JPH0764721B2 - Liposomal formulation - Google Patents

Description

TECHNICAL FIELD OF THE INVENTION The present invention relates to liposomal formulations. More specifically, the present invention relates to a liposome preparation in which a drug is stably retained inside a liposome and the therapeutic effect of the drug is sustained or a targeting effect on a specific organ is more easily exerted.

2. Description of the Related Art When using a liposome as a carrier for a drug, it is necessary for the drug to be stably retained inside the liposome as much as possible in order to enhance the therapeutic effect. Although many studies have been conducted on the stability of liposomes,
The relation between the stability of liposomes and the osmotic pressure at the time of preparation is not considered so much. Conventionally, for example, cell engineering 2 , 1,
136 (1983), it is argued that “it is essential in principle that the solution at the time of preparation and the solution to be used thereafter be isotonic”. No consideration is given to the case where the solution at the time of liposome preparation is lower than the osmotic pressure of the dispersion liquid used thereafter.

Problems to be Solved by the Invention Although various attempts have been made to stabilize liposome preparations, a simple and highly practical method has not yet been developed.

Means for Solving the Problems In view of the above situation, the present inventors have completed various aspects of the present invention as a result of various studies on a method for allowing a drug to stably exist in a liposome, particularly in relation to osmotic pressure. . That is, the present invention is a liposome preparation prepared by dispersing a REV liposome encapsulating a drug-containing liquid having an osmotic pressure of about 50 to 240 mOsm in an aqueous dispersion that is substantially isotonic with a biological fluid.

The liposome preparation of the present invention can be produced by the following method.

REV liposomes in which the drug is encapsulated can be prepared by removing the solvent from the W / O emulsion containing the drug.The emulsion is obtained by emulsifying the drug encapsulating liquid and the organic solvent in which the phospholipid is dissolved by a conventional method. To be

The phospholipid that can be used in the present invention may be one that is usually used for preparing liposomes. For example, phosphatidylcholine, phosphatidylethanolamine,
Egg yolks such as phosphatidic acid, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, those derived from soybeans and other animal and plant tissues, or mixtures of these yolk phospholipids, soybean phospholipids and hydrogenated products thereof, Alternatively, those obtained by the synthesis of dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine and the like can be mentioned. These may be used alone or in combination of two or more. In addition, for the purpose of stabilizing liposomes and the like, for example, cholesterol, α-tocopherol,
You may add dicetyl phosphate, stearyl amine, etc.

Next, the enclosed liquid uses water as a medium, and in adjusting the osmotic pressure,
An aqueous solution in which an appropriate water-soluble substance is dissolved is used, but in some cases, the drug may simply be dissolved in water. As the water-soluble substance, various buffer solutions (eg, phosphate buffer solution, citrate buffer solution), various salts (eg, sodium chloride, monosodium phosphate, disodium phosphate), sugars (eg, glucose, galactose) , Maltose, maltotriose, mannose, sorbitol), amino acids (eg, glycine) and the like can be used alone or in combination. A preservative (eg, paraben) or the like may be added to the enclosed liquid, if necessary. The amount of the water-soluble substance dissolved is appropriately adjusted so that the osmotic pressure of the enclosed liquid is generally about 50 to 240 mOsm. The specific osmotic pressure is appropriately selected depending on the intended use of the liposome preparation. For example, when administered as an injection, eye drop, nasal drop, etc., the osmotic pressure is about 50 to 50% as described above.
It is preferably adjusted to 240 mOsm, while it is not particularly limited when used as a diagnostic agent.

As the drug used in the present invention, either a hydrophilic drug or a lipophilic drug or a mixture of both can be used. Examples of hydrophilic drugs include various antiphlogistic and analgesic agents, lymphokines, anticancer agents, immunostimulants, bioactive peptide agents, antibiotics, antiprotozoal agents, enzyme agents, antiallergic agents, and the like.

Examples of anti-inflammatory analgesics include manganese-superoxide dismutase (SOD) or its derivative, superoxide dismutase-PEG (SOD-PEG.PEG).
A molecular weight of 5000) (JP-A-58-16685), lipomodulin and the like can be mentioned. Examples of lymphokines include natural or recombinant interferon (α, β, γ) and natural or recombinant interleukin II. Examples of anticancer agents include adriamycin, actinomycin, 1-β-arabinofuranosylcytosine, bleomycin, cisplatin and the like. Examples of the immunostimulant include muramyl dipeptide and muramyl tripeptide. Examples of the physiologically active peptide include tyroid releasing hormone (TRH), rheuprolide, insulin, DN-1417 (JP-A-59-21216) and the like. Examples of the antibiotics include β-lactam antibiotics such as sulbenicillin, cefotiam, cefmenoxime, and sulfazecin, and aminoglycoside antibiotics such as gentamicin, streptomycin, and kanamycin. Examples of antiprotozoal drugs include antimony anthelmintic drugs such as meglumine antimonate. Examples of enzyme agents include alkaline phosphatase, and examples of antiallergic agents include amoxanox.

Examples of lipophilic drugs include anti-cancer agents such as ansamitocin, TMD-66 (Gann 74 (2) 192-195 (1983)),
Examples include immunostimulatory and phospholipid derivatives (JP-A-59-163389) such as MTP-PE (JP-A-59-163389).

Generally, when preparing an emulsion, it is preferable that a hydrophilic drug is contained in the above-mentioned mounting liquid, and a lipophilic drug is dissolved in an organic solvent.

The organic solvent is not particularly limited as long as it can dissolve phospholipids, and is, for example, one of chloroform, ethers (eg, diethyl ether, isopropyl ether), alcohols (eg, methanol, ethanol), or the like. Two or more kinds of mixed solvents may be mentioned.

In the present invention, the preparation of liposomes from the W / O type emulsion is carried out according to the Procedures of the National Academy of the United States of
America (Proc.Natl.Acad.Sci.USA) 75, 4194 (1978 )
Alternatively, it can be carried out according to the method described in JP-A-55-118415. The organic solvent used in the emulsion preparation is generally about 2 to 10 times the amount of the enclosed liquid.
The amount of phospholipid used is about 10 to 100 μmol per 1 ml of the enclosed liquid, and it is generally preferable to dissolve it in advance in an organic solvent. On the other hand, the amount of the drug used is appropriately selected in consideration of the amount necessary for exhibiting the drug effect, the dose, and the like.

As an emulsification method for obtaining the W / O type emulsion, a conventional method such as a stirring method, a pressure method, or an ultrasonic irradiation method can be adopted. In the case of ultrasonic irradiation, a uniform emulsion can be obtained by ultrasonic irradiation with a 20 KHz probe type for about 1 to 20 minutes.

The solvent is removed from the W / O type emulsion thus obtained by a conventional method. For example, using a rotary evaporator,
Approximately 10-100 of the volume of the enclosed liquid containing the emulsion
The solvent may be distilled off by placing it in a double volume eggplant-type Kolben. The temperature at this time is about 10 ° C higher than the phase transition temperature of the phospholipid.
It is desirable to raise the temperature above 0 ° C or higher, and it is preferable to carry out under reduced pressure of about 100 to 400 mmHg in the initial stage of distillation, and after the formation of a gel in the contents, reduced pressure of about 60 to 700 mmHg. If distillation is continued and the solvent is removed, REV (Rever
se-phase evaporation vesicles) are obtained. The liposome has a unilamellar or oligolamellar (usually composed of a lipid bilayer having about 10 layers or less), and a drug is encapsulated therein.

The liposome obtained by the above-mentioned preparation step is dispersed in an aqueous solution having an osmotic pressure almost isotonic with the biological fluid to obtain the liposome preparation of the present invention. This dispersion liquid can be appropriately selected and prepared from each water-soluble substance used for the preparation of the mounting liquid, but may be the same dissolved substance as the mounting liquid or different. What is essential is that the osmotic pressure is adjusted to be higher than that of the enclosed liquid as described above. In the case of a liposome preparation intended for administration to a living body, the osmotic pressure of the dispersion liquid is adjusted to be substantially isotonic with the biological fluid.

The liposome solution prepared by the above method may be added as it is to the above dispersion solution containing the drug not encapsulated,
In addition, a previously prepared higher-concentration dispersion liquid and the water-soluble substance itself constituting the above-mentioned dispersion liquid were added to adjust the final osmotic pressure of the dispersion liquid containing liposomes to fall within the above range. You may.

On the other hand, when it is desired to remove the drug not encapsulated inside the liposome, this removal and the dispersion in the dispersion liquid having a specific osmotic pressure according to the present invention can be performed simultaneously.
Examples of the removal method include a dialysis method, a filtration method (eg, gel filtration method), a centrifugation method, and the like. In the case of the dialysis method, the liposome preparation of the present invention can be obtained by repeating the operation of putting the prepared liposome solution in a dialysis membrane and immersing it in the above dispersion solution to dialysis and remove the drug. In the case of gel filtration, the liposome preparation liquid may be subjected to filtration using the above-mentioned dispersion liquid as an eluent to separate into a non-encapsulated drug-containing fraction and a liposome-containing fraction. In the case of centrifugation, the prepared liposome solution may be centrifuged, and the resulting precipitated portion may be redispersed in the above dispersion liquid, and centrifugation and redispersion may be repeated several times as necessary. In addition, when removing the unencapsulated drug, the unencapsulated drug is removed under the isotonic condition with the encapsulating liquid by the means as described above, and the resulting liposomes are immediately added to the dispersion liquid having the osmotic pressure specified in the present invention. You may employ the method of adding to.

The amount ratio of the dispersion liquid and the liposome is appropriately determined in consideration of, for example, the encapsulation amount of the drug and the amount required for manifesting the drug effect.

EXAMPLES Hereinafter, the present invention will be described with reference to Experimental Examples, Experimental Examples and Examples.
Will be specifically described. The osmotic pressure measurement below
The osmometer is Osmet (Precision Systems Inc
Manufactured) was used.

Experimental Example 1 1) 0.05M 6-carboxyfluoroscein (manufactured by Eastman Kodak Company, abbreviated as 6CF) was added to 0.02M phosphate buffer (p
H7.2) was mixed with sodium chloride to obtain an aqueous solution adjusted to have an osmotic pressure of 289 mOsm. 5 ml of this solution is mixed with 5 ml of distilled water and the enclosed aqueous solution (14
5mOsm) was prepared.

This aqueous solution is treated with dipalmitoylphosphatidylcholine.
(DPPC) 210mg and distearoylphosphatidylco
Chloroform-isopropyl ether containing 90 mg of phosphorus (DSPC)
Ultrasonic homogenizer in addition to liquid (20ml / 20ml) solution
ー (Otake, 20KHz) at room temperature 50W; 30 seconds, 6 times super sound
Wave irradiation was performed to obtain a W / O type emulsion. This Emma
Rouge rotary evaporator in water bath (55 ℃)
The solvent was distilled off using to prepare REV liposomes.
Next, 6CF not encapsulated in liposomes was dialyzed (Spe
ctrapor , Molecular weight cutoff 6000 ~ 8000, Spectrum Medical
Industries) in saline (287 mOsm)
After removing by analysis, 5 μm filter (Acrodisc , G
Elman) was used to remove large particles of liposomes. Or
Disperse liposomes containing 6CF into physiological saline.
The resulting formulation was obtained.

2) Add 5 ml of 0.05M6CF-0.02M phosphate buffer (pH 7.2) to the aqueous solution with osmotic pressure adjusted to 193 mOsm by adding salt to it.
A liposome preparation was prepared in the same manner as in the above 1) except that the enclosed aqueous solution (97 mOsm) obtained by mixing ml was used.

3) A liposome preparation was prepared by the same method as in 1) above, except that 10 ml of 0.05M6CF-0.02M phosphate buffer (pH7.2) was added to the salt to adjust the osmotic pressure to 193 mOsm and used as the enclosed aqueous solution. Prepared.

4) A liposome preparation was prepared in the same manner as in 1) above, except that 10 ml of 0.05M6CF-0.02M phosphate buffer (pH7.2) was added to the salt to adjust the osmotic pressure to 289 mOsm and used as the encapsulated aqueous solution. Prepared.

Test Example 1 Each liposome preparation obtained in Experimental Example 1 was tested for 6CF retention by the following method.

Test method (1) 0.067M phosphate buffered saline (p
H7.2,284mOsm) 9.9 ml was added to dilute, and the mixture was gently stirred (solution A).

Add 0.2 ml of the above dilution to 0.067 with various osmotic pressures.
Add to 9.8 ml of M phosphate buffered saline (dispersion), dilute, and gently stir (solution B). 6CF fluorescence intensity at this time (Hitachi F30
00 Using fluorescence spectrometer: Excitation wavelength 494nm, Measurement wavelength
515 nm) was designated as FB.

Next, 5 ml of solution B was placed in a polystyrene round-bottom spitz roll and shaken for 3 minutes (Iwaki shaker VS type).
The subsequent 6CF fluorescence intensity was designated as FC.

To 0.1 ml of solution A, 0.1 ml of 0.02% Triton X-100 was added, and the mixture was heated in a 45 ° C water bath for 10 minutes to destroy the liposomes, and then 4.8 ml of 0.067 M phosphate buffer (pH 7.2) was added. , And mix (solution T). The 6CF fluorescence intensity at this time was designated as FT.

In the above test, 6CF from liposomes after shaking for 3 minutes
The amount released is expressed as (FC-FB) / FT x 100 (%).

FIG. 1 shows the amount of 6CF released from each liposome when shaken for 3 minutes in a dispersion having various osmotic pressures. As is clear from this figure, by reducing the osmotic pressure of the encapsulating liquid during liposome preparation to be less than the osmotic pressure of the dispersion, 6C
It was found that the amount of F released was reduced and the stability was improved.

FIG. 2 shows the relationship between the amount of 6CF released when liposomes were shaken for 3 minutes under isotonic biological fluid and the osmotic pressure during preparation. As a result, it was confirmed that when the osmotic pressure of the encapsulated liquid at the time of liposome preparation is lower than that of the dispersion, the retention of the encapsulated drug increases even under severe shaking.

Test method (2) Each of the liposome preparation liquids of solution B and solution T was placed in a polystyrene round-bottom Spitz roll, shielded from light by an aluminum foil, and allowed to stand at room temperature for 4 days. After that, the fluorescence intensity of 6CF is F
If B ', FT', 6CF emission amount (%) in these 4 days
Is calculated by (FB / FT-FB '/ FT') x 100 (%).

The above test results are shown in FIG. As is clear from this figure, even when the liposomes were allowed to stand at room temperature for 4 days, the liposomes at 6 C
This indicates that the amount of F released is reduced and the amount of F is kept stable.

Experimental Example 2 1) 0.05M6CF-0.02M phosphate buffered saline (pH7.2,289mO
sm) 5 ml and 5 ml of distilled water (150 mOsm) as an enclosed aqueous solution, egg yolk phosphatidylcholine (Sigma, P27)
72) 300 mg of chloroform-isopropyl ether (20 m
1/20 ml) solution was used to prepare REV liposomes by the same method as described in 1) of Experimental Example 1.

2) 0.05M6CF-0.02M phosphate buffered saline (pH7.2,289mO
sm) 5 ml and 0.067 M phosphate buffered saline (pH 7.2,284 mOsm) 5 m
A liposome preparation (control sample) was obtained in the same manner as in 1) above using 287 mOsm of a mixed aqueous solution of 1 l.

The following tests were conducted on the liposome preparations obtained in 1) and 2) above. That is, 0.1 ml of each liposome preparation liquid
Add to 9.9 ml of phosphate buffered saline (pH 7.2,284 mOsm), mix gently, and then take 2 ml of this and place it in the dialysis instrument Centrisart I (fraction molecular weight 20,000) of Sartorius 3000
Centrifuge at rpm 15 minutes (Hitachi 05PR-22 type centrifuge) to obtain a liposome-free solution, and 0.1 ml of this solution was added to 4.9 ml 0.067M phosphate buffered saline (pH7.2) in polystyrene round-bottom spitz roll. And gently mixed, and the fluorescence intensity FO was measured. Separately, 0.1 ml of solution (A) was added to 4.9 ml of 0.067 M phosphate buffered saline (pH 7.2), and 10 ml of spitz roll was inverted three times to mix the contents (solution B).

The measured fluorescence intensity at this time is FB. On the other hand, (A) liquid 0.
The Triton X-100 treatment was performed on 1 ml, and the fluorescence intensity at this time was designated as FT. The amount of 6CF released by dilution of the liquid (B) is expressed by (FB-FO) / FT x 100 (%).

The amounts of 6CF released upon dilution of the liposomal formulations 1) and 2) above were 7.7% and 24.5%, respectively.

Example 1 1) 0.067 M phosphoric acid containing 25 mg of manganese SOD-PEG (5000)
Buffered saline (pH 7.2 287 mOsm) solution 5 ml and distilled water 5 ml
Mixed aqueous solution (148mOsm), DSPC 30mg and DPPC 270mg
Chloroform / isopropyl ether (30ml / 30ml) dissolved
Mix the liquids and use an ultrasonic homogenizer for 50W-30 seconds for 6 seconds.
Ultrasonic irradiation was performed to prepare an emulsion. Then 55
The solvent was distilled off with a rotary evaporator on a water bath at ℃.
REV liposomes were obtained. High-speed centrifugation (50.000
g, 20 minutes, Sorval), then 0.067M phosphate buffered saline (pH7.2)
 , 287mOsm) was added and dispersed. Repeat this centrifugation twice
After returning, 5μm Acrodisc Large particles in the filter
The liposomes were removed and purified. In this way SOD-PEG
We obtained a REV liposome formulation in which (5000) was stably encapsulated.
It was

2) On the other hand, 0.067M containing 25 mg of manganese SOD-PEG (5000)
Liposomes were prepared in the same manner as above using a 10 ml solution of phosphate buffered saline (pH 7.2, 288 mOsm) and dispersed in the same dispersion to prepare a liposome preparation (control sample).

The following tests were carried out using each of the liposome formulations 1) and 2) obtained above.

The above liposomes on a polystyrene round-bottom spitz roll
Add 0.02% Triton X-100 0.2 ml to each 0.1 ml of the formulation,
After leaving it in a water bath at 0 ° C for 10 minutes, 4.7 ml of 2.6% sodium borate
An aqueous solution of triumnium was added and mixed (solution T). Separately each liposo
Take 0.2 ml of the drug preparation and add 9.8 ml of 2.6% sodium borate water.
Add to solution and mix gently. Divide this mixture into two
Place in a styrene round-bottom spitz roll, one at room temperature.
(B solution), the other was left in a 50 ° C water bath for 5 minutes (W
liquid). 2.6% Natri borate for T.B.W.solutions and blanks
About um solution (BL liquid) Pyrex glass
Transfer to a 10 ml centrifuge tube made of stainless steel and fluoresce with thorough stirring.
Minacetone solution (Roche, Fulham "Roche"
0.1mg of 1mg / ml) to give an excitation wavelength of 390nm and a measurement wavelength of 475nm.
The fluorescence intensity (F) was measured. Soak for 5 minutes at 50 ° C
Release amount of manganese SOD-PEG (5000)
(%) Was calculated by the following formula.

As a result, the SOD obtained by heating the liposome dispersion obtained by each of the methods 1) and 2) above in a water bath at 50 ° C for 5 minutes
-PEG (5000) release amount is 2.7% and 25.4% respectively
It was found that the liposome preparation of the present invention has excellent stability.

Example 2 1) 0.067M phosphate saline solution containing 30 mg of manganese-SOD (p
H7.2,289mOsm) 5 ml and distilled water 5 ml mixed aqueous solution (147 mOs
m) was used to prepare a liposome preparation by the same method as described in 1) of Example 1.

2) A liposome preparation (control sample) was prepared in the same manner as in 1) above, except that the liposome was prepared using 10 ml of 0.067 M phosphate buffered saline (pH 7.2,289 mOsm) containing 30 mg of manganese-SOD.

For each of the above liposome preparations 1) and 2), the amount of SOD released from the liposomes by immersing in a water bath at 50 ° C. for 5 minutes in the same manner as in Example 1 was 9.6% and 86.0%, respectively. It was

Example 3 1) A mixed solution of 5 ml of an injectable solution containing 2.5 mg of CDDP (cisplatin) (Randa Note manufactured by Nippon Kayaku) and 5 ml of distilled water was mixed with a phospholipid solution (DSPC / DPPC = 30 mg / 270 mg, chloroform / isopropyl ether =). 30 ml / 30 ml) was used to obtain a liposome preparation in the same manner as in 1) of Example 1.

2) CDDP injection 5ml (2.5mg, Nippon Kayaku Landa injection),
Using a mixed solution of 5 ml of physiological saline for injection (Otsuka Pharmaceutical Co., Ltd., 284 mOsm), a liposome preparation (control sample) was obtained in the same manner as in 1) of Example 1.

Prepare 0.5 ml of each of the above liposome preparation solutions 1) and 2)
Add 9.5 ml of saline (287 mOsm) and mix gently (A
liquid). Take 0.2 ml of solution A and use 9.8 ml of physiological saline (287 mOsm)
And gently mixed. Divide this into two polices
Put it in a round bottom spitz roll made of ethylene, and let it stand at room temperature.
(Solution B), and the other was shaken at room temperature for 3 minutes (Solution C).
Separately, take 0.1 ml of solution A and add 0.02% Triton X-100 water solution.
Add 0.1 ml of the liquid, mix, heat (45 ° C, 10 minutes), and add 4.8 ml of raw solution.
Add saline and mix to prepare the liquid that destroyed the liposomes.
(T solution). Release from B, C and T liquid liposomes
CDDP adduct form with diethyldithiocarbamate
Post-generation HPLC (column Zorbax CN , Eluent; n-hexane /
Isopropyl alcohol = 8/2 UU · 254 nm).
If the quantitative value at this time is H, then CDDP after shaking for 3 minutes
The amount released from liposomes isIt is calculated by. As a result, the liposo of 1) and 2) above
The amount of CDDP released when shaken for 3 minutes was 6.4% each.
And 82.7%. That is, the osmotic pressure of the enclosed liquid is dispersed
It can stabilize the liposome by lowering it than the liquid
all right.

Example 4 Water and sodium chloride are added to an α-interferon aqueous solution.
10 μl of 200 μg protein / ml, 143 mOsm aqueous solution
Obtained. A W / O emulsion is prepared in the same manner as in 1) of Example 1.
After removing the solvent, the REV liposome was obtained. this
Acrodisc  Large particles can be removed with a filter of 5 μm
After removing the posomes, a dialysis membrane (Spectropor Fractionated molecule
25000), dialyzed in physiological saline and not included
Of the α-interfacial solution that removes the drug from the
A liposome preparation containing a heron was obtained.

Example 5 Water and sodium chloride were added to an aqueous solution of interleukin-2.
In addition, an aqueous solution of 143 mOsm was prepared with 308 μg protein / ml.
Then, add 5 ml of the egg yolk phosphine in 100 ml capacity eggplant type Kolben.
Organic solvent solution containing 150 mg of fatidylcholine (chloroform
15 ml + 15 ml of isopropyl ether), and Example 1
A W / O emulsion is obtained by the same method as 1) above, and the solvent is distilled off.
After that, REV liposomes were obtained. The obtained liposomes are
Centrifuge (50000g, 20 minutes), then 5ml of saline
Redistributed into After repeating centrifugation and redispersion
5 μm spectrotor Filter with a filter and
Interleukin 2 stably encapsulated in a liposome preparation
Obtained.

EFFECTS OF THE INVENTION According to the present invention, the drug is stably retained inside the liposome, and the release amount is reduced even during storage and shaking. Therefore, the liposome can be practically and advantageously used as a drug carrier. INDUSTRIAL APPLICABILITY The liposome preparation of the present invention can be used in the form of injections, eye drops, nasal drops, etc. when administrating drugs having various therapeutic effects to a living body for the purpose of treatment, and stabilizes the drug, prolongs the effect and specifies the effect. It is useful for enhancing the therapeutic effect by targeting to other organs. Furthermore, for example, in the case of a liposome diagnostic agent that utilizes an antigen-antibody reaction, etc., a formulation with high storage stability can be obtained.

[Brief description of drawings]

FIG. 1 shows the effect of the osmotic pressure ratio between the encapsulated aqueous solution and the dispersion upon the preparation of 6CF-encapsulated liposomes on the amount of 6CF released. Figure 2 shows the amount of 6CF released under shaking when 6CF liposomes were prepared using various osmotic pressure encapsulated aqueous solutions and dispersed in physiological saline. The amount of 6CF released is shown below.

Claims (1)

[Claims] 1. A liposome preparation prepared by dispersing REV liposomes containing a drug-containing liquid having an osmotic pressure of about 50 to 240 mOsm in an aqueous dispersion that is substantially isotonic with biological fluid.