared with others (Journal of Biotechnology). Logical Chemistry: J. Biol. Chem. 268, 6415-6422, 1993
Year). In particular, fibrous aggregates are rapidly formed in a solution having a pH of 5 or more, particularly in a neutral or alkaline solution.
The instability of human calcitonin itself can be solved by solidifying it by a freeze-drying method or the like. Such an unstable drug in an aqueous solution is generally used as a ready-to-use formulation in which a lyophilized powder is dissolved in water before administration. However, this method requires a dissolution operation every time of administration, and is complicated in handling. If the storage stability of a drug that is unstable in an aqueous solution can be improved in an aqueous solution, dissolving operation is not required by preparing a solution, and convenience is improved. In addition, even in the case of a dissolving type before use, the storage period during repeated administration can be extended, so that there is an advantage that the frequency of dissolving operation can be reduced.

As described above, in order to stably store an aqueous solution of human calcitonin for a long period of time, the solution must be acidified due to the above-mentioned characteristics. However, there have been problems such as the fact that there are sites in the human body where it is difficult to administer an acidic aqueous solution, and it is not possible to use additives having poor solubility or stability in acidic conditions.

The inventors of the present invention have conducted various studies to overcome the above-mentioned problems. As a result, it has been found that the storage stability of human calcitonin under neutral or alkaline conditions can be improved by encapsulating human calcitonin in liposomes. I found it.

[0006] Here, "liposome" is a closed vesicle in which the aqueous phase is enclosed by a lipid bilayer composed mainly of phospholipid, and "encapsulation in liposome" means that a certain drug is contained inside. Refers to the production of liposomes incorporated into the aqueous phase or lipid membrane.

[0007] The most important factor in the production of liposomes is the drug encapsulation rate. The “encapsulation rate” refers to the ratio of the drug encapsulated in the liposome among the drugs charged during the production of the liposome. If the encapsulation rate is high, it becomes possible to produce drug-encapsulated liposomes with a smaller amount of drug,
Manufacturing costs can be greatly reduced. At the same time,
Since the amount of drug per liposome is also increased, there are also pharmaceutical advantages such as a reduced administration volume and a high-concentration formulation.

However, generally, the encapsulation rate of peptide drugs in liposomes is low.

[0009]

SUMMARY OF THE INVENTION The present inventors have repeatedly studied to produce human calcitonin-encapsulated liposomes having a high encapsulation rate, and as a result, the encapsulation rate has been drastically improved by devising the lipid composition and liposome composition. And found that the present invention was completed.

[0010]

The present invention provides a liposome preparation comprising (1) a liposome containing human calcitonin dispersed in an aqueous solution having a pH of 5 to 10; (2) a lipid having a negative charge of 5 to 35 mol%; The liposome preparation according to (1), which comprises 40 to 60 mol% of cholesterol as a constituent, and (3) the liposome preparation according to (2), which is obtained by performing a freeze-thaw treatment in a liposome production step. (4) The lipid having a negative charge is one or two or more lipids selected from the group consisting of phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, phosphatidic acid, cardiolipin, and dicetylphosphate. 2) or (3)
Or a liposome formulation as described above.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The liposome of the present invention comprises a lipid having a negative charge of 5 to 35 mol% and
Contains 60 mol% cholesterol. Preferable examples of the lipid having a negative charge include phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, phosphatidic acid, cardiolipin, and dicetyl phosphate. These can be used alone or in combination.

As other liposome constituents, for example, naturally occurring or synthetically obtained phospholipids such as egg yolk lecithin, soybean lecithin, phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, sphingomyelin and the like are used as required. can do.

[0013] Liposomes containing human calcitonin are produced using the above lipids. Examples of the production method include a mechanical dispersion method, a thin film method, a reverse phase evaporation method, an ethanol injection method, an ether injection method, and a dehydration method. Known methods such as a rehydration method can be mentioned (DD Lasik, liposomes:
From physics to application / DDLa
sic, Liposomes: from physics to application, Elsev
ier Science Publishers, 1993).

The lipid constituting the liposome for encapsulating human calcitonin is 10 to 10 times the weight of human calcitonin.
It is preferable to use 100 times the amount.

In the liposome dispersion produced by the above method, a part of human calcitonin that is not encapsulated remains outside the liposome. The liposome dispersion is freeze-thawed to further increase the encapsulation rate. Can be improved. Freeze-thaw treatment is a method in which a liposome dispersion is rapidly frozen in liquid nitrogen or the like and then thawed at around 40 ° C (DD Lasic, Liposomes: from physics)
to application, Elsevier Science Publishers, 1993
reference).

The calcitonin-encapsulated liposome and human calcitonin remaining in the dispersion without being encapsulated in the liposome can be separated by a method such as gel filtration or ultracentrifugation.

Since human calcitonin encapsulated in the liposome is stably stored even in a neutral or alkaline solution in which human calcitonin alone aggregates, it can be dispersed in an aqueous solution adjusted to be neutral or alkaline. it can.

In the liposome preparation, in addition to components for adjusting neutrality or alkalinity, pharmaceutically acceptable additives such as isotonic agents such as sodium chloride and glucose, benzethonium chloride, if necessary. And a preservative such as benzalkonium chloride.
The human calcitonin-encapsulated liposome preparation of the present invention can be used for injection preparations for subcutaneous administration, intramuscular administration, etc., parenteral preparations for rectal administration, nasal administration, and oral preparations.

The concentration of human calcitonin in the preparation varies depending on the administration route and the number of administrations, but it is 0.01 to 10 mg / mL.
It is preferable that

[0020]

The present invention will be described below in more detail with reference to experimental examples and comparative examples. Example 1 Dimyristoyl phosphatidylcholine in molar ratio 4/1/5 (DM
80 mg of a lipid mixture consisting of PC, manufactured by NOF Corporation / dimyristoyl phosphatidylglycerol (DMPG, manufactured by NOF Corporation) / cholesterol (Chol, manufactured by Tokyo Chemical Industry Co., Ltd.)
Was dissolved in 10 mL of chloroform in an eggplant flask, and the solvent was distilled off with a rotary evaporator to prepare a thin film. 1 mL of 0.001 N hydrochloric acid in which 3 mg / mL human calcitonin (manufactured by Peptide Research Institute Co., Ltd.) was dissolved was added to hydrate the lipid. Thereafter, liposomes were formed by probe-type sonicator treatment, and then frozen with liquid nitrogen and thawed in a 40 ° C water bath were repeated three times to prepare human calcitonin-encapsulated liposomes.

The calcitonin-encapsulated liposome dispersion obtained by the above method is subjected to ultracentrifugation (140,000 g (20 min),
The liposomes were sedimented and separated from unencapsulated human calcitonin. When human calcitonin in the liposome fraction and the supernatant was quantified, the encapsulation rate of human calcitonin in the liposome was 99%.

The method of quantifying human calcitonin in the liposome is as follows.

9 mg / mL N per 100 μL of dispersion containing liposomes
Add 900 μL of 0.01 N hydrochloric acid containing aCl, and add 2 mL of chloroform /
Methanol (1/1) was added and shaken to dissolve the lipid. Thereafter, the mixture was centrifuged to separate the aqueous phase and the organic solvent phase, and human calcitonin in the aqueous phase was quantified by HPLC under the following conditions. Column: ODP-50 6D (Showa Denko KK) Size: inner diameter 6.0 mm, length 150 mm Mobile phase: distilled water / acetonitrile / trifluoroacetic acid (70/3
0 / 0.05) Detection wavelength: 225 nm Flow rate: 1.5 mL / min Retention time: about 6.1 min Example 2 Human calcitonin-encapsulated liposome composed of DMPC / DMPG / Chol at a molar ratio of 2/1/3 was used in the same manner as in Example 1. As a result, the encapsulation rate was 99%. Example 3 A human calcitonin-encapsulated liposome comprising DMPC / DMPG / Chol at a molar ratio of 1/2/3 was prepared by the same method as in Example 1, and the encapsulation rate was 100%. Example 4 A human calcitonin-encapsulated liposome composed of DMPC / disetyl phosphate (DP, manufactured by Sigma) / Chol at a molar ratio of 4/1/5 was prepared in the same manner as in Example 1, and the encapsulation rate was 96%.
Met. Example 5 DMPC / phosphatidylinositol in a molar ratio of 4/1/5 (PI,
A human calcitonin-encapsulated liposome composed of (Sigma) / Chol was prepared by the same method as in Example 1, and the encapsulation rate was 100%. Example 6 Human calcitonin-encapsulated liposomes composed of DMPC / DMPG / Chol at a molar ratio of 4/1/5 were prepared in the same manner as in Example 1, subjected to ultracentrifugation to precipitate the liposomes, and the supernatant was used as a preservative.
The liposomes were redispersed by replacing with 50 mM citrate buffer (pH 6) or 50 mM borate buffer (pH 9) containing 0.1 mg / mL benzethonium chloride.

The human calcitonin-encapsulated liposome dispersion was stored in a constant temperature room at 25 ° C. over time, and after a certain period of time, the remaining human calcitonin in the liposome dispersion was quantified.
About 80% remained at H6 and about 100% remained at pH9.

The method for quantifying human calcitonin in the liposome dispersion is as follows.

The liposome dispersion was appropriately diluted with physiological saline, and the liposomes were destroyed with 1% polyethylene glycol monooctyl phenyl ether (manufactured by Tokyo Chemical Industry Co., Ltd.). 0.2μm pore size filter (Millipore Dimex 13,
After filtering through human Millipore to remove human calcitonin aggregates, human calcitonin was quantified under the HPLC conditions shown in Example 1. Comparative Example 1 A human calcitonin-encapsulated liposome comprising only DMPC was prepared in the same manner as in Example 1, but no human calcitonin was encapsulated in the liposome. Comparative Example 2 A human calcitonin-encapsulated liposome composed of DMPC / Chol at a molar ratio of 1/1 was prepared in the same manner as in Example 1, and the encapsulation rate was 16%, which was lower than that in Example 1. Comparative Example 3 A human calcitonin-encapsulated liposome composed of 4/1 DMPC / DMPG at a molar ratio was prepared in the same manner as in Example 1.
The encapsulation rate was 77%, which was lower than that in Example 1. Comparative Example 4 Neutral human calcitonin not encapsulated in liposomes,
When the storage stability under alkaline conditions was examined by the method described in Example 6, human calcitonin was reduced to about 10% after 6 days at pH 6, and to about 10% after 1 day at pH 9. It was of lower stability than human calcitonin encapsulated in liposomes. Comparative Example 5 A dispersion of human calcitonin not encapsulated in liposomes and empty liposomes not encapsulating human calcitonin having the same lipid composition as in Example 4 was neutralized and alkalined by the method shown in Example 6. Examination of storage stability under conditions showed that human calcitonin outside the liposome was pH6.
The pH decreased to about 30% after 6 days, and at pH 9 to about 10% after 1 day. It was of lower stability than human calcitonin encapsulated in liposomes.

[0027]

As described above, the liposome preparation of human calcitonin of the present invention has a high encapsulation rate and has an effect of improving the storage stability of human calcitonin under neutral or alkaline conditions.

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Claims (4)

[Claims] Claims 1. A liposome containing human calcitonin is expressed as p
A liposome preparation dispersed in an aqueous solution of H5 or more and 10 or less. 2. A lipid having a negative charge of 5 to 35 mol% and 4
2. The liposome preparation according to claim 1, comprising 0 to 60 mol% of cholesterol. 3. The liposome preparation according to claim 2, which is obtained by performing a freeze-thaw treatment in a liposome production process. 4. The lipid having a negative charge is one or more lipids selected from the group consisting of phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, phosphatidic acid, cardiolipin, and dicetylphosphate. 4. The liposome preparation according to claim 2 or 3.