JPH0348614A - Epithelial cell growth factor-bound liposome - Google Patents

Abstract

PURPOSE:To obtain a liposome, selectively incorporating a large amount of a drug into cells excessively expressing an epithelial cell growth factor(EGF) receptor and enabling excellent targeting therapy of the drug by binding the EGF to the surface thereof. CONSTITUTION:An EGF-bound liposome, obtained by using a constituent material of a liposome and a substance prepared by binding the aforementioned material to a crosslinking agent, preparing a liposome, reacting the resultant liposome with an EGF or reactive derivative thereof and containing the EGF bound to the surface thereof. Various drugs (e.g. anti-inflammatory agent, anticaner agent or antibiotic substance) can be dissolved in a mother liquor in preparing the liposome to afford an excellent targeting therapeutic agent.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to liposomes that serve as excellent targeting carriers and are useful for cancer treatment and the like.

[Conventional technology]

Nowadays, the ability to impart target-directedness to drugs,
That is, targeting is a drug delivery system (
DO3> is the most important issue in research.

Known methods for targeting drugs include molecular modification using synthetic chemical methods such as prodrugs, and pharmaceutical methods using particulate carriers such as emulsions, microspheres, or liposomes. .

Among these, liposomes are capable of 1) incorporating water-soluble drugs into their inner aqueous layer and lipid-soluble drugs into their lipid membranes; 2)
) when administered intravenously, liver mallet, ell! 1) It is easily distributed in the reticuloendothelial system such as the lungs, 3) It can prevent the decomposition and deactivation of drugs and reduce toxicity by 1 Irfa, and 4) It has low immunogenicity and can be decomposed in vivo. It has unique characteristics and is expected to have a variety of applications. In addition, recently active targeting, in which the surface of liposomes is modified with physiologically active substances such as antibodies, glycoproteins, glycolipids, and lectins, and attempts to actively reach specific target sites by utilizing these physiological activities, has been developed. There are many attempts being made. Especially in the field of cancer treatment, many studies have been reported on liposomes modified with antibodies against specific antigens.

[Problem to be solved by the invention]

However, there are many problems such as antigen expression differing even in the same tissue and the number of antigen expression per cell being small, and the reality is that sufficient targeting effects are not achieved.

[Means to solve the problem]

In view of the above-mentioned circumstances, the present inventors conducted intensive studies with the aim of obtaining liposomes with more efficient targeting properties, and found that liposomes with epidermal growth factor (8Gl') bound to their surface were superior. The present inventors have discovered that it can be used as a targeting carrier, and have completed the present invention.

That is, the present invention provides a liposome having epidermal growth factor bound to its surface.

The liposome of the present invention is produced by binding BGF to the surface of the liposome by means known per se.

As a method for binding HGF to the surface of liposomes,
Any method can be used as long as it can bind BG+' without losing its affinity for the BGF receptor. A preferred method is to prepare liposomes using 136F and react with 136F or a reactive derivative thereof.

CFG can be found in the urine of humans, horses, rabbits, etc.
A polypeptide isolated from rat and mouse submandibular glands and known to exist in mammals across species (8dv, Metab, Dis, 2003).

8, 265 (1975), Japanese Unexamined Patent Publication No. 56-25112, etc.). In the present invention, the oral GF is not limited to those naturally occurring, but also has any substitution, addition or deletion of amino acids constituting them, and the above-mentioned IEGI'
So-called fragments and derivatives that are different from BGF receptor but have similar affinity to BGF receptor [for example, human F! ,
Among the 53 amino acid residues constituting GF (t+BGf'),
Leucine was substituted for the 21st methionine from the N-terminus ((Leu”) hBGF: JP-A-602
8994), etc.].

Methods for preparing 06F include, for example, methods for isolating it from biological components (JP-A-58-99418, JP-A-58-2191).
No. 24, Publication No. 59-204123, etc., Special Publication No. 441
No. 2744, No. 53-4527, No. 59-50315
No. 5950316, No. 59-42650, etc.), chemical synthesis methods (Japanese Patent Laid-Open No. 59-27858, etc.), and genetic engineering methods (Japanese Patent Laid-open No. 57-122096, No. 58-216697,
59-132892, etc.).

As for the constituent materials of liposomes, all the constituent materials normally used in the production of existing liposomes can be used, such as natural lecithin, dipalmitoylphosphatidylethanolamine (DPP), dipalmitoylphosphatidylinositol (DPPI), etc. , dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine ([1uPC)
, cystearoylphosphatidylcholine (O8PC)
Examples include one or more types of phospholipids such as, or a mixture of these and cholesterols.

Examples of the crosslinking agent include monovalent crosslinking agents, divalent crosslinking agents, and other known crosslinking agents, but preferred crosslinking agents include:
5PDP [:N-succimidyl-3-(2-pyridyldithio)propionate], SMP [N-succimidyl-4-(p-maleimidophenyl)butyrate], [
! MC8CN-maleimidocaproyloxy)succimide] and the like.

In addition, as a liposome preparation method, all known liposome preparation methods such as port swing method, surfactant removal method, and reversed phase evaporation method (1 RU V method) can be mentioned.

DPPB as a liposome constituent material and S as a crosslinking agent.
An example of the method for preparing liposomes of the present invention using P. foliage is as follows.

1) DPP [! SP Kano is reacted with Kano PR-5PD, which is a reactant in the liposome membrane.
Prepare P conjugate ((]PPB-[]TP).

2) Mouth Gf'-D obtained by reacting BGF and SP leaf
BGI'-3ll which reduces TP and has an active SH group
Prepare.

3) Prepare liposomes containing DPP-low DTP,
This is reacted with the previously obtained EIGP-SH to form BGI.
? Combined liposome: Al! ! do.

Furthermore, it goes without saying that the liposome according to the present invention can encapsulate various drugs such as anti-inflammatory agents, anti-cancer agents, antibiotics, anti-viral agents, and hormone agents.

In order to encapsulate these drugs in the liposomes of the present invention, for example, the drugs may be dissolved in the mother liquor during liposome preparation, and the liposomes may be prepared by the above-described means.

〔Effect of the invention〕

The BGP-binding liposome of the present invention allows a large amount of a drug to be selectively taken up into cells that overexpress 86F receptors, thereby enabling excellent drug targeting therapy.

〔Example〕

EXAMPLES The present invention will be described in detail below with reference to Examples and Test Examples.

Example I Production of BGF-conjugated liposomes (1) DPPE! -Preparation of DTPDPP [110μmOl chloroform/methanol (
9: 1) Dissolve in 700 μβ mixture, add 12 μmo
300 μβ of methanol containing 1 of 5PDP and 20 μmol of triethylamine were added and incubated at 25° C. for 2 hours. The solution was washed with 2 ml of 0.1M phosphate buffer (PBS), pH 7.4, and the organic phase was washed twice with distilled water again, and then the organic phase was dried and smoked with DPPB.
-DTP was obtained.

(2) Introduction of SR group into BGP 500 μg of BGF was dissolved in 0.1 M PBS (pH 17,5
) 0.5 mj2 and added a tracer amount of 5P to this solution.
Add 20 μm of ethanol solution containing DI′ and heat at 25°C.
After incubation for 30 min at 0.145 M Na
Cj2 including 0. I M El acid buffer (pl! 4
．． 5) If 20mN with 0.02% NaL
-) Sephadex G-2 equilibrated with lis(hydroxymethyl)methyl 2-aminoethanesulfonic acid buffer (pH 7, 4) (TBS buffer)
5 column, unreacted substances and N-hydroxysuccinimide (Its) were separated, and BGF-DTP in which a 2=pyridyl disulfide group was introduced into EGI' was obtained.

Next, E! The Gfl-DTP solution was subjected to ultrafiltration under nitrogen pressure (3.5 kg/cm') using an Amicon 8010 type stirred cell equipped with a diaflow membrane YM2 of about 1.0 mJ! Concentrated into This concentrate contains I
After adjusting the pH to 4.5 by adding N-hydrochloric acid, the final concentration was 50mM.
Add dithiothreitol (DTT) so that 2
Incubated for 20 minutes at 5°C. This solution was added to TB
Loaded onto a Sephadex 6-25 column equilibrated with S buffer and containing active Sll groups [! GF-3l+ was obtained.

(3) Preparation of BGF-binding liposomes DPPC22,
6 μmol, :l Restyrol 13.2 μmol and DPPB-DTP prepared in (1) 1.4 μm
ol in 4 ml of chloroform, and in an eggplant-shaped flask (50 mf), the solvent was distilled off under reduced pressure under a nitrogen stream to form a uniform lipid film on the inner wall.
was prepared.

Chloroform 4mj on this liquid film? Add and dissolve 0.27% glucose or 100μci'
11 ml of TBS containing H-inulin [20mM TBS buffer - 0.1M NaCl (pH 8.0) was added and stirred. The w10 emulsion was subjected to fVia by ultrasonication at ℃ for 5 minutes.The emulsion was gelled by distilling off the solvent at 42℃ under a reduced pressure of 400 mmHg using a rotary evaporator, and then suspended by portic swing. 73 hours until the chloroform odor disappears.
The solvent was distilled off at 42°C under reduced pressure of 0 mmHg for about 2 hours. The suspension is transferred to a polycarbonate membrane (pore size:
After uniformizing the particle size by performing sequential extrusion at 10 to 25 kg/cm2 under nitrogen flow using an extruder equipped with 0.4, 0.2 μm), the inverted layer liposomes ( RE!V
).

Adding BGF-SH solutions of various concentrations to this liposome,
t, 0. 2 M T[! containing I M NaC1 S
Adjust the pl+ of the reaction solution to 8.0 with a buffer solution and heat at 25°C.
After incubation at 4° C. for 1 hour, further incubation was performed at 4° C. for 18 hours. This was loaded onto a Sepharose 4B column, and BGF-bound liposomes were fractionated.

A chromatogram obtained by loading onto a Sepharose 4B column is shown in FIG.

From Figure 1, the position corresponding to the void volume (fraction N
Since inorganic phosphorus derived from the phospholipid of the liposome and +25j radioactivity derived from BGF were eluted in α15-18), it was confirmed that the [lGF-binding liposome was successfully prepared].

Test Example I Stability of BGF-bound liposomes (1) Release of enclosed substances over time: 3 (1-inulin as an internal aqueous phase marker in Dulbecco's modified Eagle's medium (DMBM) or 10% tallow serum (Fe2) Control liposomes in DME!M containing [!GF-unmodified liposomes]
The stability of [! DM of GF-conjugated liposomes [! The stability in M was investigated. The results are shown in FIGS. 2 and 3.

Control liposomes had a retention capacity of about 90% after 48 hours of incubation regardless of whether the medium contained Fe3. On the other hand, BGF-conjugated liposomes had a retention capacity of 80% after 48 hours.

(2) Dissociation of BGF: [!] in DMBM or DMBM containing 10% FC3. [!] from GF-conjugated liposomes We investigated the dissociation of GF.

The results are shown in Figure 4.

DM [! In M, almost no dissociation was observed even after 24 hours of incubation. On the other hand, 10% PC8
Even in DMBM containing , almost no dissociation was observed, although there was some variation.

Test Example 2 Uptake of BGF-bound liposomes by mouse breast cancer cells (MM 102) (1) Culture of MM 102 10% FC3, 100 μg/m! streptomycin,
RoswellPa containing 100 ro/mA penicillin G
rk Memorial In5titude's M
edium 1640 (RPM11640) 10
mj! Breast cancer cells MM102 derived from mouse mammary gland were added to
Culture bottles (
Plant in a CO2 incubator (bottom area 25cm').
The cells were cultured in air:CO,=95:5.37°C). Note that passage was performed every 4 days.

(2) Uptake of BGF-conjugated liposomes by MM 102 MM 102 cell suspension (1,7X1
0'cells/mi'), add BGF-conjugated liposomes (PL; 280 nmol) containing 314-inulin or control liposomes (PL; 280 nmol) to each, and incubate in a C02 incubator ( Air co C02=95:
5.37°C). 1 m over time
l was collected and cooled on ice at RPM+1640 (5mj!)
was added and washed three times (2,200×g, 3 minutes).

The obtained cells were solubilized with 1 ml of 1% Triton
To determine the radioactivity of BGF, transfer this solution to a vial, add 7.0 mL of Aquasol-2, and measure the radioactivity of 'H-inulin with a liquid scintillation counter. The survival rate of MM 102 cells after completion of this experiment was over 99%.

The results are shown in Figure 5.

Uptake of control liposomes into MM 102 cells was very low and hardly increased over the incubation period. For this, E! Uptake of Gl'-linked liposomes increased over time, and a significant difference was observed compared to control liposomes.

[Brief explanation of drawings]

FIG. 1 is a drawing showing the elution pattern of the BGF-bound liposome prepared in Example 1 from a Sepharose 4B column. Figure 2 shows control liposomes encapsulating “H-inulin in DMBM and DMBM containing 10% FC3 [!M
3 is a drawing showing the retention ability of 311-inulin in the cell. FIG. 3 is a drawing showing the retention ability of 3H-inulin in DMBM of BGF-bound liposomes encapsulating 3]1-inulin. Figure 4 shows DM[t in DMBM and containing 10% FC3.
FIG. 3 shows the dissociation of BGIi from BGF-bound liposomes in M. FIG. 5 is a diagram showing the amount of 13GI'-conjugated liposomes taken into MM 102 cells. that's all

Claims (1)

[Claims] 1. Ribosomes with epidermal growth factor bound to their surface.