JP2611307B2 - Liposome preparation and method for producing the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a liposome preparation and a method for producing the same.

Conventional technology Drug Delivery System that targets liposomes containing a drug intravenously and targets the drug to a limited site
The idea of (DDS) has already been generalized [Di Gregoriadis et al .: Receptor-Mediated Targeting of Drugs, Plenum Press (G.
Gregoriadis et al., Receptor-madiated targeting of
drugs, Plenum Press, New York, p243-266 (1980)].
In such DDS, after intravenous administration of liposomes, it is primarily required to stably circulate in the body with blood for a longer time. However, liposomes are not very stable in blood due to the interaction between lipids, which are membrane components, and components such as lipoproteins in blood. In addition, liposomes administered intravenously have properties such that they are recognized as foreign substances by the reticuloendothelial system (RES) and easily disappear from the blood due to their physical shape and biochemical properties. Therefore, even if a drug-encapsulated liposome is intravenously administered, the liposome disappears from the blood faster than expected. Therefore, how to stabilize liposomes in the blood, avoid recognition by RES, and prolong the disappearance time of liposomes from the blood has been an important study topic. For example, it has been reported that the stability of liposomes in blood is increased by adding cholesterol to the membrane composition of liposomes [Di J. Knight, "Liposomes: From physical structure to therapeutic applications" (C.
G. Knight, “Liposomes; from physical structure to th
erapeutic applications ", Elsevier, North Holland p31
0-311 (1981)]. However, it can be said that the effect of the addition differs greatly depending on the membrane composition of the originally used liposome [Biochemica et Biophysica Acta 839 , 1-8 (198
Five)]. In addition, there is a report that the distribution of liposomes to RES can be suppressed by coating the liposome membrane surface with sialic acid using a glycoprotein having a sialic group [Chemical and Far Matthewal Bretane (Chem. Pharm. Bull.), 34 , 2
979-2988 (1986)]. On the contrary, it has been reported that such glycolipids having sialic acid are well distributed in the liver, which is one of the RESs [Biochemica et Biophysica Acta, 49
7 , 760-765 (1977)].

On the other hand, there is almost no case where a surfactant is used as a component of a liposome membrane. This is because surfactants are generally considered to destabilize the membrane structure of liposomes [Cell Engineering, 2 , 1136 (1983)], and are often used to break down membranes [BioChemica].・ Biochemica et Biophysica Acta,
551 , 295 (1979)]. The only possible liposome preparation method using a surfactant is to disperse a homogeneous mixture of an ionic surfactant and a lipid in the aqueous phase at a concentration lower than the critical micelle concentration of the surfactant in the aqueous phase, thereby producing a monoleaf liposome. A method is known (JP-A-59-8963).
3). The liposome obtained by this method, when administered intravenously, is rapidly eliminated from the blood and cannot necessarily be expected to have a sufficient effect for the purpose of DDS.

Problems to be Solved by the Invention As described above, although there is an idea that a drug-encapsulated liposome is used as DDS by intravenous administration,
The liposome preparation according to the conventional method has a fast elimination time from the blood after intravenous administration, and a highly practical means for effectively achieving the purpose of DDS has not yet been developed.

Means for Solving the Problems In view of the above circumstances, the present inventors have studied various methods for stably circulating a liposome preparation administered intravenously with blood for a longer period of time in the body. As a result, in preparing liposomes, a phospholipid having a saturated acyl group as an acyl group is used to form a liposome membrane in the presence of a specific surfactant, that is, an anionic surfactant having a Krafft point of 37 ° C. or higher. The inventors have found that the stability of the obtained liposome preparation in the blood of the body is increased by doing this, and have further studied to complete the present invention.

That is, the present invention provides (1) a liposome preparation comprising a drug encapsulated in a liposome comprising a phospholipid having a saturated acyl group as an acyl group and an anionic surfactant having a Krafft point of 37 ° C. or higher as a membrane component. (2) The phospholipid wherein the acyl group is a saturated acyl group in the preparation of the preparation according to the above (1), wherein the anionic surfactant is an acyl taurine salt, and (3) a liposome preparation containing a drug. A method for producing a liposome preparation, comprising forming a liposome membrane using an emulsion or dispersion of an aqueous liquid containing an anionic surfactant having a Krafft point of 37 ° C. or higher and a critical micelle concentration or higher. It is.

Examples of the phospholipid whose acyl group is a saturated acyl group (hereinafter, may be simply referred to as phospholipid) used in the production of the liposome preparation of the present invention include a glycerophospholipid or a sphingolipid whose acyl group is a saturated acyl group. Is raised. Examples of the present phospholipid include those whose two acyl groups are saturated alkyl having 8 or more carbon atoms and at least one of which is a saturated alkyl having 10 or more carbon atoms, preferably 12 to 18 carbon atoms. Further, those in which both saturated acyl groups are saturated alkyl having 12 to 18 carbon atoms are more preferably used. Such phospholipids include:
Hydrogenated lecithin or lauryl obtained by hydrogenating lecithin of animal or plant origin (eg, egg yolk lecithin, soy lecithin),
Examples include phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and sphingomyelin obtained by semisynthesis consisting of a combination of myristoyl, palmitoyl, stearoyl, and the like. More specifically, those having a phase transition temperature of about 20 to 80 ° C. can be preferably used. For example, dimyristoyl phosphatidylcholine (DMPC, 23.9 ° C.), palmitoyl myristoyl phosphatidyl choline (PMPC, 27.2 ° C.), myristoyl palmitoyl phosphatidyl choline (MPPC, 35.3 ° C.) whose measured values of phase transition temperature are shown in parentheses below.
° C), dipalmitoylphosphatidylcholine (DPPC, 41.
4 ℃), stearoyl palmitoyl phosphatidylcholine (SPPC, 44.0 ℃), palmitoyl stearoyl phosphatidylcholine (PSPC, 47.4 ℃), distearoyl phosphatidylcholine (DSPC, 54.9 ℃), dimyristoyl phosphatidylethanolamine (DMPE, 50 ℃), dipalmitoyl phosphatidylethanol Amine (DPPE, 60
° C), distearoyl phosphatidylethanolamine (DSPE, 60 ° C or higher), dimyristoyl phosphatidylserine (DMPS, 38 ° C), dipalmitoylphosphatidylserine (DPPS, 51 ° C), distearoylphosphatidylserine (DSPS, 50 ° C or higher), Dimyristoyl phosphatidyl glycerol (DMPG, 23 ° C), dipalmitoyl phosphatidyl glycerol (DPPG, 41 ° C), distearoyl phosphatidyl glycerol (DSPG, 55 ° C), dipalmitoyl sphingomyelin (DPSM, 41 ° C), distearoyl sphingomyelin (DSSM) , 57 ° C).

In the present invention, an anionic surfactant having a Krafft point of 37 ° C. or higher (hereinafter, may be simply referred to as an anionic surfactant) usually has a sulfate group or a sulfonic group. Available to The upper limit of the craft point is not particularly limited, but is usually selected from those up to 90 ° C. As the anionic surfactant, for example, those represented by the following general formula can be used.

R-Xm- (Y ₁ or Y ₂₎ n-Z [wherein, R represents 12 carbon atoms which may be substituted with a sulfate group
X represents -CONH-, -COO-, Y ₁ is —CH ₂ CH ₂ —, Or -CH ₂ CH ₂ CH _2- , and Y ₂ is -OCH ₂ CH _2- , Or -OCH ₂ CH ₂ CH ₂ - one of the, Z is -SO ₃ ^- M ^+, -S
O ₄ ^- M ⁺ (M represents an alkali metal element) indicates, m is 0 (direct bond) or 1, n is 0 (direct bond) ~
It is an integer of 2. Provided that X is -CONH- or , N of Y ₂ is 0, and X is In this case, n is 0 (direct bond). The alkyl group represented by R usually has 12 to 25 carbon atoms, and when substituted with a sulfate group, preferably has 16 to 25 carbon atoms, and the sulfate group has an alkali metal ion (sodium, Potassium, lithium).

Hereinafter, specific examples of the anionic surfactant are shown.

As the anionic surfactant having a sulfate group, for example, sodium hexadecyl sulfate (Kraft point; 43
℃), sodium octadecyl sulfate (Kraft point; 58
C) and other sodium alkyl sulfates; sodium hexadecyldisulfate (Kraft point; 39 ° C), sodium octadecyldisulfate (Kraft point; 45 ° C)
Alkyl disulfate salts such as sodium oketadecyl ether sulfate (Kraft point; 46 ° C), sodium octadecyl diether sulfate (Kraft point; 40 ° C), and sodium palmitoyl ethanolamide sulfate (Kraft point; 40 ° C) Kraft point; 42 ° C), sodium stearoylethanolamide sulfate (Kraft point; 53 ° C), sodium palmitoylpropanolamide sulfate (Kraft point; 47 ° C), sodium stearoylpropanolamide sulfate (Kraft point; 57 ° C) Fatty acid alkanolamide sulfates and the like. Examples of the anionic surfactant having a sulfonic acid include sodium dodecane sulfonate (Kraft point; 38 ° C.), sodium tetradecane sulfonate (Kraft point; 48 ° C.), and sodium pentadecane sulfonate (Kraft point; 48 ° C.) Alkanesulfonates such as sodium hexadecanesulfonate (Kraft point; 57 ° C), sodium heptadecanesulfonate (Kraft point; 62 ° C), sodium octadecanesulfonate (Kraft point; 70 ° C); sodium dodecylbenzenesulfonate ( Kraft point; 40 ° C), Sodium tetradecylbenzenesulfonate (Kraft point; 43 ° C), Sodium hexadecylbenzenesulfonate (Kraft point; 46
C)), alkylbenzene sulfonates such as sodium octadecylbenzenesulfonate (Kraft point; 56 ° C); sodium myristoyloxyethanesulfonate (Kraft point; 39 ° C), sodium palmitoyloxyethanesulfonate (Kraft point; 51 ° C); Sodium stearoyloxyethanesulfonate (Kraft point; 51
℃) and other acyloxyethane sulfonates; sodium palmitoyl taurine (Kraft point; 43 ° C.), sodium stearoyl taurine (Kraft point; 58 ° C.), sodium palmitoyl methyl taurine (Kraft point; 43 ° C.)
C)), and acyltaurine salts or acylmethyltaurine salts such as sodium stearoylmethyltaurine (Kraft point; 58 ° C). Among the above anionic surfactants, acyltaurine salts or acylmethyltaurine salts are particularly preferred in consideration of the fact that the obtained liposomes have high stability in blood and that industrial supply is easy. Can be used. Next, a method for preparing the liposome preparation of the present invention will be described.

The liposome in the present invention is prepared so that the phase transition temperature of the membrane generally indicates about 37 to 60 ° C, preferably about 40 to 55 ° C. The adjustment of the phase transition temperature can be performed by appropriately selecting the types and blending ratios of the phospholipid and the anionic surfactant to be used.

The phase transition temperature of the liposome membrane can be confirmed by calorimetry, such as DSC, or by measuring the release of the enclosed drug. Usually, in order to achieve the above-mentioned phase transition temperature of the liposome membrane, the phospholipid and the anionic surfactant are used in an amount of about 0.5 to 50 parts by weight, preferably about 0.5 to 50 parts by weight, based on 100 parts by weight of the phospholipid. Is used in a proportion of about 5 to 20 parts by weight. By adjusting the phase transition temperature of the liposome membrane to the range as described above, the object of the present invention to prolong the disappearance time of the obtained liposome preparation in blood is advantageously achieved.

In preparing the liposome preparation of the present invention, an aqueous liquid containing an anionic surfactant at a critical micelle concentration or higher is used. The critical micelle concentration here can be measured by a usual method. For example, by examining the relationship between physicochemical properties such as surface tension, osmotic pressure coefficient, and electrical conductivity, and the concentration of an anionic surfactant. [Masayuki Nakagaki and Naofumi Koga, "Pharmaceutical Physics and Chemistry", 3rd edition, p.111 (1969), Nankodo]. Therefore, using these measured values as indices, an aqueous liquid may be prepared so that the concentration of the anionic surfactant is equal to or higher than the critical micelle concentration and the amount used for the phospholipid is the above-described ratio. In preparing the present aqueous liquid, the anionic surfactant may be dissolved in the aqueous medium at a temperature higher than the Kraft point or may be dispersed at a lower temperature lower than the Kraft point. When the drug is water-soluble, it is preferable that the drug is contained in the aqueous liquid, and if necessary, other additives (eg, sugars and salts as an osmotic pressure adjusting agent, buffers as a pH adjusting agent) Agent).
The content of the drug is appropriately determined according to the purpose of treatment and the degree of efficacy.

An emulsion or dispersion is prepared using the aqueous liquid and the phospholipid obtained as described above to form liposomes, and the method itself is performed by a known method for preparing REV, MLV, SUV or other liposomes. It can be implemented according to. For example, to prepare a liposome from an emulsion, first, a phospholipid is dissolved in an organic solvent (eg, diethyl ether, isopropyl ether, chloroform or a mixed solvent of two or more of these), and then the above-mentioned anionic surfactant is dissolved. An aqueous liquid is added, and a W / O emulsion is prepared by a conventional method. This W
Preparation of liposomes from / O emulsions is based on Procedures of the National Academy of the United States of America (Proc. Natl. Ac.
ad.Sci.USA) 75 , 4194 (1978) or JP-A-55-11841.
It can be carried out according to the method described in 5. The organic solvent used in the preparation of the emulsion generally ranges from 2 to
About 10 times is used. The amount of the phospholipid is about 10 to 100 μmol per 1 ml of the encapsulating solution, and it is generally preferable to dissolve it in an organic solvent in advance.

An emulsification method for obtaining a W / O emulsion is a conventional method,
For example, a stirring method, a pressure method, and an ultrasonic irradiation method can be adopted.
In the case of ultrasonic irradiation, a uniform emulsion can be obtained by ultrasonic irradiation for about 1 to 20 minutes with a 20 KHz probe type. According to the method for producing an anionic surfactant of the present invention, emulsification is easy, and a uniform and fine emulsion can be obtained.

The solvent is removed from the W / O emulsion obtained in this manner by a conventional method. This may be done by, for example, using a rotary evaporator to distill off the solvent. The temperature at this time is 40 ° C
The above is desirable, and about 60 to 400 m at the beginning of distillation
Perform under reduced pressure of mHg, and after the contents have formed a gel,
The pressure is preferably reduced to about 00 to 700 mmHg. Further evaporation is continued to remove the solvent, and REV (Reverse-phase Evap
oration Vesicle) liposomes are obtained. The present liposome has the form of unilamellar or oligolamellar (usually composed of about 10 or less lipid bilayer membranes), and a drug is encapsulated therein.

On the other hand, the organic solvent solution of phospholipids prepared by the same method as above is evaporated under a reduced pressure to remove the organic solvent to form a phospholipid thin film, and then an aqueous liquid of an anionic surfactant containing a drug is added. By dispersing at a temperature of 40 ° C. or higher, a liposome preparation of a multilamellar vesicle (MLV) containing a drug can be obtained. Further, by shaking the MLV thus obtained with a probe-type ultrasonic oscillator, a liposome preparation which is a small unilamellar vesicle (SUV) can be obtained.

Further, the liposome production method of the present invention is based on the Stable Pull Llamella Vesicle (SPLV) method (JP-A-59-500952).
And the dehydration-rehydration vesicle method [C. Kirby et al., Biotechnology, Nov., 979 (198
4)]. In the case of a drug that is fat-soluble and has low solubility in water, the drug can be dissolved in the lipid organic solvent solution to obtain a liposome encapsulating the drug. The thus obtained drug-encapsulated liposome may be used as it is, but if necessary, can be adjusted to a preferable particle size. As the preparation method, a Nuclepore filter or gel filtration can be used. On the other hand, when using liposomes, it is preferable to separate and remove free drugs that are not encapsulated, for example, by centrifugation, gel filtration, or dialysis.

Next, the drug used in the present invention is not particularly limited as long as it is used for the purpose of DDS. For example, platinum compounds (eg, cisplatin, carboplatin, spiroplatin, etc.), adriamycin, mitomycin C, Anticancer drugs such as actinomycin, ansamitocin, bleomycin, 5-FU, methotrexate; natural or recombinant interferons (α, β,
γ) and lymphokines such as natural or recombinant interleukin 2; manganese superoxide desmutase (SOD) or its derivative superoxide desmutase PEG (PEG-500) (JP-A-5
Physiologically active peptides such as 8-16685, EPC Publication No. 0210761); beta-lactam antibiotics such as sulfazecin; gentamicin; streptomycin;
Antibiotics such as aminoglycoside antibiotics such as kanamycin; Vitamins such as cyanocobalamin and ubiquinone; Antiprotozoal drugs such as meglumine antimonate; Enzyme agents such as alkaline phosphatase; Anticoagulants such as heparin; Amoxanox Antiallergic agents such as muramyl dipeptide, muramyl tripeptide, TMD-66 (Gann 74
(2), 192 to 195 (1983)); cardiovascular drugs such as propranolol; and metabolic drugs such as glutathione. The present invention can be preferably applied to water-soluble drugs for that purpose. For example, a drug having a logarithmic value of the partition ratio between octanol / water of 10 or less can be mentioned. The amount of the drug to be used may be appropriately selected in consideration of the efficacious amount, dosage, and the like of the target drug so that the therapeutic purpose can be achieved.

The liposome preparation of the present invention is generally used in the form of a solution or an emulsion dispersed in physiological saline or the like in an appropriate amount depending on the purpose of treatment, and administered intravenously by injection or infusion.

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

Example 1 270 mg of DPPC and 30 mg of DSPC were dissolved in 70 ml of a 1: 1 mixed solution of chloroform and isopropyl ether in a 1-liter beaker. On the other hand, 30 mg of sodium stearoylmethyltaurine (SM) was added at room temperature to 10 ml of an aqueous solution of 6-carboxyfluorescein (6-CF) at pH 7 which had been prepared to have the same osmotic pressure as that of a physiological saline solution.
T) was added. At this time, the SMT was almost insoluble, but formed a micelle at a temperature higher than the Kraft point and rapidly dissolved. Next, this solution was added to the above-mentioned phospholipid organic solvent solution and emulsified by a probe-type ultrasonic oscillator (Ohtake) to prepare a w / o-type emulsion. Ultrasonic irradiation is 30 at 50 watts
It was repeated 10 times per second. The emulsion thus obtained was applied to a rotary evaporator, and the organic solvent was distilled off at 60 ° C. under reduced pressure to obtain REV. The degree of vacuum of the evaporator was initially high, and as the evaporation of the organic solvent proceeded, the degree of vacuum was reduced to prevent bumping. Then further RE
A small amount of the organic solvent remaining in V was distilled off by wiping with nitrogen gas. An appropriate amount of physiological saline was added to the obtained REV to make 10 ml, and a 1.2 micron filter (A
crodisc, Gelman) and dialysis membrane (Spectrapor, Spect)
The liposome in which 6-CF was encapsulated was obtained by dialysis for 24 hours in physiological saline using rumMedical). Furthermore, by quantifying the amount of 6-CF encapsulated in the liposome (Note 1), the encapsulation rate in this liposome was 33.2%.
%.

(Note 1) Measurement of 6-CF content in liposome and calculation of encapsulation rate 0.1 ml of liposome was added to phosphate buffered saline (PBS, pH 7.
2) After diluting 100 times, further dilute 100 times with PBS containing 0.02% Triton X-100, heat at 60 ° C for 30 minutes to break down the liposomes, and measure the fluorescence intensity of the solution (Hitachi, F300
0 Fluorescence spectrometer, excitation wavelength 494 nm, measurement wavelength 51
5 nm) to determine the total amount of 6-CF in the liposome dispersion. Separately, 0.1 ml of liposome is
Dilute 2,000-fold and centrifuge 2.5 ml of the filter (Cent
(Risart, SM13249E, Sartorius), and the amount of free 6-CF remaining in the liposome dispersion without being encapsulated was determined by measuring the fluorescence intensity of the filtrate.

Encapsulation rate = [(total 6-CF amount in liposome)-(free 6-CF amount in liposome)] / (6-
(CF amount) x 100 Example 2 The same method as in Example 1 was used, except that 15 mg of SMT was used instead of 30 mg SMT used in Example 1, and the encapsulation rate of 6-CF was 34.9%.
Of liposomes.

Example 3 6-CF encapsulation rate was 39.4% in the same manner as in Example 1, except that 45 mg of SMT was used instead of 30 mg SMT used in Example 1.
Of liposomes.

Example 4 Using 60 mg of SMT instead of 30 mg SMT used in Example 1, the encapsulation rate of 6-CF was 46.3% in the same manner as in Example 1.
Of liposomes.

Example 5 Using 30 mg of sodium palmitoylmethyltaurine (PMT) instead of the 30 mg SMT used in Example 1,
In the same manner as in Example 1, liposomes having a 6-CF encapsulation rate of 32.3% were obtained.

Example 6 Liposomes having a 6-CF encapsulation rate of 33.3% were obtained in the same manner as in Example 1 except that 30 mg of sodium octadecanesulfonate (ODS) was used instead of 30 mg SMT used in Example 1.

Example 7 Encapsulation rate of 6-CF was 24.1% in the same manner as in Example 1, except that 15 mg of ODS was used instead of 30 mg ODS used in Example 1.
Of liposomes.

Example 8 Using 45 mg of ODS instead of 30 mg ODS used in Example 1, the encapsulation rate of 6-CF was 38.3% in the same manner as in Example 1.
Of liposomes.

Example 9 6-CF encapsulation rate was 40.1% in the same manner as in Example 1 except that 60 mg of ODS was used instead of 30 mg ODS used in Example 1.
Of liposomes.

Example 10 Instead of 270 mg DPPC and 30 mg DSPC used in Example 1, 210 mg DPPC and 90 mg DSPC were used in Example 1
A liposome having an encapsulation ratio of 6-CF of 24.1% was obtained in the same manner as described above.

Example 11 Liposomes having an encapsulation rate of 6-CF of 35.2% were obtained in the same manner as in Example 1 except that 300 mg of DPPC was used instead of 270 mg of DPPC and 30 mg of DSPC used in Example 1.

Example 12 Example 1 was replaced by 210 mg DPPC, 90 mg DSPC instead of 270 mg DPPC, 30 mg DSPC used in Example 6.
A liposome having an encapsulation rate of 6-CF of 28.3% was obtained in the same manner as described above.

Example 13 Liposomes having an encapsulation rate of 6-CF of 34.6% were obtained in the same manner as in Example 1 except that 300 mg of DPPC was used instead of 270 mg of DPPC and 30 mg of DSPC used in Example 6.

Example 14 Example 1 was repeated using 30 mg of sodium palmitoyltaurine (PT) instead of the 30 mg SMT used in Example 1.
A liposome having a 6-CF encapsulation rate of 22.4% was obtained in the same manner as described above.

Example 15 360 mg of DPPC and 40 mg of DSPC were dissolved in 40 ml of chloroform in a 1 liter beaker. Further, the organic solvent was distilled off using a rotary evaporator to form a lipid thin film on the glass wall. A trace amount of the organic solvent remaining in the thin film was removed by wiping with nitrogen gas. At a temperature of 60 ° C.,
40 mg of Example 1 previously stored at 60 ° C.
10 mL of a 6-CF solution containing SMT was added, and the mixture was dispersed by vortex to obtain MLV liposomes. The MLV thus obtained was irradiated with ultrasonic waves for about 10 minutes at 50 watts using the probe type ultrasonic oscillator used in Example 1 to obtain an SUV. Example 1
Filtration and dialysis were performed in the same manner as in
5.7% liposomes were obtained.

Example 16 Instead of 360 mg of DPPC and 40 mg of DSPC used in Example 15, using 280 mg of DPPC and 120 mg of DSPC, the procedure of Example 15 was repeated.
A liposome having an encapsulation rate of 6-CF of 6.3% was obtained in the same manner as described above.

Example 17 Liposomes having a 6-CF encapsulation rate of 6.0% were obtained in the same manner as in Example 15 except that 400 mg of DPPC was used instead of 360 mg of DPPC and 40 mg of DSPC used in Example 15.

Example 18 Liposomes having an encapsulation rate of 6-CF of 6.8% were obtained in the same manner as in Example 15, except that 40 mg of PMT was used instead of 40 mg SMT used in Example 15.

Example 19 6-CF encapsulation was 6.5% in the same manner as in Example 15, except that 40 mg of ODS was used instead of 40 mg SMT used in Example 15.
Of liposomes.

Example 20 Example 15 was repeated using 40 mg of sodium palmitoyltaurine (PT) instead of the 40 mg SMT used in Example 15.
A liposome having a 6-CF encapsulation rate of 6.0% was obtained in the same manner as described above.

Example 21 Instead of the 6-CF solution obtained in Example 1, 500 μg / m
Using a 1% cisplatin (CDDP) saline solution,
A liposome preparation having a CDDP encapsulation rate of 23.5% and a phase transition temperature of 42.7 ° C. was obtained in the same manner as in Example 1.

(Note 2) Measurement method of CDDP content in liposome 0.1 ml of liposome was dispersed in 5 ml of physiological saline solution, 2.5 ml of the liposome was frozen and heated, and about 2.5 ml of the obtained liposome disrupted solution was filtered through Centrisalt. And the filtrate 0.1
2 ml of a 0.1N NaOH solution containing 10% of diethyldithiocarbamate (DDTC) was added to each ml, and the adduct obtained after standing at room temperature for 30 minutes was extracted with 5 ml of n-hexane, and the extract was subjected to HPLC (column; Zorbax (CN, solution; n-hexane / isopropyl alcohol = 8/2; UV = 250 nm), and the total CDDP amount of the liposome dispersion was determined. Separately, add about 2.5 ml of the remaining liposome saline to Centrisal
Under the same conditions as above, the amount of free CDDP present without being encapsulated in the liposome was quantified.

Example 22 Instead of the 6-CF solution used in Example 2, 500 μg
A liposome preparation having a CDDP encapsulation rate of 21.4% was obtained in the same manner as in Example 2 using a physiological saline solution of cisplatin (CDDP) at a concentration of 1 / ml.

Example 23 Instead of the 6-CF solution used in Example 3, 500 μg
A liposome preparation having a CDDP encapsulation rate of 25.8% was obtained in the same manner as in Example 3 using a physiological saline solution of cisplatin (CDDP) at a concentration of / ml.

Example 24 Instead of the 6-CF solution used in Example 5, 500 μg
A liposome preparation having a CDDP encapsulation rate of 24.0% was obtained in the same manner as in Example 5 using a physiological saline solution of cisplatin (CDDP) at a concentration of / ml.

Example 25 Instead of the 6-CF solution used in Example 6, 500 μg
A liposome preparation having a CDDP encapsulation rate of 21.8% and a phase transition temperature of 43.9 ° C. was obtained in the same manner as in Example 6 using a cisplatin (CDDP) physiological saline solution having a concentration of / ml.

Example 26 Instead of the 6-CF solution used in Example 7, 500 μg
A liposome preparation having a CDDP encapsulation rate of 21.9% was obtained in the same manner as in Example 7 using a physiological saline solution of cisplatin (CDDP) at a concentration of / ml.

Example 27 Instead of the 6-CF solution used in Example 8, 500 μg
A liposome preparation having a CDDP encapsulation rate of 24.9% was obtained in the same manner as in Example 8 using a cisplatin (CDDP) physiological saline solution having a concentration of 1 / ml.

Example 28 Instead of the 6-CF solution used in Example 10, 500 μg
A liposome preparation having a CDDP encapsulation rate of 23.3% was obtained in the same manner as in Example 10 by using a physiological saline solution of cisplatin (CDDP) at a concentration of / ml.

Example 29 Instead of the 6-CF solution used in Example 11, 500 μg
A liposome preparation having a CDDP encapsulation rate of 27.7% and a phase transition temperature of 41.9 ° C. was obtained in the same manner as in Example 11 using a cisplatin (CDDP) physiological saline solution having a concentration of / ml.

Example 30 Instead of the 6-CF solution used in Example 12, 500 μg
A liposome preparation having a CDDP encapsulation rate of 24.0% was obtained in the same manner as in Example 12 using a physiological saline solution of cisplatin (CDDP) at a concentration of / ml.

Example 31 Instead of the 6-CF solution used in Example 13, 500 μg
A liposome preparation having a CDDP encapsulation rate of 24.5% and a phase transition temperature of 42.5 ° C. was obtained in the same manner as in Example 13 using a cisplatin (CDDP) physiological saline solution having a concentration of / ml.

Example 32 Instead of the 6-CF solution used in Example 14, 500 μg
A liposome preparation having a CDDP encapsulation rate of 25.0% was obtained in the same manner as in Example 14 using a cisplatin (CDDP) physiological saline solution having a concentration of / ml.

Example 33 Instead of the 6-CF solution used in Example 15, 500 μg
A liposome preparation having a CDDP encapsulation rate of 4.8% was obtained in the same manner as in Example 15, using a cisplatin (CDDP) physiological saline solution having a concentration of / ml.

Example 34 Instead of the 6-CF solution used in Example 16, 500 μg
A liposome preparation having a CDDP encapsulation rate of 5.0% was obtained in the same manner as in Example 16 using a cisplatin (CDDP) physiological saline solution having a concentration of / ml.

Example 35 Instead of the 6-CF solution used in Example 17, 500 μg
A liposome preparation having a CDDP encapsulation rate of 5.2% was obtained in the same manner as in Example 17 using a cisplatin (CDDP) physiological saline solution having a concentration of / ml.

Example 36 Instead of the 6-CF solution used in Example 18, 500 μg
A liposome preparation having a CDDP encapsulation rate of 4.3% was obtained in the same manner as in Example 18 using a cisplatin (CDDP) physiological saline solution having a concentration of / ml.

Example 37 Instead of the 6-CF solution used in Example 19, 500 μg
A liposome preparation having a CDDP encapsulation rate of 4.9% was obtained in the same manner as in Example 19 using a cisplatin (CDDP) physiological saline solution having a concentration of / ml.

Example 38 Instead of the 6-CF solution used in Example 20, 500 μg
A liposome preparation having a CDDP encapsulation rate of 4.7% was obtained in the same manner as in Example 20, using a cisplatin (CDDP) physiological saline solution having a concentration of / ml.

Example 39 Instead of the 6-CF solution used in Example 11, 308 μg
Using a protein / ml aqueous solution of interleukin 2 (IL-2) (solution type: 25 mM ammonium acetate solution, pH 6), a liposome preparation of the present invention was obtained in the same manner as in Example 11.

Example 40 Instead of the 6-CF solution used in Example 13, 308 μg
A liposome preparation of the present invention was obtained in the same manner as in Example 13 using an aqueous IL-2 solution of protein / ml.

Example 41 Instead of the 6-CF solution used in Example 11, 100 μg
using ansamitocin saline solution at a concentration of / ml
In the same manner as in Example 11, a liposome preparation of the present invention encapsulating ansamitocin was obtained.

Example 42 Instead of the 6-CF solution used in Example 13, 100 μg
Using an ammacytocin saline solution at a concentration of / ml,
In the same manner as in Example 13, a liposome preparation of the present invention encapsulating ansamitocin was obtained.

Example 43 Instead of the 6-CF solution used in Example 11, 5 mg / ml
A liposome preparation of the present invention encapsulating methotrexate was obtained in the same manner as in Example 11 using a methotrexate physiological saline solution having a concentration of

Example 44 Instead of the 6-CF solution used in Example 13, 5 mg / ml
In the same manner as in Example 13, a liposome preparation of the present invention containing methotrexate was obtained using a methotrexate physiological saline solution having a concentration of

Example 45 Instead of the 6-CF solution used in Example 11, 200 μg
A liposome preparation of the present invention encapsulating mitomycin C was obtained in the same manner as in Example 11 using a mitomycin C physiological saline solution having a concentration of / ml.

Example 46 Instead of the 6-CF solution used in Example 13, 200 μg
A liposome preparation of the present invention encapsulating mitomycin C was obtained in the same manner as in Example 13 using a mitomycin C physiological saline solution having a concentration of / ml.

Example 47 Instead of the 6-CF solution used in Example 11, 1 mg / ml
A liposome preparation of the present invention in which adriamycin is encapsulated was obtained in the same manner as in Example 11 using an adriamycin physiological saline solution having a concentration of

Example 48 Instead of the 6-CF solution used in Example 13, 1 mg / ml
A liposome preparation of the present invention in which adriamycin is encapsulated was obtained in the same manner as in Example 11 using an adriamycin physiological saline solution having a concentration of

Example 49 Instead of the 6-CF solution used in Example 11, 3 mg / ml
Example using bleomycin saline solution at a concentration of
A liposome preparation of the present invention containing bleomycin was obtained in the same manner as in 11.

Example 50 Instead of the 6-CF solution used in Example 13, 3 mg / ml
Example using bleomycin saline solution at a concentration of
A liposome preparation of the present invention encapsulating bleomycin was obtained in the same manner as in 13.

Experimental Example 1-1 A liposome containing no anionic surfactant was prepared as a control corresponding to the liposomes of Examples 1, 10, 11, 15, 16, and 17, respectively. In addition, 270 m of the first embodiment
200m instead of g DPPC, 30mg DSPC and 30mg SMT
A control liposome was prepared in the same manner as in Example 1 using g of phosphatidylcholine made of egg yolk having an unsaturated acyl group, 100 mg of cholesterol and 30 mg of SMT. In addition, a liposome containing no SMT corresponding to this liposome was also prepared as a control. On the other hand, the SMT of Example 15
Was replaced with sodium dodecyl sulfate (SDS, Krafft point; 9 ° C.) to prepare control liposomes.

Experimental Example 1-2 Each of the liposome obtained in Example 1 and the liposome prepared in the same manner without adding an anionic surfactant was 0.1% each.
When -0.5 ml was intravenously administered to a rat and the disappearance of liposomes from the blood was examined (Note 3), the results shown in FIG. 1 were obtained. As shown in FIG. 1, the blood concentration of a liposome containing an anionic surfactant (indicated by-●-in the figure)
Is a control liposome not containing it (in the figure, ... X
(Indicated by *). Similarly, 0.1-0.5 ml of each of the liposomes obtained in Examples 1, 6, 10, 15, 18, 19 and 20 was intravenously administered to rats, and
The liposomes remaining in the blood after time were 9.7, 11.9, 26.4, 2.7, 2.3, 2.8, and 2.2, respectively, compared to similarly prepared liposomes without the addition of an anionic surfactant.
It was twice. On the other hand, as shown in FIG. 2, a liposome containing an anionic surfactant prepared from egg yolk phosphatidylcholine and cholesterol (in the figure,-●-
) Disappeared from the blood as quickly as the control liposomes (indicated by... X in the figure). FIG. 3 shows a liposome containing SDS obtained in Experimental Example 1-1 (indicated by... X in the figure).
And the results of examining the disappearance of liposomes from the blood by intravenously administering 0.2 ml of each of the liposomes prepared in the same manner without addition of SDS (indicated by-in the figure) to rats. . As is clear from the results shown in these figures, the liposome of the present invention prepared using a phospholipid having a saturated acyl group as the liposome membrane composition and an anionic surfactant having a Krafft point of 37 ° C. or higher. The preparation is characterized in that the elimination time from blood after intravenous administration is extremely long as compared to control liposomes.

Experimental Example 1-3 The liposomes obtained in Examples 1, 15, 18, 19 and Experimental Example 1-1 were intravenously administered to rats, and 1 hour later, the concentration of 6-CF in the liver was measured to determine the RES of the liposomes. Investigation of the distribution to (Note 2) gave the results in Table 1. These results indicate that the time required for liposomes to disappear from the blood is prolonged,
This indicates that the distribution to S has decreased.

(Note 3) Method for measuring blood concentration and liver concentration of 6-CF liposome A blood dispersion was prepared by adding 10 ml of PBS to 0.2 ml of heparin-treated blood obtained from the tail vein. Then centrifuge (3000
rpm, 10 minutes) and Triton X-100 is added to 5 ml of the separated supernatant.
Was added, and the liposome was broken under heating at 60-70 ° C.
The liposome concentration in blood was determined by measuring the fluorescence of the released 6-CF. The liver obtained after laparotomy was immersed in PBS containing 0.02% Triton X-100, and 100 ml
After breaking the tissue with a tissue disrupter (Polytron, Kinematica) and heating at 60-70 ° C., a homogenate from which all 6-CF was released was obtained. The homogenate was ultracentrifuged (50000 g, 10 minutes), diluted 20-50 times, and then diluted to 0.
45 micron membrane filter (Acrodisk, Gelma
After filtration in n), the fluorescence was measured to determine the liposome concentration in the liver.

EXPERIMENTAL EXAMPLE 1-4 The liposomes of Examples 1, 2 and 6 and the liposomes containing no anionic surfactant for these liposomes were diluted 10,000 times with PBS (2% plasma), and the heat release property was connected to a temperature raising system. The amount of released 6-CF from the liposome was determined by continuously measuring the amount of 6-CF released from the liposome, and the phase change (change from gel to liquid crystal) of the liposome membrane was examined. Table 2 shows the heat release onset temperature and phase transition temperature determined from this release curve. Phase transition temperature is thermal analysis system (SEIKOI & E, SSC 5000 type, 2 ℃ / min)
Was used for measurement. As shown in the table below, the two correspond well.

Table 2 Phase transition temperature of the liposome membrane (° C) and heat release temperature of 6-CF from the liposome (° C) Type of liposome Phase transition temperature Thermal release start temperature Example 1 42.3 36.1 Example 6 43.7 39.3 Example 10 42.8 36.3 No Surfactant 41.9 37.9 Experimental Example 2-1 A liposome preparation containing no anionic surfactant was prepared as a control, corresponding to the liposome preparations of Examples 21, 28, 29, 33, 34 and 35.

Experimental Example 2-2 6-CF concentration in blood for up to 6 hours when the liposome preparation of Example 1 was intravenously administered to rats and CDDP when the liposome preparation of Example 15 was intravenously administered to rats By comparing the concentrations (Note 4), the results in FIG. 4 were obtained. At any time, CDDP showed a concentration similar to that of 6-CF, and the CDDP liposome preparation (indicated by-●-in the figure) was not treated with 6-CF liposome preparation (in the figure, ... X
..).
In addition, the liposome preparations obtained in Examples 21, 22, 25, 26, and 29 also showed high values as in the case of the 6-CF liposome. These results also show that the liposome preparation of the present invention using a phospholipid having a saturated acyl group as a liposome membrane composition and an anionic surfactant having a Krafft point of 37 ° C. or higher was intravenously administered as compared to control liposomes. It has the characteristic that the time after which it disappears from the blood is extremely long.

(Note 4) Measurement method of blood concentration of CDDP Obtain a blood dispersion containing 2 ml of PBS in 0.2 ml of heparin-treated blood obtained from the tail vein. Further, 1 ml of the DDTC solution was added to 1 ml of the separated supernatant obtained by centrifugation, and the above-mentioned CDDP was added.
The amount of total CDDP in blood was quantified in the same manner as in the method of quantification.

Experimental Example 2-3 When the SMT content of Example 21 was quantified (Note 5),
About 90% of the charged amount at the time of liposome preparation remained. This amount was significantly higher than the CDDP encapsulation rate of 23.5%, indicating that SMT was clearly a membrane constituent of liposomes. Means that it exists.

(Note 5) Measurement method of SMT content Methylene blue 15mg, concentrated sulfuric acid 6g, anhydrous sodium sulfate
Dissolve 25 g in distilled water to prepare a 500 ml reaction solution. 10 ml of a liposome diluent (10000-fold) and 5 ml of chloroform were added to 5 ml of this reaction solution, and the mixture was thoroughly shaken.
The layers were separated, and the absorbance (653 nm) of the chloroform layer was measured. The absorbance was measured using an SMT solution (10 ppm or less) to prepare a calibration curve. A blank test was performed using the liposome containing no SMT in Experimental Example 2-1.

Effect of the Invention The liposome preparation of the present invention is characterized by using an anionic surfactant having a high Krafft point together with a saturated phospholipid at a critical micelle concentration or higher.

The liposome preparation of the present invention can stably circulate in the body with blood for a long time after intravenous administration, thereby alleviating the toxicity of the drug, increasing the target effect of the drug on a specific lesion, and sustaining the drug. It is useful for enhancing the therapeutic effect. In particular, the liposome preparation of the present invention in which an anticancer agent is encapsulated can be expected to improve the therapeutic effect by administering it at the time of cancer therapy, in which case the liposome membrane has a phase transition temperature of about 40 to 55 ° C. Can be preferably used.

[Brief description of the drawings]

FIGS. 1, 2 and 3 show the relationship between the time course and the blood 6-CF concentration when a liposome preparation encapsulating 6-CF was intravenously administered to rats in Experimental Example 1-2. FIG. 4 shows the relationship between the time course and the blood concentration of each drug when a liposome preparation encapsulating 6-CF or CDDP was intravenously administered to rats in Experimental Example 2-2. Here, it is assumed that the blood volume of the rat occupies 10% of the body weight.

Claims (3)

(57) [Claims] 1. A liposome having a membrane composed of a phospholipid whose acyl group is a saturated acyl group and an aqueous liquid containing an anionic surfactant having a Krafft point of 37 ° C. or higher at a critical micelle concentration or higher. A liposome preparation in which a drug is encapsulated. 2. The preparation according to claim 1, wherein the anionic surfactant is an acyltaurine salt. 3. A method for producing a liposome preparation containing a drug, wherein the phospholipid wherein the acyl group is a saturated acyl group,
A method for producing a liposome preparation, comprising forming a liposome membrane using an emulsion or dispersion of an aqueous liquid containing an anionic surfactant having a Kraft point of 37 ° C. or higher at a critical micelle concentration or higher.