JPH0899897A - Interleukin 2 absorbing type lipid globule - Google Patents

Abstract

PURPOSE: To obtain a kit for readily preparing a pharmaceutical preparation obtained by bringing a specific amount of interleukin-2 (IL-2) into contact with a lipid globule in an aqueous medium in high retaining ratio. CONSTITUTION: This kit contains (A) a lipid of a lipid globule, preferably consisting of a liposome or the lipid microsphere and (B) IL-2 in an amount of 1-0.0001 pts.wt. based on 100 pts.wt. of the component A. Furthermore, the lipid in the component A contains preferably a neutral phospholipid as a main component and the lipidincludes distearoyl phosphatidylcholine, dimyristoylphosphatidylcholine, dilignoceroylphosphatidyl-choline, sphingomyelin, egg yolk lecithin or hydrogenated soybean lecithin as a preferred example.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a preparative method for preparing an interleukin-2 adsorbing lipid microsphere preparation by contacting interleukin-2 (IL-2) with a lipid microsphere in an aqueous medium. The present invention relates to a kit, a method for preparing an IL-2 adsorbing lipid microsphere preparation by the kit, and an IL-2 adsorbing lipid microsphere preparation prepared thereby.

[0002]

BACKGROUND OF THE INVENTION Interleukin-2 (IL-2) is
It is a glycoprotein molecule with a molecular weight of about 15,000 derived from T cells, including T cells, B cells, natural killer (NK) cells,
It is known to have various actions such as promoting proliferation, differentiation or functional activation of monocytes, macrophages and the like. At present, IL-2 is mass-produced using genetic engineering, and clinical application such as antitumor immunotherapy is performed. However, there is a problem that the systemic administration of the high dose causes serious side effects.

In order to solve this problem, IL-2 is used for the purpose of reducing systemic toxicity by changing the distribution and absorption of IL-2 such as sustained release to prolong the half-life in blood. A method of encapsulating the liposome membrane has been devised. Anderson et al.
As a method of encapsulating syrup, a method of repeating freeze-thawing and sonication several times has been disclosed, but these steps are not suitable for industrialization because they are complicated (Cancer Res., 50 , 1853-18
56 (1990)). Further, Bergers et al. Have examined the charge of liposome membrane lipid and the pH of a preparation solution as a method for increasing the encapsulation rate of IL-2.
Only 81% (Pharm. Res., 10 (12), 1715-1721 (199
3)).

In addition to the above-mentioned encapsulation type liposome preparation, a method for producing an adsorption type liposome preparation in which a charged liposome is prepared and then adsorbed with an oppositely charged drug has been reported (JP-A-4-82839, And JP-A-4-103527). In these adsorptive liposome preparations, the type of drug to be applied is limited to the oppositely charged drug, and thus the type and preparation method of the membrane lipid are limited.

[0005]

The present invention is to solve the above-mentioned conventional problems, and an object thereof is a kit for easily preparing an IL-2 adsorbing lipid microsphere preparation at a high retention rate, It is intended to provide an IL-2 adsorbing lipid microsphere preparation produced by the method and a method for producing the preparation.

[0006]

As a result of various studies, the present inventors have found that lipid globules and interleukin-2
It has been found that an IL-2 adsorbing lipid microsphere preparation having a high retention rate can be prepared by contacting (IL-2) with an aqueous medium regardless of charge.

The preparation of the interleukin-2 adsorbing lipid microsphere preparation of the present invention comprises an interleukin-2 adsorbing lipid microsphere preparation by the step of contacting interleukin-2 with a lipid microsphere in an aqueous medium. For preparation, it contains interleukin-2 and lipid globules, and interleukin-2 is contained in a ratio of 1 to 0.0001 parts by weight to 100 parts by weight of lipids in the lipid globules.

According to a preferred embodiment, the lipid microspheres are liposomes or lipid microspheres.

According to a preferred embodiment, the main component of the lipid of the above-mentioned lipid microsphere is a neutral phospholipid.

According to a preferred embodiment, the neutral phospholipid is synthetic or natural phosphatidylcholine, sphingomyelin, or a mixture thereof.

According to a preferred embodiment, the neutral phospholipids are distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, dibehenoylphosphatidylcholine,
It is at least one lipid selected from the group consisting of dilignocelloylphosphatidylcholine, sphingomyelin, egg yolk lecithin, soybean lecithin, hydrogenated egg yolk lecithin, and hydrogenated soybean lecithin.

According to a preferred embodiment, the phosphatidylcholine is a synthetic phosphatidylcholine.

According to a preferred embodiment, the lipid microspheres contain charged lipids.

According to a preferred embodiment, the lipid microspheres consist of uncharged lipids.

According to a preferred embodiment, the lipid microspheres are charged or uncharged with the same polarity as interleukin-2.

According to a preferred embodiment, at least one of the lipid microspheres and interleukin-2 is uncharged.

According to a preferred embodiment, a drug other than interleukin-2 is encapsulated, distributed or adsorbed in the lipid microspheres.

According to a preferred embodiment, the average particle size of the lipid globules is 500 nm or less.

According to a preferred embodiment, the pH of the aqueous medium is about 3-9.

According to a preferred embodiment, the pH of the aqueous medium is near the isoelectric point of interleukin-2.

According to a preferred embodiment, the kit comprises the lipid globules and / or interleukin-
2 lyophilisate.

According to a preferred embodiment, the kit further comprises an aqueous medium.

According to a preferred embodiment, the kit contains an aqueous dispersion of lipid globules and a freeze-dried aqueous solution of interleukin-2.

The method for preparing the interleukin-2 adsorbing lipid microsphere preparation of the present invention includes the step of contacting interleukin-2 with the lipid microsphere in an aqueous medium.

In a preferred embodiment, the above method uses the kit described in any of the above. The interleukin-2 adsorbing lipid microsphere preparation of the present invention is preferably prepared by the method described in any of the above.

According to a preferred embodiment, the above formulation comprises
In gel filtration, the amount of adsorbed and retained interleukin-2 on the lipid globules is 80 to 100%.

According to a preferred embodiment, the above formulation comprises
The lipid globule contains 100 parts by weight of lipid and 1 to 0.0001 parts by weight of interleukin-2.

According to a preferred embodiment, the above formulation comprises
It is freeze-dried.

[0029]

BEST MODE FOR CARRYING OUT THE INVENTION The kit for preparing an IL-2 adsorbing lipid microsphere preparation of the present invention may have a configuration in which at least IL-2 and lipid microspheres are combined as one administration system. The form of the combination does not matter. That is, the definition also includes the equivalent of a combination product. A medical device such as a syringe may be further set. The lipid microspheres are at least one of liposomes and lipid microspheres. IL-
Biadsorbent lipid globule formulations can be prepared by contacting IL-2 in the kit with lipid globule in an aqueous medium. The step for contacting IL-2 with the lipid globules is not particularly limited. For example, the kit of the present invention may be IL-
2 and lipid globules are mixed and dried (eg freeze-dried)
When the dried product is contained, the dried product can be regenerated by adding an aqueous medium at the time of use to prepare an IL-2 adsorptive lipid microsphere preparation. When the kit of the present invention contains, for example, IL-2 and lipid globules separately, both of them are dried products, or one of them is a dried product and the other is an aqueous solution or dispersion, at the time of use, It can be prepared by adding an aqueous medium as necessary and mixing them. In these cases, the kit of the present invention may include an aqueous medium adjusted to an appropriate pH. The kit of the present invention may contain a lipid globule dispersion and an IL-2 aqueous solution. Further, it also includes a kit produced by preparing an aqueous medium containing lipid microspheres and IL-2, once forming an IL-2 adsorbing lipid microsphere preparation, and then freeze-drying. With such a kit, the adsorbable lipid microsphere preparation can be easily regenerated by simply returning it to an aqueous medium. The lipid microspheres contained in the kit for preparing the IL-2 adsorbing lipid microsphere preparation of the present invention may be prepared as an aqueous dispersion as described below, or may be a freeze-dried preparation or a spray prepared by a usual method. It may be prepared as a dry preparation.

The lipid microspheres used in the present invention include liposomes and lipid microspheres as described above. The particle size of the liposome is about 20 nm to several μ.
m, preferably about 500 nm or less, more preferably about 200 nm or less. In addition, the lipid microspheres refer to small fat particles obtained by suspending vegetable oil such as soybean oil in water together with phospholipids (such as lecithin). Generally, the average particle size of lipid microspheres is about 200 nm.

As the main component of the phospholipid of the liposome or lipid microsphere used in the present invention, a neutral phospholipid can be used. The neutral phospholipid is not particularly limited as long as it is a lipid having no charge in the neutral region as a whole molecule. For example, synthetic or natural phosphatidylcholine, sphingomyelin, etc., or mixtures thereof may be used. The natural phosphatidylcholine may also include various lecithins, and may be unsaturated or saturated (hydrogenated). For example, distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, dibehenoylphosphatidylcholine, dilignocelloylphosphatidylcholine, sphingomyelin, egg yolk lecithin, soybean lecithin, hydrogenated egg yolk lecithin, hydrogenated soybean lecithin, and the like. 1 selected from the group
One kind or two or more kinds of lipids can be used, but one having a high phase transition temperature is preferable. When using two or more lipids,
The mixing ratio is not particularly limited.

The phospholipids of liposomes or lipid microspheres used in the preparation of the IL-2 adsorbing lipid microsphere preparation of the present invention are mainly composed of the above-mentioned uncharged lipids, and if necessary, may contain charged lipids. It The positively charged lipid is not particularly limited as long as it is a positively charged substance having no toxicity. For example, stearylamine, basic lipids such as oleylamine, N ^a - such as basic amino acid-based surfactants such as acyl -L- arginine. The negatively charged lipid is not particularly limited as long as it is a negatively charged substance having no toxicity. For example, phosphatidylserine, naturally occurring phosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, cardiolipin, phosphatidylinositol, acidic phospholipids such as phosphatidic acid, ganglioside GM ₁ , ganglioside GM _{3, and the} like sialic acid. And acidic lipids such as dicetyl phosphate, acidic amino acid surfactants such as N-acyl-L-glutamic acid, and fatty acids such as oleic acid and stearic acid.

When the above lipid globules contain charged lipids, for example, the total weight of lipids used in the lipid globules is 100%.
The content is 0 to 100%, preferably 0 to about 30%.

The lipid microspheres used in the preparation of the IL-2 adsorbing lipid microsphere preparation of the present invention, which are mainly used in combination with the above phospholipids, include vegetable oils (eg, soybean oil, sesame oil, Safflower oil) or synthetic or semi-synthetic medium chain fatty acid triglycerides (eg Miglyol, ODO®) may be used. The ratio of the phospholipid to the vegetable oil or the medium chain fatty acid triglyceride is 5 to 50 parts by weight, preferably 10 to 25 parts by weight, based on 100 parts by weight of the vegetable oil or the medium chain fatty acid triglyceride.

As the lipid component of the above lipid globules, in addition to the above various phospholipids and charged lipids, phosphatidylethanolamine, sterols such as cholesterol as a membrane stabilizer, and α-tocopherol as an antioxidant. It may be added. The addition ratio of such an additional substance is, for example, 0 to 30 for phosphatidylethanolamine when the total lipid is 100 parts by weight.
Parts by weight, sterols used as a stabilizer are 0 to
100 parts by weight, preferably 20 to 55 parts by weight, and 0 to 20 parts by weight, preferably about 1 part by weight, for α-tocopherol and the like used as an antioxidant.

The method for preparing the lipid microspheres used in the present invention is not particularly limited and can be prepared by various known methods.
Examples of the method for preparing liposomes include ultrasonic preparation method, vortex method, surfactant method, ethanol injection method, ether injection method, French press method, and reverse phase evaporation method. The structures of liposomes obtained by these preparation methods are multilamellar liposomes (MLV),
Examples include small unilamellar vesicles (SUV), large unilamellar vesicles (LUV), reverse phase liposomes (REV) and the like. On the other hand, in the case of preparation of lipid microspheres, unlike the case of preparation of liposomes, vegetable oil such as soybean oil and sesame oil, or synthetic or semi-synthetic medium chain fatty acid triglyceride is used in addition to phospholipid. The preparation method is not particularly limited, for example, vegetable oil or medium chain fatty acid triglyceride 2 to 20% to water,
Then, the lipid microspheres can be prepared by applying the phospholipid to which 12% is added to a high pressure emulsifying machine. The particle size of the lipid microspheres used in the present invention is not particularly limited, but generally 20 on average.
It is 0 nm. Further, the surface of these lipid globules is, for example, galactose, mannose, lactose, a monoclonal antibody, a molecule having tissue affinity or organ affinity such as amino acid, or, for example, polyethylene glycol, pullulan, mannan, polyamino acid, etc. It may be chemically or physically modified with the hydrophilic polymer of.

The lipid microspheres used in the present invention include drugs other than IL-2 or physiologically active substances such as 5-fluorouracil, doxorubicin, γ-interferon,
It may be one in which mitomycin C, a platinum complex, cyclophosphamide, etc. are included, distributed, or adsorbed.

The pH of the aqueous medium used in the present invention is usually about 3 to 9, preferably 6 to 9, from the stability of lipid globules, and more preferably about the isoelectric point of IL-2. 7-8. The acid that can be used to adjust the pH is an inorganic acid such as hydrochloric acid, nitric acid or hydrobromic acid, or an organic acid such as lactic acid, glyceric acid, acetic acid or citric acid, preferably hydrochloric acid, acetic acid or lactic acid. As the base, potassium hydroxide, sodium hydroxide, monovalent hydroxide such as lithium hydroxide, or triethylamine,
Examples of amines such as trimethylamine, diisopropanolamine, diethanolamine, triethanolamine, tetramethylamine, and trisaminomethane are preferably potassium hydroxide and sodium hydroxide. Sodium diphosphate, sodium carbonate and the like can also be used. The ion concentration of the aqueous medium is, for example, 0 to 500 m.
It is M.

The origin of IL-2 used in the present invention may be any origin, but in general, commercially available recombinant IL-2, preferably recombinant human IL.
-2 is used. IL-2 is mixed at the time of preparation at a ratio of 1 to 0.0001 parts by weight, preferably 0.1 to 0.001 parts by weight, based on 100 parts by weight of lipids in the lipid microspheres (I
L-2 1mg = 10 ⁷ national standard unit (JRU) conversion). Alternatively, the IL-2 / lipid microsphere mixing ratio (JRU / nmole) is 1
0.0-0.01, preferably 5.0-0.05, more preferably 1.
It is 0 to 0.1.

In the aqueous medium used in the present invention, as a stabilizer, sugars (eg, monosaccharides such as glucose and galactose, disaccharides such as maltose and sucrose, polysaccharides such as dextran), glycerol, mannitol, sorbitol, Additives such as polyols such as xylitol, proteins (eg, human serum albumin), water-soluble polymers (eg, gelatin, hydroxyethyl starch, etc.), surfactants and the like may be contained.

In the IL-2 adsorbing lipid microsphere preparation of the present invention, (1) gel filtration of a mixed system of IL-2 and lipid microspheres is performed as described in Examples 1 to 11 below. , The majority of IL-2, preferably 8 in the eluate fraction of lipid globule, which is different from the eluate fraction in the case of IL-2 alone.
Since 0 to 100%, and more preferably 90 to 100% are eluted together, (2) the persistence of the blood level of IL-2 is demonstrated as described in Examples 15 and 16. Therefore, the lipid globules and IL-2 are not simply mixed in the aqueous medium, but the lipid globules are not mixed with IL-.
2 was found to be in the supported or adsorbed form.
In the IL-2 adsorbing lipid microsphere preparation of the present invention, IL-2
Are not encapsulated in the lipid globules but are adsorbed on their outer surface.

[0042]

The present invention will be described in more detail by the following examples.

Example 1 5 ml of a chloroform solution containing 100 mg of distearoylphosphatidylcholine (DSPC), which is a neutral phospholipid, was placed in a 300 ml eggplant-shaped flask.
The solvent was distilled off under reduced pressure using a rotary evaporator to form a thin film of lipid (DSPC) on the inner wall of the eggplant-shaped flask. This was further transferred to a desiccator, and the residual solvent was removed under reduced pressure overnight. Next, 2 ml of phosphate buffered saline was added to this lipid film, and the mixture was shaken by hand on a hot water bath at 60 ° C. to emulsify, to obtain a crude dispersion liquid of liposomes (MLV). Next, nu with a pore size of 0.4 μm
The crude liposome dispersion was subjected to pressure filtration using an extruder (Lipex Biomembranes, Inc.) equipped with a clepore membrane (Nucrepore Corporation). Furthermore, this filtrate was added to a Nuclepore membrane (Nucr
By pressure filtration using epore Corporation), a DSPC-liposome dispersion liquid having a particle diameter of about 100 nm was obtained. Then, the particle diameter thus obtained is about 100 nm.
DSPC-liposome dispersion (lipid concentration: 50 μmol / ml) 1 m
For l, recombinant interleukin-2 (I
L-2) 50 mM acetic acid aqueous solution (8.4 million domestic standard units (JRU) / m
l) 42 μl was added and gel filtration was performed using Sephadex G-75 (Pharmaci
a, column: diameter x length = 1.0 cm x 25 cm, developing solvent: 1% BS
Phosphate buffered saline containing A, sample: 0.1 ml) was performed. IL-2 contained in each of the fractions collected was analyzed by enzyme-linked immunosorbent assay (EIA) (Mori et al., Basic and Clinical, 22 (17), 5955 (19).
88)), and the ratio of IL-2 adsorbed on the liposome (retention rate) was determined from the content of IL-2 contained in the liposome fraction. The results are shown in Table 1 together with the results of other examples. Free IL-2 not retained in the liposome was separated and eluted in another fraction.

(Example 2) 42 μl of an aqueous solution of IL-2 in acetic acid
Was the same as in Example 1 except that 21 μl was used instead of.

Example 3 Instead of DSPC 100 mg, DSPC was used.
Same as Example 1 except that a mixture of 100 mg and negatively charged lipid distearoylphosphatidylglycerol (DSPG) 10 mg was used.

(Example 4) 42 μl of an aqueous solution of IL-2 in acetic acid
The same as Example 3 except that 1.2 μl was used instead of.

Example 5 Instead of DSPC, neutral phospholipid dipalmitoylphosphatidylcholine (DPPC)
The same as Example 1 except that was used.

(Example 6) 42 μl of an aqueous solution of IL-2 in acetic acid
The same as Example 5 except that 1.2 μl was used instead of.

Example 7 Instead of DPPC 100 mg, DPPC
Same as Example 5 except that a mixture of 100 mg and negatively charged lipid dipalmitoylphosphatidylglycerol (DPPG) 10 mg was used.

Example 8 42 μl of an aqueous solution of IL-2 in acetic acid
The same as Example 7 except that 1.2 μl was used instead of.

[0051]

[Table 1]

(Embodiment 9) In the same manner as in Embodiment 1, DS
Liposomes consisting of PC and DSPG (molar ratio 10: 1) (ML
V) A crude dispersion liquid (lipid concentration: 50 μmol / ml) was prepared, and 20 ml of this preparation liquid was applied to a high pressure emulsifier (Nanomizer LA-10).
H: SAYAMA TRADING CO. LTD.) To obtain fine particles having an average particle size of about 20 nm. With 5 ml of this micronized liposome dispersion, Immunase Note 35 (350,000 domestic standard units (JRU) /
2.5 vials (Shionogi Pharmaceutical Co., Ltd.) were dissolved, and a part thereof was subjected to gel filtration by the method described in Example 1. As a result, the IL-2 retention rate of the liposome was calculated to be 97.6% from the content of IL-2 contained in the liposome fraction.

The results of Examples 1 to 9 are all results when the lipid concentration was kept constant, and by appropriate selection of the lipid composition or the mixing ratio of lipid and IL-2,
It shows that an IL-2 adsorbing liposome preparation having a retention rate of 90% or more can be obtained. For all lipid compositions, the lower the mixing ratio of IL-2 to lipid, the higher the adsorption retention rate. It also shows a high retention rate with or without charged lipids, and this experiment shows that IL-
Since it was performed at pH near the isoelectric point of 2 (about 7.4),
It is considered that the main cause of this high retention is adsorption due to the hydrophobic interaction between IL-2 and the liposome.

Example 10 IL-2 Concentration (4.2 × 10 ⁵ JR
U / ml) and lipid composition (DPPC / DPPG = 10/1) were kept constant, and the mixing ratio of IL-2 and liposome was changed, and the same procedure as in Example 1 was performed. The elution profile of IL-2 by gel filtration with Sephadex G-75 is shown in FIG. 1, and the retention rate of IL-2 calculated from the results of gel filtration is shown in Table 2.
From these results, it was found that, as with the results of Examples 1 to 9 described above, the lower the mixing ratio of IL-2 to lipid, the higher the adsorption retention rate of IL-2.

[0055]

[Table 2]

Example 11 Egg yolk PC / DPPG = 3 in lipid composition
/ 1, DSPC / DPPG = 10/1, and DPPC / DPPG = 10/1 IL−
2 Adsorption-type liposome dispersion (IL-2 concentration: 4 × 10 ⁵ JRU /
ml, lipid concentration: 47 μmol / ml) were respectively obtained in the same manner as in Example 1 and subjected to gel filtration with Sephadex G-75. Figure 2 shows the elution profile of IL-2 by gel filtration.
Table 3 shows the retention rate of IL-2 calculated from the results of gel filtration. From this result, it was found that the synthetic phosphatidylcholine has stronger IL-2 adsorption force than the natural phosphatidylcholine.

[0057]

[Table 3]

(Example 12) A crude dispersion of liposomes was prepared as follows.
Various pore sizes (1.0 μm, 0.4 μm, 0.2 μm, and 0.1 μm)
Of nucleopore membrane (Nucrepore Corporation) under pressure filtration to obtain particles of 1000 nm, 400 nm, 200 nm,
And 100 nm liposomes, 30 nm liposomes are
Example 3 is the same as Example 3 except that it was prepared by the high-pressure emulsifying machine used in Example 9. The lipid concentration is 50 μmole / ml, but only when the liposome particle size is 30 nm, 30 μmole / m
I went with l. The results are shown in Table 4. Particle size of liposome is 1
In the case of 000 nm, the retention rate of IL-2 was slightly low, but in all other cases, the retention rate was 90% or more.

[0059]

[Table 4]

(Embodiment 13) From the results of the above embodiment, I
It is considered that the adsorption power of IL-2 can be increased by decreasing the mixing ratio of L-2 to lipid and decreasing the particle size. Therefore, the relationship between the mixing ratio and the particle size was examined in the same manner as in Example 9 when a commercially available IL-2 preparation, Immunase, was used. However, the particle size is 30
The lipid concentration in the case of nm was 30 μmole / ml. The results are shown in Table 5. It was shown that an IL-2 adsorbing liposome preparation having a retention rate of 90% or more can be obtained by appropriately selecting the mixing ratio of IL-2 and lipid and the particle size of the liposome. This is Imuneth 35 (3.5 × 10 ⁵ JR
IL-2 was prepared by dissolving 1 vial of U) in 1 to 2 ml of liposome dispersion liquid (lipid concentration: 50 μmole / ml).
It is shown that an adsorption type liposome preparation can be easily obtained.

[0061]

[Table 5]

(Example 14) DSPC 216 mg, DSPG 24 mg,
Mix 500 mg of glycerin and 20 ml of distilled water for injection,
A homogenizer (Polytron (registered trademark)) was dispersed, 2 g of soybean oil was added, and the mixture was further treated with a homogenizer to obtain a crude emulsion. This is a high pressure emulsifier (Nanomizer LA-10H: SAYAMA TRADING CO. LT
D.) for pulverization to obtain lipid microspheres having an average particle size of about 200 nm. To 120 μl of this Lipid Microsphere, add 120 μl of Immunase Injection dissolved in 1 ml of distilled water for injection / vial. About 200 μl of this Sephad
Gel filtration was performed with ex G-75. The conditions and the calculation method of the retention rate at this time are the same as those in the first embodiment. As a result, IL
The retention rate of -2 was 89%.

Example 15 An IL-2 adsorptive liposome preparation having a lipid composition DSPC / DSPG = 10/1 obtained by the same method as in Example 3 (average particle size: about 100 nm, IL-2 concentration) : 9.1 × 10 ⁴ JRU / ml, lipid concentration: 50 μmol / ml) 0.2 ml
Was subcutaneously administered to the back of a mouse (Jcl-BDF1, 4 weeks old, about 20 g), and blood was collected over time. The blood level of IL-2 was measured by EIA. In parallel with this, IL-2 alone (1.9 × 10 ⁵ JRU / ml, 0.2 ml) was administered to
The changes in the blood concentration of The result is shown in FIG. I
In the case of L-2 alone (● in the figure), the blood concentration rises quickly and disappears rapidly, whereas in the case of the IL-2 adsorbing liposome preparation (○ in the figure), the blood concentration is high. The sustainability of

Example 16 The lipid composition is DSPC / DPPG = 10 /
The liposome of 1 (average particle size: about 100 nm) was prepared in the same manner as in Example 3 except that DPPG was used instead of DSPG. A mixed solution of IL-2 and liposome (IL-2: 7.
0.2 ml of 5 × 10 ⁴ JRU, lipid concentration: 50 μmol / ml) was administered to the tail vein of mice (slc-BDF1, 4 weeks old, about 20 g), and the concentration of IL-2 in each organ after administration Was measured by EIA.

FIG. 4 shows the results of comparison of changes in blood levels of IL-2 in the administration of the IL-2 / liposome mixture or IL-2 alone (IL-2: 0.955 × 10 ⁴ JRU / mouse). IL by mixing IL-2 and liposome
When administered as a -2 adsorbable liposome preparation (○ in the figure), the area under the blood concentration-time curve (AUC) is increased as compared with the administration of IL-2 alone (● in the figure),
The blood concentration was persistent. FIG. 5 shows the results of comparison of AUC up to 4 hours after the administration of IL-2 in each organ when the IL-2 / liposome mixture or IL-2 alone was administered. Under this administration condition, the IL-2 / liposome mixture was found to have a high distribution in the liver and spleen. In particular, accumulation in the spleen was remarkable. It has been reported so far that a high dose of IL-2 is effective for renal cancer and melanoma cancer patients, but there has been a problem of side effects due to the high dose of IL-2. However, I of the present invention
The characteristics of the L-2 adsorbable liposome preparation, such as increased AUC, sustained blood concentration, and accumulation in specific organs, indicate the possibility of lowering the dose of IL-2 and reducing side effects.
It also shows the possibility of expanding the application of item 2.

Example 17 Lipid composition (DSPC / DSPG = 10 /
1), liposomes having an average particle size of about 20 nm were prepared as an aqueous dispersion. Separately, a lyophilized product of IL-2 was prepared as a vial and set as a liposome dispersion liquid to prepare IL-
A kit for preparing 2 adsorbable liposomes was manufactured. IL-
2 is 0.01 per 100 parts by weight of lipid in the liposome
Includes parts by weight. If IL-2 contained in this kit was dissolved in the liposome dispersion liquid as in Example 9, IL-
A two-adsorption lipid microsphere formulation is prepared.

[0067]

EFFECT OF THE INVENTION By using the kit of the present invention, an IL-2 adsorbing lipid microsphere preparation having a high retention rate can be prepared only by contacting lipid microspheres with IL-2 in an aqueous medium. In particular, the method using the kit of the present invention can obtain an IL-2 adsorbing lipid microsphere preparation simply by mixing, regardless of the charge of the lipid microspheres, and therefore, the method is significantly superior to conventional methods. It's simple. Further, since it can be easily formulated into a preparation, it can be prepared at the time of use and is excellent in the stability of the preparation. The obtained IL-2 adsorbing lipid microsphere preparation was
Since it is sustained-released as compared with the case of administration of L-2 alone, the blood concentration half-life of IL-2 can be prolonged, the drug effect of IL-2 can be sustained, and side effects can be reduced. Furthermore, by intravenously or intraarterially administering the preparation of the present invention, selective delivery to a target tissue is possible, and IL-2 can work locally and effectively.

[Brief description of drawings]

FIG. 1 shows the elution profile of an IL-2 adsorbing lipid microsphere preparation of the present invention or IL-2 alone by gel filtration on Sephadex G-75.

FIG. 2 shows the elution profile of an IL-2 adsorbing lipid microsphere preparation of the present invention or IL-2 alone by gel filtration on Sephadex G-75.

FIG. 3 is a graph showing changes in blood levels of IL-2 when the IL-2 adsorbing lipid microsphere preparation of the present invention or IL-2 alone is subcutaneously administered to mice.

FIG. 4 shows the IL when the IL-2 adsorbing liposome preparation of the present invention or IL-2 alone is administered to the tail vein of mice.
It is a figure which shows the transition of the blood concentration of -2.

FIG. 5 is a graph showing AUC ratios up to 4 hours after administration of IL-2 in each organ when the IL-2 adsorbing liposome preparation of the present invention or IL-2 alone is administered to the tail vein of mice. .

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI Technical indication location // C07K 14/55 8318-4H (72) Inventor Koichiro Hirano 3 Matsuodai Inagawa, Kawabe-gun, Hyogo Prefecture -2-5 (72) Inventor Watsaku Kawakami 3-1-76-306 Asahigaoka, Kashiwara City, Osaka Prefecture

Claims (24)

[Claims] 1. Interleukin-2 by the step of contacting interleukin-2 with lipid globules in an aqueous medium.
An interleukin-2 and a lipid microsphere for preparing an adsorbable lipid microsphere preparation,
2 is 1 to 0.0 with respect to 100 parts by weight of lipid in the lipid globule.
Interleukin-containing at a ratio of 001 parts by weight
2 Kit for preparation of adsorbable lipid microsphere preparation. 2. The kit according to claim 1, wherein the lipid microspheres are liposomes or lipid microspheres. 3. The kit according to claim 1, wherein the main component of the lipid of the lipid globule is a neutral phospholipid. 4. The kit according to claim 3, wherein the neutral phospholipid is synthetic or natural phosphatidylcholine, sphingomyelin, or a mixture thereof. 5. The neutral phospholipid is distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, dibehenoylphosphatidylcholine, dilignocelloylphosphatidylcholine, sphingomyelin, egg yolk lecithin, soybean lecithin, hydrogenated egg yolk lecithin, and The kit according to claim 3, which is at least one lipid selected from the group consisting of hydrogenated soybean lecithin. 6. The kit according to claim 4 or 5, wherein the phosphatidylcholine is a synthetic phosphatidylcholine. 7. The kit of claim 1, wherein the lipid microspheres contain charged lipids. 8. The lipid globule comprises an uncharged lipid.
The kit according to claim 1. 9. The lipid microspheres are interleukin-
The kit according to claim 1, which is charged or uncharged with the same polarity as 2. 10. The kit according to claim 1, wherein at least one of the lipid globules and interleukin-2 is uncharged. 11. Interleukin-on the lipid globule
The kit according to claim 1, wherein a drug other than 2 is included, distributed, or adsorbed. 12. The average particle size of the lipid globules is 500.
The kit according to claim 1, which is less than or equal to nm. 13. The kit of claim 1, wherein the pH of the aqueous medium is about 3-9. 14. The kit according to claim 1, wherein the pH of the aqueous medium is near the isoelectric point of interleukin-2. 15. The kit according to claim 1, which comprises a freeze-dried product of the lipid microspheres and / or interleukin-2. 16. The method of claim 1, further comprising the aqueous medium.
The kit described in. 17. The kit according to claim 1, which contains an aqueous dispersion of lipid globules and lyophilized interleukin-2. 18. A method for preparing an interleukin-2 adsorbing lipid globule preparation, which comprises a step of contacting interleukin-2 with a lipid globule in an aqueous medium. 19. The method according to claim 18, wherein the kit according to any one of claims 1 to 17 is used. 20. An interleukin-2 adsorbing lipid microsphere preparation. 21. The formulation of claim 20, prepared by the method of claim 18 or 19. 22. The preparation according to claim 20, wherein the amount of adsorbed and retained interleukin-2 on the lipid globules in gel filtration is 80 to 100%. 23. The preparation according to claim 20, which comprises 100 parts by weight of lipids in lipid microspheres and 1 to 0.0001 parts by weight of interleukin-2. 24. The formulation of claim 20, which is lyophilized.