JPH0482839A - Conjugated lipid corpuscle with protein - Google Patents

Abstract

PURPOSE:To obtain the subject conjugated corpuscle having prolonged half-life of protein in blood to effect sufficient exhibition of the activity of the protein by bonding a protein to the surface of a lipid membrane composed of a lipid and a positively charged lipid and/or negatively charged lipid. CONSTITUTION:The objective conjugated corpuscle can be produced by bonding a protein to a surface of a lipid membrane composed of a lipid (e.g. phospholipid, glycolipid and derived lipid) and a positively charged lipid (e.g. a lipid produced by bonding a fatty acid or cholesterol to a 12-22C aliphatic amine or basic amino acid) and/or a negatively charged lipid (e.g. acidic phospholipid). The amount of the positively and/or negatively charged lipid is about 1-1,000mumol per 100mumol of the phospholipid. The combined use of a positively charged lipid and a negatively charged lipid is preferable. The amount of protein to be bonded to the lipid surface is about 0.01-10 pts.wt. based on 100 pts.wt. of lipid used in the formation of the lipid membrane.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a protein/lipid body complex whose purpose is to extend the blood half-life of proteins and fully exhibit their efficacy.

[Conventional technology and its issues]

Proteins have a short half-life in the blood, so they have the problem of not being fully effective. On the other hand, when a protein wN-lipid body complex containing protein fj is made, the blood half-life is extended, but there is a problem that the efficacy is not fully exerted because the proteins are not released from the lipid body. Ta.

An object of the present invention is to provide a protein/lipid body complex that can extend the blood half-life of proteins and exhibit its efficacy.

[Failure to solve the problem]

As a result of various studies in view of the above circumstances, the present inventors found that by binding proteins to the surface of a lipid membrane consisting of lipids and positively charged and/or negatively charged lipids, the amount of proteins in the blood can be halved. We have discovered that it is possible to extend the period and fully demonstrate the effect, and have completed the present invention.

That is, the protein-lipid complex of the present invention is a protein-lipid body characterized by binding proteins to the surface of a lipid membrane consisting of a lipid and a positively charged lipid and/or a negatively charged lipid. It is a complex.

The structure of the protein/lipid body complex will be described in detail below.

(i) Lipid bodies In the present invention, lipid bodies include liposomes, fat emulsions,
Preferably, it is a W10/- type emulsion. In the present invention, lipid bodies (liposomes) have a structure of multiple concentric bilayers, a single layer, or a multilayer, and the lipid membrane surface has the ability to bind various proteins, such as lipids and It is prepared by contacting a lipid membrane formed from positively charged lipids and/or negatively charged lipids with a solution containing proteins, and binding the proteins to the membrane lipids and the membrane surface of the lipid body. .

The lipids for forming the lipid bodies include endoplasmic reticulum (
It is not particularly limited as long as it can produce liposomes, and examples include phospholipids, glycolipids, derived lipids, and fatty acids.

As the phospholipid, any phospholipid that is physiologically acceptable and metabolized can be used in the present invention. For example, phosphatidylcholine, phosphatidylserine, phosphatidic acid, phosphatidylglycerin, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, dicetyl phosphate, lysophosphatidylcholine (lysolecithin), or a mixture thereof such as soybean phospholipid, egg yolk phospholipid, etc. are used. . Among these, the most preferred phospholipid is phospholipid from soybean or egg yolk.

Glycolipids include cerebroside, sulfate
ide) and gangliosides.

Examples of the derived lipid include cholic acid, deoxycholic acid, dehydrocholic acid, and the like.

The positively charged lipid is not particularly limited as long as it is a positively charged lipid. Examples include saturated or unsaturated aliphatic amines having 12 to 22 carbon atoms, basic amino acids bound to fatty acids, cholesterol, etc., and quaternary amines containing fatty acids. Specifically, oleylamine, stearylamine, etc. Illustrated.

The negatively charged lipid is not particularly limited as long as it is a negatively charged lipid. For example, acidic phospholipids such as oleic acid, phosphatidic acid, and phosphatidylserine (ether type is also possible), synthetic phospholipids such as dicetyl phosphoric acid, and synthetic phospholipids with 1 carbon number.
Examples include 4 to 22 saturated or unsaturated higher fatty acids.

The amount of positively charged and/or negatively charged lipid added is phospholipid 100
, about 1 to 1000 μmO1 for I/110+.

The positively charged lipid and/or the negatively charged lipid are used in combination with the phospholipid, and the ratio (molar ratio) is phospholipid:positively charged lipid and/or negatively charged lipid -100:1 to 1:10, preferably 2: It is about 1.

The positively charged lipid and/or the negatively charged lipid may be a positively charged lipid or a negatively charged lipid alone, but a combination of a positively charged lipid and a negatively charged lipid is more preferable.

(11) Proteins The proteins used in the present invention include not only proteins but also polypeptides and peptides. There are no particular restrictions on the protein, but the effects of the present invention are particularly noticeable when the protein has a short half-life in the blood and therefore cannot fully exert its activity. Furthermore, the concept of proteins is not limited to those listed below, but also includes derivatives thereof.

Specifically, as proteins, urokinase (UK)
, urokinase precursor (PPA), globulin (Ig
L interleukins (TL), colony stimulating factor (
csF), erythroboetin (EPO), interferon (IFN), lymphokines, and the like.

The origin of such proteins is not particularly limited, and examples thereof include those prepared by cell culture methods, genetic engineering methods, etc. In addition, proteins are not limited to those mentioned above,
This concept also includes its derivatives.

An example of the amount of protein added is about 0.01 to 10 parts by weight per 100 parts by weight of the lipid used to form the lipid membrane described in (i).

(iii) Structure of lipid bodies The structures of lipid bodies (liposomes) include multilamellar vesicles (MLV), small unilamellar vesicles (SU
V), large lamellar hecicle (LUV), rehearse phase eruption hecicle (REV), and the like.

(1) Method for producing lipid bodies The outline of the method for producing lipid bodies is described below, but this is an example and does not limit the present invention.

The solvent is distilled off from a solution containing a suitable lipid and a positively charged lipid and/or a negatively charged lipid (a solution in a solvent that does not denature lipids, such as chloroform, ethanol, etc.) to form a thin film of lipid, and the solution is applied to this thin film. In addition, the mixture is vigorously shaken and preferably subjected to ultrasonication to uniformly disperse the lipids to prepare a lipid suspension.

The solvent used in the solution may be any solvent that does not denature or decompose lipid bodies and is physiologically acceptable, such as a buffer adjusted to pH 4 to 11, preferably 6 to 9.
For example, citrate buffer, phosphate buffer, acetate buffer,
(physiological saline solution), ethanol, etc.

Proteins to be bound to lipid vesicles may be added after the lipid suspension is prepared.

Note that cholesterol may be added as a stabilizer when preparing the lipid membrane. Preferably, an antioxidant such as tocopherol (vitamin E) is also added to stabilize the phospholipids, preferably in a weight percent of about 0% relative to the phospholipids.
．． 01 to 0.5% (W/W)).

Furthermore, before adding proteins, heat treatment may be performed at 40 to 90° C. for several minutes to one hour. When a urokinase precursor is used as the protein, the amount of the urokinase precursor-containing solution added is 0.0001 to 1 part by weight based on 1 part by weight of phospholipid used to form the lipid membrane.

In addition, the preparation of the present invention also contains monofluorinated F, such as glucose and mannitol.
Disaccharides such as maltose, sucrose, dextran, etc. A polymer substance selected from the group consisting of vinyl polymers, nonionic surfactants, gelatin, and hydroxyethyl starch may be blended as a stabilizer.

The polymer stabilizer may be incorporated into the voids within the lipid body, or may be added to a lipid body preparation to which proteins are bound (i.e., added/blended outside the lipid body). good. Of course, it goes without saying that it may be incorporated both inside and outside the lipid body.

The amount of the stabilizer added is 0.5 to 1 part by weight of lipid.
0 parts by weight, preferably 1 to 5 parts by weight.

In the present invention, liposomes can be prepared using the ultrasonic treatment method [Yatori Shoshichi et al., Liposomes, 27 (1988)], the French press method [Ibid., 30 (1988, Surfactant method]
Ibid., 32 (1988)), extrusion method [(Hope et al., Chemist.
y and Physics of Lipids.

40-102 (1986)], 2-microfluidization method [Talsma et al., Drug development
Lopment and Industrial Ph
armacy, 15 (2) 197-207
(1989N, reverse phase evaporation method (Szoka et al.
, Proc, Natl, Acad, Sci,, 75.
4194 (1978)] Calcium-EDTA chelation method Papahadjopoulos et al.
,, Biochem, Biophys.

Acta, 394.483. (1975)] may be prepared by various methods depending on the purpose.

A protein/lipid body complex having an average diameter in the range of about 0.02 to 0.1 μm can be provided, and if necessary, impurities or free substances can be further removed by molecular sieving or centrifugation. .

The thus obtained protein-lipid body complex has 80% or more of the added amount of protein bound to the lipid body, and its particle size is uniform and extremely small, making it preferable as a medical preparation.

The binding rate of proteins to lipid bodies can be determined by dissolving the prepared lipid bodies in ethanol and performing liquid chromatography (
The values obtained from gel filtration chromatography (gel filtration chromatography) analysis are shown.

Lipid bodies bound to proteins can be recovered as a precipitate. For example, lipid bodies can be recovered by subjecting a medium containing lipid bodies to ultracentrifugation or sucrose density gradient centrifugation.

This product is then washed, if necessary, with a physiologically acceptable aqueous solution and prepared as a pellet or suspension preparation. Formulation follows a widely known method for manufacturing pharmaceuticals. Further, the present complex is provided as a lyophilized preparation by freezing the liquid preparation and then drying it under reduced pressure.

The complex of the present invention is used as an injection or oral preparation for boat fishing. When used, it is generally dissolved or diluted with a physiologically acceptable aqueous solution before use, but it may also be made into tablets, capsules, or enteric fluid capsules depending on the formulation.

〔Effect of the invention〕

Compared to the protein itself, the complex of the present invention can
In particular, it is possible to suppress the disappearance of proteins in the blood, increase the blood concentration, and furthermore, it is possible to control the half-life in the blood.

Therefore, the efficacy of the protein can be enhanced in combination with the original properties of the protein.

[Examples] Examples and experimental examples are given to explain the present invention in more detail, but the present invention is not limited by these in any way.

Example 1 1.5 g of egg yolk lecithin, 282.5 mg of oleic acid and 1.5 mg of DC-α-tocopherol with 0.11
By adding 1.0 mfl of Squirrel buffer and performing ultrasonication while cooling with ice water, the average particle size was reduced to 50 nm.
The following liposomes were prepared. The pH of the liposome solution is 0.
．． p+ (adjusted to 8,0 by adding IM Tris buffer or IN hydrochloric acid. Also, the lecithin sensitivity of the liposome solution is 0.1M) Lis buffer, pII8.
The concentration was adjusted to 100 mg/ml by adding 0.

1000 units (0.5 mf) of urokinase precursor was mixed with the thus prepared liposome 'a, 1.5 aff (referred to as liposome 1).

Example 2 Egg yolk lecithin 1.5g, oleylamine 267.5m
g and DI, -α-tocopherol 0.1 to 15 mg
M) By adding 7.0% of Lith buffer and performing ultrasonic treatment while cooling with ice water, the average particle size was reduced to 50 nm.
The following liposomes were prepared. The pII of the liposome solution was adjusted to pII 8.0 by adding 0.1 portion of Tris buffer or IN hydrochloric acid. In addition, the lecithin concentration of the liposome solution is 0.1M) Lis-HCl buffer, pI
By adding I8.0, it was adjusted to 100 μ/d.

1000 units (0.5 mfl) of urokinase precursor was mixed with 1.5 ml of the liposome solution prepared in this way (
(referred to as liposome 2).

Example 3 7.0 ml of O,I-Tris buffer was added to 1.5 g of egg yolk lecithin, 282.5 μg of oleic acid, 267.5 mg of oleylamine, and 15 mg of DL-α-tocopherol, and the mixture was ultrasonicated while cooling with ice water. By performing the treatment, liposomes with an average particle diameter of 50 nm or less were prepared.

The pII of the liposome solution is 0. ], M) The pII was adjusted to 8.0 by adding Lis-HCl buffer or IN-HCl. In addition, the lecithin concentration of the liposome solution is 0.1
M Tris buffer, pII8. By adding O, the concentration was adjusted to 100 mg/form.

1000 units of urokinase precursor (0.5 mff1) was mixed with 1.5 ml of the liposome solution prepared in this way (
(referred to as liposome 3).

Comparative Example 1 A 0.1M hydrochloric acid buffer (pH 8, approximately 1.0 m, By adding e) and performing ultrasonication while cooling with ice water, liposomes with an average particle size of 50 nm or less were prepared. The pH of the liposome solution was adjusted to ρ118.0 by adding 0.1 M'l-Lis buffer or IN hydrochloric acid. In addition, the lecithin concentration of the liposome solution was reduced to 75 mg by adding 0.1M) Lis-HCl buffer, pII8゜0.
/d (referred to as liposome 4).

Experimental Example I PPA activity of liposomes 1 to 4 was measured. PPA activity was determined by injecting a liposome fluid sample into a gelatin-containing buffer (p
After appropriately diluting PPA to urokinase (H8,4), weigh out a certain amount, add plasmin and heat to activate PPA to urokinase, and then convert the peptide fluorescent substrate (Gl
It was measured using t-Gly-Arg-MCA, V-3097, Peptide Institute). In addition, the same measurement was performed on a sample treated with the surfactant Triton X-100 before adding plasmin.

Table 1 summarizes the PPA activities of liposomes in a system in which the liposomes were not treated with a surfactant and in a system in which the liposomes were treated with a surfactant.

Note that the numerical values are expressed as percentages relative to the PPA activity added to the liposomes.

[Margin below] Liposome PPA PPA activity (%) No 1], 00.5
100.7No 2 100.3
], OO, INo, 3
98.4 102.1 No.
4 32.9 46.4 Experimental Example 2 0.1. m! 0.1M+-Lis-HCl buffer containing 1 and 2.5M sucrose, pl+8.0
0.2 precipitates were mixed, and on top of that, 0.2 precipitates containing 0.5M sucrose were mixed.
4.3 mfi of 1M) Lis-HCl buffer, pH 8.0, was overlaid. This was carried out using an ultracentrifuge at ζ50.00Orpm.
The mixture was centrifuged for G time, fractionated into 5 fractions starting from the lower layer, and the PPA activity and lecithin concentration of each fraction were measured.

Experimental Example 3 A certain amount of human plasma to which 1251-labeled human fibrinogen was added was taken into a test tube, and Ca11 was added. and 1 thrombin was added for coagulation. After thoroughly washing the clot thus prepared with physiological saline, it was suspended in human plasma, and ribomes 1 to 4 and liposomes 1 to 4 as test drugs were separated by sucrose density gradient centrifugation. Five fractions (constant amount of lipids) were added and incubated. Plasma was collected after 5 hours, and the clot lysis rate was calculated from its radioactivity. The results are shown in Table 2.

No, 1 88.3No.
, 2 84.6No, 3
87.5No, 4
44.9No. 5th fraction of 1
67.4 No. 5th fraction of 2 18.4
No. 5 fraction of 3 44.6 No. 4 of 5
10.6 Experimental Example 4 Liposomes 1 to 4 were administered through the tail vein of male Wistar rats weighing 200 to 250 g, blood was collected over time from the carotid artery, and PPA activity in the plasma was measured.

Experimental Example 5 1251-labeled fibrinogen was added to rat plasma, and CaC1□ and human thrombin were added thereto for coagulation. The clot thus prepared was thoroughly washed with physiological saline and then ground in a mortar cooled with liquid nitrogen.

NaI was injected into the tail vein of male Wistar rats weighing 200 to 250 g, and then a 125I-labeled fibrinogen suspension was injected to create a pulmonary embolus. Five minutes after the administration of the 1'I-labeled fibrinogen suspension, liposomes 1 to 4 (300 υ/kg as PPA) were administered as a bolus through the tail vein as test drugs. 55 minutes after drug administration, the rats were sacrificed and the clot lysis rate was calculated from the radioactivity remaining in the lungs. The results are shown in Table 3.

(Margin below) No, 2 NOl 3 No 4 78.5 44.2 61.3 42.3 44.8 57.8 63.4 76.7

[Brief explanation of the drawing]

FIG. 1 is a graph showing the PPA activity and lecithin concentration in each fraction when liposomes 1 to 4 prepared in Examples 1 to 3 and Comparative Example 1 were fractionated by sucrose density gradient centrifugation. be. FIG. 2 is a graph showing the change in blood PPM activity when liposomes 1 to 4 and PPA were intravenously injected to rats. ■ (ILLI/l0LLIn) Entai f%+41 (+uuln) ■ dd plate (+ul+ou+n) 誼＠/neeH<< 口(+111711) Yirou dd 口(ILLI/l0LLIn) Joejo X 壬44 (+LLltn) Satokai dd Book (+uul+ouun) Vice/%+41 (+uuIn) ■ dd

Claims (1)

[Claims] 1. A protein/lipid body complex, characterized in that the protein is bound to the surface of a lipid membrane consisting of a lipid and a positively charged lipid and/or a negatively charged lipid.