JPH069374A - Liposome pharmaceutical preparation and its production - Google Patents

Abstract

PURPOSE:To obtain a liposome pharmaceutical preparation advantageously utilizable for using the hyperthermia for cancer and an antitumor agent in combination due to its small particle diameter and ability to maintain a high level in blood and excellence in temperature sensitivity CONSTITUTION:The liposome pharmaceutical preparation is obtained by sealing a hypertonic medicine-containing liquid having an osmotic pressure of 2.0-5.0 times based on that of a biological humor of a warm-blooded animal in a liposome (having <=0.1mum average particle diameter) which is a small unilamellar vesicle having 40-45 deg.C phase transition temperature of the membrane.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a liposome preparation containing a drug useful in the field of medicine, and a method for producing the same.

[0002]

2. Description of the Related Art Liposome preparations having various characteristics, aiming at targeting treatment for cancer lesions, are being developed as drug delivery systems. A temperature-sensitive liposome preparation known as one of them is a thermotherapy for cancer (locally heating a lesion with an appropriate device (around 43 ° C.) to specifically damage only cancer cells. The effect of the chemotherapeutic agent can be enhanced by the combined use with a therapeutic method that gives Examples of the report are (1) J. N. Wei
nstein, R.A. L. Magin, M .; B. Ya
tvin and D.D. S. Zaharko, Sci
ence, 204 , 188 (1979), (2)
J. N. Weinstein, R.S. L. Magin,
R. L. Cysyk and D.M. S. Zahark
O, Cancer Res. , 40 , 1388 (1
980), (3) M.M. B. Yatvin, T .; C.
Cree, I.D. M. Tegmo-Larsson a
ndJ. J. Gipp, Strahlentera
pie 160 , 732 (1984) and the like.

After the liposome preparation is administered intravenously,
It gets on the bloodstream and reaches the cancer lesion. When the cancer lesion is heated, only the temperature-sensitive liposomes distributed in that portion are disintegrated by sensing the temperature difference from normal body temperature, and the encapsulated drug is locally released. Since this liposome is stable in normal tissues that are not heated and does not release the drug, it can be expected to reduce side effects. Thus, the temperature-sensitive liposome preparation is a preparation that can enhance the targeting effect of the chemotherapeutic agent.

[0004]

As mentioned above, it is considered useful to apply a temperature-sensitive liposome preparation to hyperthermia of cancer. Conventional liposome preparations are large unilamellar vesicles (LUV; generally diameter 0.1-2.0
μm) is used, but there are two possible drawbacks.
One is that LUV administered intravenously has the property of being easily taken up by reticuloendothelial tissues such as liver and spleen.
There is concern that chemotherapeutic agents encapsulated in LUVs (which are generally said to have side effects) may adversely affect normal tissue. Another reason is that LUV is easily taken up by the reticuloendothelial system tissue, so that it is difficult to maintain the blood concentration thereof, and the targeting efficiency to the cancer lesion is lowered. Based on the principle of action of temperature-sensitive liposomes, it is important to keep the blood concentration as high as possible,
Furthermore, A. As pointed out by Gabizon et al. (Pro
c. Natl. Acad. Sci. , 85 , 6949
(1988)) It is also known that liposome preparations that remain in circulating blood for a long time tend to passively accumulate in cancer lesions, and a higher synergistic effect of targeting is expected.

As a clue to solve these problems,
It is known that if the particle size of the liposome is reduced, it will be difficult for the liposome to be taken up by the reticuloendothelial system tissue and the blood concentration will be kept high. Examples of this report include (1) R. L. Juli
an and D. Stamp, Biochem.
Biophys. Res. Commun. , 63 , 6
51 (1975), (2) T.I. M. Allen an
d J. M. Everest, J.M. Pharmaco
l. Exp. Ther. , 226 , 539 (198
3), (3) Y. Sato, H .; Kiwada a
nd Y. Kato, Chem. Pharm. Bul
l. , 34 , 4244 (1986), (4) R.I. M.
Abra and C.I. A. Hunt, Biochi
m. Biophys. Acta, 666 , 493 (1
981), (5) H. Kiwada, S .; Obar
a, H.A. Nishiwaki and Y. Kato,
Chem. Pharm. Bull. , 32 , 124
9 (1986), (6) C.I. A. Hunt, Y.
M. Rustum, E .; Mayhew and D.M.
Papahadjopoulos, Drug. Met
ab. Dispos. , 7 , 124 (1979),
(7) Y. E. Rahman, E .; A. Cerny,
K. R. Patel, E .; H. Lau and
B. J. Wright, Life Sci. , 3
1 , 2061 (1982) and the like. But,
It is said that when the particle size of the liposome is reduced, the temperature sensitivity is extremely lowered. An example of this report is (1) I
nt. J. Pharmaceut. , 57 , 241 (1
989), (2) Cancer Drug. Del
Ivy, 3 , 223 (1986), (3) Yasuaki Ogawa, Hajime Toguchi, Cancer and Chemotherapy, 17 , 1127 (199)
0) and the like. Therefore, at present, there is a demand for the development of a liposome preparation having a small particle size of liposome, capable of maintaining a blood concentration, and having excellent temperature sensitivity.

The present invention provides a small unilamellar vesicle (SUV; diameter: 0.1 μm) having a small particle size such that the uptake into the reticuloendothelial tissue is small and the blood concentration can be maintained.
m or less) and is intended to prepare a liposome preparation having excellent temperature sensitivity.

[0007]

In view of the above situation, the inventors have selected a liposome membrane composition and a drug solution to be encapsulated in the liposome in order to produce a liposome having a small particle size and excellent temperature sensitivity. The present invention has been completed by various studies from the viewpoint of adjusting the osmotic pressure. That is, according to the present invention, the liposome membrane has a phase transition temperature of 40 to 45 ° C. and is stable at 37 ° C. in SUV having a hypertonicity of 2.0 to 5.0 times higher than the biological fluid osmotic pressure of a warm-blooded animal. The present invention relates to a liposome preparation containing a drug-containing liquid and a method for producing the same.

First, the composition of the membrane constituting the liposome preparation of the present invention will be described. In the present invention, the constituent material of the liposome membrane and its constituent ratio are selected so as to cause a phase transition at the temperature of hyperthermia, that is, the phase transition temperature of the membrane is 40 to 45 ° C, preferably 40 to 43 ° C. To be done. As a material for this membrane, it is advantageous to use various phospholipids in which the acyl group is a saturated acyl group, alone or in combination. The two saturated acyl groups of glycerophospholipid are acyl groups derived from higher saturated fatty acids, and usually have a saturated acyl group having 12 to 20 carbon atoms, preferably 1 carbon atom.
Those having 4 to 18 saturated acyl groups can be used. Examples of such phospholipids include hydrogenated lecithin obtained by hydrogenating egg yolk lecithin and soybean lecithin, and phosphatidylcholine obtained by total synthesis or semisynthesis, which is composed of a combination of lauryl, myristoyl, palmitoyl, stearoyl, and the like. Etc. can be used. Also, depending on the type of drug to be encapsulated, phosphatidylglycerol having an acyl group as a saturated acyl group can be used. Specifically, phosphatidylcholine obtained by total synthesis or semisynthesis is advantageously used, of which the phase transition temperature of the membrane is close to the temperature of hyperthermia,
Dipalmitoylphosphatidylcholine having a saturated acyl group having 16 carbon atoms (DPPC; phase transition temperature 41.4 ° C.),
Alternatively, distearoylphosphatidylcholine having 18 carbon atoms (DSPC; phase transition temperature 54.9 ° C.) is preferably used.

The phase transition temperature of the prepared liposome membrane is reported to be close to the phase transition temperature determined from the weight proportional distribution of the saturated phospholipid used in combination, but the liposomes used in the present invention having a small average particle size. In the case of, the phase transition temperature of a common liposome having the same weight proportion is lower by about 1 to 4 ° C. The fact that such a temperature difference is observed is considered to be one of the proofs which are different from those in which the liposome of the present invention has been conventionally used. As a combined example of the saturated phospholipid used in the present invention, it is used so that the molar ratio of DPPC and DSPC (DPPC / DSPC) is in the range of 90/10 to 50/50, preferably 85/15 to 65/3.
It is advantageous to use it in the range of 5.

In the liposome preparation of the present invention, a membrane stabilizer and a charge control agent may be used as long as they do not significantly impair the temperature sensitivity. Similarly, a device for further suppressing uptake by reticuloendothelial tissues such as liver and spleen (eg, with a water-soluble polymer such as polyethylene glycol or a ganglioside such as GM1) unless temperature sensitivity is significantly impaired. Coating can also be applied.

Next, the drug-containing liquid encapsulated in the liposome in the present invention will be described. The osmotic pressure of the drug-containing liquid encapsulated in the liposome is 2.0 to 5.0 times higher than the osmotic pressure of the biological fluid of a warm-blooded animal, preferably 2.5 to
Adjusted to be 4.5 times hypertonic. In consideration of the solubility of the drug, this liquid can reach the above-mentioned osmotic pressure with the drug alone, but if necessary, an appropriate osmotic pressure adjusting agent can be used. The osmotic pressure regulator may be used without particular limitation as long as it is water-soluble and physiologically acceptable to warm-blooded animals, and salts (sodium chloride etc.), saccharides (sucrose etc.), amino acids ( Glycine etc.) are preferably used.

[0012] The drug used in the present invention is a drug which is expected to have a synergistic effect in combination with hyperthermia, and is selected for the purpose of encapsulating in a liposome to enhance the targeting effect. As a specific example of such a drug, an antitumor agent is a preferred target drug. Specific examples thereof include metal complexes such as cisplatin, carboplatin, tetraplatin and iproplatin, adriamycin, ansamitocin or its derivatives, antitumor antibiotics such as bleomycin, Ara-C and daunomycin, 5-FU and methotrexate. Antimetabolites, alkylating agents such as BCNU and CCNU, and other anticancer agents such as melphalan and mitoxantrone. Among them, cisplatin is considered to be useful as a drug expected to have a particularly high targeting effect. Report on this is as follows: (1) Tetsuzo Agishi's “Targeted Therapy” 1st edition, Japan Medical Center, Tokyo,
P. 165 (1989) (2) Yasuaki Ogawa, Hajime Toguchi, Cancer and Chemotherapy, 17 , 1127 (1990), and the like.

Since the liposome used in the present invention is SUV, the retention volume of the drug becomes small and the relative amount of phospholipid increases, but it is generally known that the raw material phospholipid has low toxicity and administration The required dose can be obtained by adjusting the density of liposomes in the liquid.

The method for producing the liposome preparation of the present invention will be described below.

Regarding the method for producing liposomes, known methods such as ultrasonic method, French press method, extrusion method and the like can be applied as long as they are methods for producing SUV. For example, in the ultrasonic method used in the present invention, first, an organic solvent such as chloroform is added to a mixture of phospholipids having a saturated acyl group as described above or a mixture thereof, and the mixture is sufficiently stirred, and then the solvent is distilled off to remove phosphorus. Form a thin film of lipids.
This thin film is thoroughly dried, a preparation solution containing a drug is added, and the mixture is vigorously stirred to produce multilamellar vesicles, which are then subjected to ultrasonic treatment in a nitrogen stream for SUV.
To get The liposomal formulation thus obtained generally needs to be separated from the unencapsulated (free) drug. As a method for removing the drug, a gel filtration method or a dialysis method is generally applied, but in the case of separating an SUV that does not have the same charge as the charged drug, a method using an ion exchange resin is preferable.

The liposome of the present invention can be applied without particular limitation as long as it is a cancer disease to which hyperthermia can be applied, and it can be applied not only to humans but also to tumor-bearing animals.

[0017]

EXAMPLES The present invention will be described in more detail below by showing Examples, Comparative Examples and Test Examples in which a fluorescent substance is enclosed, but the present invention is not limited to the Examples and Test Examples.

Example 1 3 ml of a chloroform solution containing 21 micromoles of DPPC and 9 micromoles of DSPC was placed in a 50 ml pear-shaped flask and mixed thoroughly, and then the solvent chloroform was distilled off to form a thin film of the above phospholipid. . The thin film is sufficiently dried, 2 ml of 0.094 M calcein-0.200 M sucrose (pH 7.4) solution (osmotic pressure 2.0 times) is added, and the mixture is vigorously stirred to produce multilamellar vesicles. Furthermore, a probe-type ultrasonic generator (BRA
NON SONIFIER Cell Disr
SUV is obtained by sonicating for 30 minutes under a nitrogen stream using an uptor 185). Then, by passing through a diethylaminoethyl Sephadex A-25 column saturated with sodium chloride / phosphate buffer (pH 7.4) having an osmotic pressure equal to that of the liquid encapsulated in the liposome, calcein not encapsulated in the liposome was obtained. Adsorb and remove.

The particle size of the manufactured SUV was measured by a light scattering method (NICOMP model 370), and the average particle size was 0.10 μm or less.

Example 2 A liposome preparation was prepared in the same manner as in Example 1 except that 0.094 M calcein-0.400 M sucrose solution (pH 7.4) was used as the solution to be encapsulated in the liposome, and A susceptibility test was performed. The osmotic pressure of this solution was 3.0 times, and the average particle size of the produced liposomes was 0.10 μm or less.

Example 3 A liposome preparation was prepared in the same manner as in Example 1 except that 0.094 M calcein-0.560 M sucrose solution (pH 7.4) was used as the solution to be encapsulated in the liposome, and the temperature was adjusted. A susceptibility test was performed. The osmotic pressure of this solution was 4.0 times, and the average particle size of the produced liposomes was at least 0.10 μm.

Example 4 A liposome preparation was prepared in the same manner as in Example 1 except that 0.094 M calcein-0.705 M sucrose solution (pH 7.4) was used as the solution to be encapsulated in the liposome, and A susceptibility test was performed. The osmotic pressure of this solution was 5.0 times, and the average particle size of the produced liposomes was 0.10 μm or less.

Example 5 3 ml of a chloroform solution containing 42 micromoles of DPPC and 18 micromoles of DSPC was placed in a 50 ml pear-shaped flask and thoroughly mixed, and then the solvent chloroform was distilled off to form a thin film of phospholipid. The thin film was sufficiently dried, and 0.2% cisplatin (CDDP) -0.9% sodium chloride-0.230M sucrose solution (pH 5.0,
Multilamellar vesicles are manufactured by adding 4 ml of osmotic pressure 2.0 times) and stirring vigorously. Furthermore, a probe type ultrasonic generator (BRANSON SONIFIE
Using Cell Disruptor 185 manufactured by R Co., Ltd., ultrasonic treatment is performed for 30 minutes under a nitrogen stream to obtain SUV. Next, the CDDP not encapsulated in the liposome was passed through a Sulfopropyl Sephadex C-25 column saturated with sodium chloride / phosphate buffer (pH 5.0) having an osmotic pressure equal to that of the liquid encapsulated in the liposome. To remove.

The particle size of the manufactured SUV is measured by a light scattering method (NICOMP model 370).
The average particle diameter was 0.10 μm or less.

Example 6 0.2% CDDP as a solution to be encapsulated in liposomes
Using -0.9% sodium chloride-0.439 M sucrose solution (pH 5.0), a liposome preparation was produced in the same manner as in Example 5 except that, and a temperature sensitivity test was conducted. The osmotic pressure of this solution was 3.0 times, and the average particle size of the produced liposomes was 0.10 μm or less.

Example 7 As a solution to be encapsulated in liposomes, 0.2% CDDP was used.
Using -0.9% sodium chloride-0.620 M sucrose solution (pH 5.0), a liposome preparation was produced in the same manner as in Example 5 except for the above, and a temperature sensitivity test was conducted. The osmotic pressure of this solution was 4.0 times, and the average particle size of the produced liposomes was 0.10 μm or less.

Example 8 A liposome preparation having an average particle size of 0.10 μm or less and an osmotic pressure of 2.0 times was obtained in the same manner as in Example 5 except that 48 micromoles of DPPC and 12 micromoles of DSPC were used. .

Example 9 48 μmol of DPPC and 12 μmol of DSPC were used as a solution to be encapsulated in liposomes at 0.2
% CDDP-0.9% sodium chloride-0.439 M sucrose solution (pH 5.0), otherwise Example 5
A liposome preparation having an average particle size of 0.1 μm or less and an osmotic pressure of 3.0 times was obtained by the same method as described above.

Example 10 48 μmol of DPPC and 12 μmol of DSPC were used and 0.2
% CDDP-0.9% sodium chloride-0.620 M sucrose solution (pH 5.0), otherwise Example 5
A liposome preparation having an average particle diameter of 0.1 μm or less and an osmotic pressure of 4.0 times was obtained by the same method.

[0030]

[Comparative example]

Comparative Example 1 A 0.094 M calcein solution (pH 7.4) was used as a solution to be encapsulated in liposomes, and other than that, Example 1 was used.
A liposome preparation was prepared by the same method as in Example 1 and subjected to a temperature sensitivity test. The osmotic pressure of this solution was 1.1 times, and the average particle size of the produced liposomes was 0.10 μm or less.

Comparative Example 2 0.2% CDDP-as a solution to be encapsulated in liposomes
Using 0.9% sodium chloride solution (pH 5.0),
Otherwise, a liposome preparation was produced in the same manner as in Example 5, and a temperature sensitivity test was conducted. The osmotic pressure of this solution is 1.
It is 0 times, and the average particle size of the manufactured liposome is 0.1.
It was 0 μm or less.

Comparative Example 3 48 micromoles of DPPC and 12 micromoles of DSPC were used, and a solution for encapsulation in liposomes was 0.2
% CDDP-0.9% sodium chloride solution (pH 5.
0) was used to produce a liposome preparation in the same manner as in Example 5 except for the above, and the temperature sensitivity test was conducted. The average particle size of the manufactured liposomes was 0.10 μm or less.

[0033]

[Test example]

Test Example 1 The liposome preparations obtained in Examples 1 to 4 and Comparative Example were subjected to a temperature sensitivity test using calcein.

The liposome solution thus prepared was diluted so that the concentration of the drug to be encapsulated was 167 μM with respect to the total solution, and subjected to an in vitro temperature sensitivity test. The in vitro temperature sensitivity test was conducted as follows. That is, 0.5 ml of blood collected from a Crj: CD-1 female mouse was placed in a constant temperature bath at 37 ° C., 41 ° C. and 43 ° C. and preincubated for 5 minutes. 50 μl
When the liposome solution was added to 0 minutes, 2, 5, 10, 1
1 after incubation for 5, 20, 30 minutes
A mixed solution of 0 μl of liposome and blood was collected, and a buffer for fluorescence measurement (0.35 M sucrose-0.01 M EDTA was used.
-0.04M Tris-HCl; pH 8.5) 1.2
Placed in ml. Immediately thereafter, the blood cells were removed by centrifugation at 1400 g for 10 minutes. After measuring the fluorescence amount of 0.9 ml of the supernatant (fluorescence amount A), 0.1 ml of 10% Triton X100 solution was added to completely destroy the liposomes to release calcein, and the fluorescence amount was measured (fluorescence amount B ). The release rate of the drug was obtained by the formula 1.

Release rate = {1- (0.9 × A / B) / self-quenching value} × 100 (1) However, the self-quenching value represents a value in a buffer solution before adding blood, Similarly, it is determined from the ratio of the fluorescence amount measured by adding 0.1 ml of 10% Triton X100 solution after measuring the fluorescence amount of 0.9 ml.

The release rate after 15 minutes of incubation at each temperature was summarized (Table 1). The time course of the release rate was shown using the liposome preparation of Example 3 as a representative example (FIG. 1). In the figure,-□-is 37 ° C,-●-is 41 ° C,-
○ -represents the release rate at 43 ° C.

[0037]

[Table 1] From Table 1, when the osmotic pressure is less than 2.0 times, the stability of the membrane at 37 ° C is extremely poor and the drug inside is released, but this can be improved by increasing the osmotic pressure of the enclosed liquid. Recognize. Moreover, the fact that the drug release rate at 43 ° C. remains unchanged and is good also indicates that the liposome of the present invention is useful in combination with hyperthermia. In addition, the drug release rate at 41 ° C was also high, and
As shown in (1), the liposomes rapidly disintegrate after addition, which indicates that the liposome preparation developed in the present invention has excellent temperature sensitivity.

Test Example 2 The liposome preparations obtained in Examples 5 to 10 and Comparative Examples 2 and 3 were subjected to a temperature sensitivity test using cisplatin.

The liposome solution thus prepared was diluted so that the concentration of the drug contained therein was 400 μM with respect to the total solution, and the solution was subjected to an in vitro temperature sensitivity test. The in vitro temperature sensitivity test was conducted as follows. That is, 0.582 ml of blood collected from a female mouse of Crj: CD-1 was placed in a constant temperature bath at 37 ° C.
Pre-incubation was performed for a minute. When 100 μl of the liposome solution was added, it was defined as 0 minutes, and 2, 5, 10, 1
After incubation for 5, 20 and 30 minutes, 6
A mixed solution of 0 μl of liposome and blood was collected and placed in 0.15 ml of sodium chloride / phosphate buffer (pH 5.0) having an osmotic pressure equal to that of the liposome internal solution. Immediately 1
The blood cell component was removed by centrifugation at 400 g for 10 minutes. 50 μl of the supernatant was taken into a test tube (T sample), and the remaining supernatant was put into Centricon 100 (Amicon Division, Grace Japan Co., Ltd.) and centrifuged at 170 g for 20 minutes to obtain a liposome-free filtrate. 50 μl of the filtrate was taken and transferred to a test tube (F sample). 0.2 ml of concentrated nitric acid solution was added to each of the F sample and the T sample, and heated at 140 ° C. for 2 hours. Then add 0.2 ml of 30% hydrogen peroxide solution to 1
It was heated at 40 ° C. for 30 minutes for ashing to obtain a colorless and transparent sample liquid. After measuring the liquid volume, platinum was determined in the F sample and the T sample using an atomic absorption spectrophotometer (Z-9000 type, manufactured by Hitachi, Ltd.). The cisplatin release rate (R ₃₇ ) at 37 ° C. was calculated from Equation 2.

R ₃₇ = PtF / PtT (2) where PtF represents the amount of platinum in the F sample and PtT represents the amount of platinum in the T sample.

In the 41 ° C. and 43 ° C. incubation tests, the collection time of the mixture of liposome and blood was 1, 2,
Changes were made after 4, 7, 10 and 15 minutes, all else followed the above method. The cisplatin release rate at 41 ° C. and 43 ° C. was calculated from Equation 3.

R ₄₁ (or R ₄₃ ) = PtF / PtT / 0.77 (3) However, the value 0.77 is a correction value, and the free cisplatin solution not encapsulated in the liposome is treated under the same conditions and the same procedure. It is the value obtained by performing the incubation test.

The release rate after 15 minutes of incubation at each temperature was summarized (Tables 2 and 3).

From Tables 2 and 3, it can be seen that a considerable amount of the drug leaks at 37 ° C. in the liposome containing the drug solution having an osmotic pressure of 1.0 times. Moreover, at 41 ° C. and 43 ° C., the drug is not released so much, and it can be said that the liposome has markedly poor temperature sensitivity. On the other hand, 3 liposomes contain hypertonic solution.
It can be seen that the drug leakage at 7 ° C. is improved in proportion to the osmotic pressure ratio, and the drug release properties at 41 ° C. and 43 ° C. are also improved.

[0045]

[Table 2]

[0046]

[Table 3] Test Example 3 S-180 cells were transplanted intramuscularly in the left foot of 7-week-old Crj: CD-1 female mice, and on day 9, the diameter of the cancer (from the diameter of the left foot in which the cancer had grown to the diameter of the normal right foot) was examined. Value after subtracting
Twenty cancer-bearing mice having a size of 0.53 cm were divided into 5 groups and used in this test. The coefficient of variation of the diameter of the cancer is 4.0
%, And mice with uniform cancer size were used. Hyperthermia treatment was performed by the following method. That is, by attaching a coating thermocouple to the skin surface of the cancer tissue and sending a skin temperature signal to the temperature controller to control the output of the DC constant voltage / constant current power source, the cord heater wrapped around the cancer tissue is heated. did. By this method, heating could be performed with an accuracy of 42.5 ± 0.1 ° C.

The CDDP-encapsulated SUV prepared by the method of Example 8 was adjusted to a CDDP concentration of 400 μM to prepare a dosing solution. The CDDP aqueous solution was prepared by diluting Randa Injection of Nippon Kayaku Co., Ltd. with physiological saline (Otsuka saline) and adjusting the CDDP concentration to 400 μM. The dose of CDDP was 1.5 mg / kg, heating was started immediately after the administration into the tail vein, and the skin surface temperature of the cancer tissue was 42.
Warming was continued for 40 minutes after reaching 5 ° C. The treatment was performed three times in total on the 9th, 13th and 17th days after transplantation.

In FIG. 2,-●-represents a group in which CDDP-encapsulated SUV was injected intravenously and combined with hyperthermia treatment, and-□-represents CDDP-encapsulated SUV.
UV-injected hyperthermia-free group,-△-is C
The group treated with hyperthermia by injecting the DDP aqueous solution intravenously,-◎-represents the group treated with hyperthermia, and-○-represents the untreated group. As is apparent from FIG. 2, the CDDP-enclosed SU of the present invention
The antitumor effect was observed in the group treated with hyperthermia by administration of V (-●-), and the effect was clearly larger than that in the group treated with hyperthermia by administration of the CDDP aqueous solution (-△-). It was The average tumor diameter 20 days after the last treatment day, in which all the mice were alive, was-●-
The group was 58% and the -Δ-group was 91%.

[0049]

INDUSTRIAL APPLICABILITY The liposome preparation of the present invention can maintain a high blood concentration due to its small particle size and is excellent in temperature sensitivity. Therefore, it can be used in combination with thermotherapy for cancer and an antitumor agent. It can be used to advantage.

[Brief description of drawings]

FIG. 1 shows the time-dependent change in the release rate of calcein encapsulated in the liposome preparation prepared in Example 3 at each temperature.

FIG. 2 shows the antitumor effect when the liposome preparation encapsulating CDDP of Example 8 was administered to mice in comparison with the control group.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Satoshi Funada 810, Saijo, Oma-gun, Tsumuma-machi, Saitama Prefectural Bank of Ari Pharmaceutical Co., Ltd. Yu Pharmaceutical Co., Ltd. R & D Lab. (72) Inventor Sueaki Ishimaru, Saitama Prefecture, Oma-gun, Tamanuma-cho, 810 Nishijo, Manyu Yu Pharmaceutical Co., Ltd. R & D Lab.

Claims (7)

[Claims] 1. A liposome, which is a small unilamellar vesicle having a membrane phase transition temperature of 40 to 45 ° C. (average particle size of 0.1 μm or less), is 2.0 to 2.0 higher than the biological fluid osmotic pressure of a warm-blooded animal. A liposome preparation containing a 5.0-fold hypertonic drug-containing solution. 2. The preparation according to claim 1, wherein a phospholipid whose acyl group is a saturated acyl group is a main constituent of the liposome membrane. 3. The liposome preparation according to claim 1 or 2, wherein a drug-containing solution having a hypertonicity of 2.5 to 4.5 times is enclosed. 4. The preparation according to claim 1, wherein the drug is an antitumor agent. 5. A small unilamella having a membrane phase transition temperature of 40 to 45 ° C., in which a drug-containing liquid whose osmotic pressure is 2.0 to 5.0 times higher than that of a biological fluid of a warm-blooded animal is enclosed. A method for producing a liposome preparation, which comprises forming liposomes (average particle diameter of 0.1 μm or less) that are vesicles. 6. The method for producing a liposome preparation according to claim 5, wherein the main component is a phospholipid whose acyl group is a saturated acyl group to form a liposome membrane. 7. The method for producing a liposome preparation according to claim 5, wherein the drug is an antitumor agent.