JPH013115A - liposome formulation - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed Description of the Invention] <Industrial Application Field> The present invention is characterized in that sugars are contained and the ion concentration is about 20mM.
The present invention relates to a liposome preparation prepared by freeze-drying or spray-drying an aqueous dispersion of liposomes as described below. The formulation of the present invention has excellent liposome stability.

<Prior art> The structure of liposomes is often not thermodynamically stable, and even when prepared as a suspension, liposome particles usually agglomerate and fuse with each other, as well as form precipitates and insoluble matter. It is known that the precipitation occurs relatively quickly.

That is, if the apparent particle size of liposomes changes due to aggregation or fusion, it is naturally expected that changes in internal dynamics, changes in the expression of drug efficacy, etc. will occur accordingly. Therefore, although many researchers are conducting clinical application research on liposomes, one major factor that has prevented the commercialization of large-scale liposome preparations is the instability of liposomes as preparations. It's there.

Research on freeze-drying an aqueous dispersion of liposomes and condensing them with water before use includes (1) a report by Crommelin et al. [International Journal of Pharmaceutics, 22, 299-310 (1984)];
], (2) Gordon et al.'s report [Drug Deherobment and Industrial Pharmacy-1
8゜455-473 [19B2) ], (3) Cr
ow et al. [Archips of Biochemistry and Biophysics, 2<2. z4o-24
7 (s985)]. Cromm
In the case of elin, etc., toxorhiscine-containing liposomes are aliquoted into a mixture of 0.01M l-lys-hydrochloric acid buffered tethered glucose solution (mixing ratio 11) and lyophilized to give 4 to 6
The drug retention rate and particle size changes after storage at ℃ for 2 months and recondensation are being investigated.

Gordon et al. prepared antimony-containing liposomes and
Freeze-dry using water as an aqueous solvent, from 4℃ to 55℃
As a result of studying the drug retention rate and particle size change after condensation after storage for 5 days, the drug retention rate was 20% compared to the initial value after 5 days.
Although the particle size decreased to ~80*, the particle size remained unchanged. Crow et al. also prepared enzyme-containing liposomes,
*001λl TES(2-([2-h
hydroxy-1, 1-b is (hydroxy
me thyl) e thyl ]-aminole
thanesulfonjc acfd), 0.2
As a result of testing using an mM EDTA-buffered (pH 7) trehalose solution, if trehalose, a trisaccharide, is present in both the inner and outer aqueous layers of the liposomes, freeze-drying-
The company claims that there is no drug leakage during condensation operations, and does not report on its stability over time. Further, as a method of use in which an aqueous dispersion of liposomes is spray-dried and condensed with water at the time of use, the method disclosed in Japanese Patent Application No. 168821/1980 can be mentioned. This method does not disclose whether high concentrations of electrolytes such as sodium chloride, sugars such as glucose or mannitol are added, or stability over time.

As described above, conventional liposome preparations prepared by freeze-drying or spray-drying an aqueous dispersion of liposomes are extremely unstable, and no one has been obtained that is stable for more than half a year.

<Configuration of the Invention> The present invention contains sugars and has an ion concentration of about 2011
The present invention relates to a liposome preparation prepared by freeze-drying or spray-drying an aqueous dispersion of liposomes with a concentration below IM.

The ions related to the present invention are preferably -valent cations and anions, and specific examples thereof include alkali metal ions such as potassium ion, sodium ion, and lithium ion, triethylammonium ion, diisopropanol ammonium ion, and jetanol. Examples include ammonium ions such as ammonium ions, triethanolammonium ions, and tetramethylammonium ions, chlorine ions, bromide ions, iodide ions, and nitronium ions, preferably sodium ions, potassium ions, and chloride ions. The lower the concentration of these ions in the aqueous dispersion, the better from the standpoint of liposome stability, and it is usually necessary to keep it at about 20 mM or less; higher concentrations are unstable from the standpoint of long-term stability of the liposome. There are many cases.

Examples of sugars related to the present invention include monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, and xylose; trisaccharides such as lactose, sucrose, cellobiose, trehalose, and maltose; trisaccharides such as raffinose and melezitose; Oligosaccharides such as dextrin, polysaccharides such as dextrin, sugar alcohols such as xylitol, sorbitol, mannitol, maltitol, etc., preferably lactose, trehalose, maltose,
You can give mannitol etc.

The amount of these saccharides used is not particularly limited, and is usually 0.001 to 0.3 parts by weight per 1 part by weight of the liposome aqueous dispersion.
Preferably, 0.01 to 0.15 parts by weight are used. Next, a method for producing the liposome preparation of the present invention will be explained.

That is, examples of various known methods can be found in Journal of Molecular Biology, Vol. 113, p. 238 (1
965), the membrane component of the liposome is dissolved in a suitable organic solvent such as chloroform, the solvent is distilled off under reduced pressure to form a ribit film, this is suspended in water, and then the saccharide is removed. It is possible to produce an aqueous dispersion of liposomes by adding Furthermore, an aqueous dispersion of liposomes can also be produced by suspending the pit film produced as described above in an aqueous solution containing sugars.

The membrane component substances mentioned above include phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, lysophosphatidylcholine, sphingomyelin, egg yolk lecithin, soybean lecithin, glycolipids, dialkyl type synthetic surfactants, etc. The main ingredient is one or a mixture of two or more of the following. Then, to this, sterols such as cholesterol and cholestanol are added as membrane stabilizers, dicetyl phosphate, phosphatidic acid, ganglioside, stearylamine, etc. are added as charged substances, and α as an antioxidant.
- A membrane component substance may be formed by adding tropherol or the like. Although the ratio of the components of such liposome membrane component substances is not limited in any way, it is preferable that
It is suitable to add about 0 to 1 part by weight of sterols and about Q to 0.2 parts by weight of charged substances based on the weight part.

The amount of the above membrane component substance used is particularly limited, usually water 1
The amount is 0.0001 to 0.05 part by weight, preferably 0.001 to 0.008 part by weight.

It is desirable to adjust the pH of the aqueous liposome dispersion prepared in this way to around neutrality, preferably about 60 to about 80, using a -valent base or acid.

The above-mentioned -valent bases include hydroxides such as sodium hydroxide, potassium hydroxide, and lithium hydroxide, and amines such as triethylamine, trimethylamine, diisopropanolamine, jetanolamine, triethanolamine, and tetramethylamine. , preferably potassium hydroxide, sodium hydroxide, etc. In addition, as -valent acids, hydrochloric acid, nitric acid, hydrobromic acid, hydroiodic acid, etc.
Preferred examples include hydrochloric acid and nitric acid.

It is desirable that the amounts of these acids and bases used be as small as possible. The ion concentration in the final aqueous dispersion of liposomes is preferably as low as possible, specifically about 20mM as described above.
The aqueous dispersion of liposomes thus prepared can be freeze-dried or spray-dried in a conventional manner to produce the desired liposome preparation. Desirable operations for the above-mentioned freeze-drying include rapid freezing at a freezing temperature of -5 to -80°C, preferably -30°C; One example of this is to sublimate water under reduced pressure conditions of ITorr or less, and a desirable operation in spray drying is to keep the population temperature at 110 -
The temperature can be 200°C, preferably 120 to 150°C.

There are no particular limitations on the drugs retained in the liposome preparation of the present invention, including anticancer drugs such as cytosine arabinosin I, methotrexate, and adriamycin; antibiotics such as amphotericin B, gentamicin, and piperazine;
Liver disease drugs such as glutathione, enzymes such as superoxide dismutase and glucoamylase, interferon,
Physiologically active substances such as interleukin, tumor necrosis factor (TNF), epidermal growth factor (EGF), and erythroboetin, hormones such as prostaglandin and steroids,
Immunostimulants such as muramilcibebutide, muramiltribebutide, lymphokine, lentinan, DNA, RNA
In addition to nucleic acids such as , vitamins typified by vitamins A, C, and H, general drugs such as sodium salicylate can be mentioned. These drugs can be retained in liposomes as follows. That is, it is sufficient to dissolve the drug in water and then suspend Lipid film therein.For membrane-compatible drugs such as chlorophyll, gramicinha S, and vitamin VIII, it is necessary to dissolve the drug in water and then suspend the Lipid film therein. A liposome aqueous dispersion with good drug retention efficiency can be obtained by mixing the liposomes with the drug in an organic solvent, then distilling off the solvent, and suspending the resulting Rivit film in water. By freeze-drying or spray-drying this as described above, a liposome preparation retaining the drug can be obtained.

<Effects of the Invention> The liposome preparation of the present invention has long-term liposome stability that has not been seen in conventional preparations. Therefore, the present invention is excellent as a stable liposome preparation.

<Examples> Next, the present invention will be explained by Examples and Test Examples, but these are not intended to limit the present invention.

Example 1 Fully hydrogenated purified egg yolk lecithin (TV-1, phospholipid 95*
(30.0 g), 150 g of cholesterol, 2.2 g of cicetyl phosphate, and 1.6 g of vitamins were weighed out and dissolved in chloroform Loomρ in an Ajihomo mixer, and nitrogen gas was added while stirring with a paddle mixer. The feed solvent was removed. At this time, the solvent was almost completely recovered using a trap cooled with liquid nitrogen. The temperature inside the Ashihomo mixer at this time is 50 to 60°C.
Met.

To the obtained dry paste-like homogeneous mixture, preliminarily add 6
5 * mannitol water m heated to 0℃? (12000mI
L was added and the mixture was sufficiently swollen. While maintaining the temperature between 50 and 60°C, the mixture was thoroughly stirred using a homomixer and a paddle mixer, and the mixture was brought to room temperature, resulting in a pale yellow aqueous dispersion of liposomes containing vitamin 1 (pi(3,6)).
I got it.

An aqueous dispersion of liposomes with sufficient nitrogen bubbling was added to
Dispense each mfl into brown vials, use a freeze dryer,
Freeze-drying was carried out for 25 hours under reduced pressure at a freezing temperature of -40°C and a degree of vacuum of 0.07 to 0.02 Torr, and the via was plugged to obtain a liposome preparation in the form of a yellowish white mass.

Example 2 L-α~dipalmitoylphosphatidylcholine 2.91
1g, cholesterol 15g, dicetyl phosphate 0
．． Cut off 22g of tusks and add chloroporm 50 in the carrier.
After dissolving it in mρ, warm it to 50-60℃ and add 5% mannitol water with 40mg of Nagashin dissolved therein.
The mixture was warmed to 60°C and added to the mixture to allow sufficient swelling. While keeping the temperature at 50 to 60°C, the solution was thoroughly stirred using a homo mixer (manufactured by Tokushu Kika Kogyo Co., Ltd.), and the liquid was further filtered through a 02 μm polycarbonate membrane filter to reduce the particle size to 02 μm or less. A milky white liposome aqueous dispersion tpu373) was obtained. An aqueous dispersion of liposomes with sufficient nitrogen bubbling was poured into 2 mρ brown vials, and heated to a freezing temperature of -28 using a freeze dryer.
Freeze-drying was carried out for 21 hours at ~-36°C under reduced pressure of 0.06 to 0.04 Torr, and the vial was capped to obtain a white bulk liposome preparation.

Example 3 L-α-dipalmitoylphosphatidylcholine 0.92
g, cholesterol 0.2 g, dicetyl phosphate [
Weigh out 1,0935g and add chloroform 5g in a beaker.
After melting to 0 mfl, the solvent was removed with nitrogen gas while heating to 50 to 60°C.

300 ml of 5* mannitol aqueous solution was preheated to 60°C and added to the obtained dry pasty homogeneous mixture.
It was sufficiently swollen. While maintaining the temperature at 50 to 60°C, the temperature was sufficiently stirred using a TK homomixer, and then 0. I
The pH was adjusted to 6.5 using N sodium hydroxide. This mixture was further subjected to extrusion filtration using a 0.4 μm membrane filter (manufactured by Fuji Photo Filter Co., Ltd.) to obtain a milky white aqueous dispersion of liposomes.

This is frozen at a temperature of -40℃ and a vacuum level of 006-0.038T.
Freeze-drying was performed for 41.5 hours to obtain a liposome preparation in the form of a white lump.

Example 4 A white blocky liposome preparation was obtained in the same manner as in Example 1, except that fully hydrogenated purified soybean lecithin (phospholipid 95% or more) was used instead of L-α-dipalmitoyl phosphatidylcholine. The pH of the prepared liposome aqueous dispersion was 652.

Example 5 Completely hydrogenated purified egg yolk lecithin (phospholipid 9596 or higher) instead of L-α-dipalmitoylphosphatidylcholine
A white lump-like liposome preparation was obtained by performing the same procedure as in Example 1 except for using . The pl-1 of the prepared liposome aqueous dispersion was 7.00, and the freezing conditions were -38 to -40°C, degree of vacuum 0, (15 to 0.C12
8 Torr, drying time 235 hours).

Example 6 Completely hydrogenated purified soybean lecithin (phospholipid 9 Li or more) 7.
37 g of cholesterol, 1.58 g of cholesterol, and 074 g of dicetyl phosphate were treated in the same manner as in Example 1 to remove chloroform.

To the dry paste-like homogeneous mixture thus obtained, 300 mu of 5*Mannitol water-soluble solution containing 409 g of dextran T was mixed and heated to 60°C to crystallize it,
Thereafter, the same procedure as in Example 1 was carried out to obtain a milky white liposome aqueous dispersion (pl-16,73). 50 mft of this liquid
After adding 350 mu of 5% mannitol aqueous solution and stirring thoroughly, use a spray dryer to lower the population temperature to 14.
0℃, outlet temperature 75℃, dry air flow rate 0.4m37m1
n, atomization pressure 1.0 kg/cm2, sample liquid feeding amount 6
Spray drying was carried out under conditions of 0 g/min, and the resulting white powder was divided into 11 Bmg portions into high-fill portions and capped to obtain liposome preparations.

Test Examples The liposome preparations obtained in Examples 1 to 6 were stored in a constant temperature room at 5° C., and changes in appearance and particle size were tested. Changes in appearance were determined by visual observation, and changes in particle size were determined by quasi-elastic light scattering.

These results are shown in Table 1.

Table 1 Appearance and particle size changes of liposome preparations Particle size; Equally weighted average particle diameter There is not much difference between the particles when they are rehydrated, and even when they are rehydrated after storage over time, there is no change in appearance, and the particle size is also not much different from that before freezing or spray drying. was not seen. Therefore, it was confirmed that the liposome formulation of the present invention has excellent stability.

Claims (1)

[Claims] A liposome preparation prepared by freeze-drying or spray-drying an aqueous dispersion of liposomes containing sugars and having an ion concentration of about 20 mM or less.