JPH04169532A - Liposome preparation containing lipo-soluble platinum complex - Google Patents

Abstract

PURPOSE:To provide the subject preparation having an average particle diameter of <= a specific value and having an excellent anti-cancer activity by adding an unsaturated fatty acid or a fatty acid ester to a liposome preparation comprising a lipo-soluble platinum complex and a natural phospholipid. CONSTITUTION:A liposome preparation having an average particle diameter of <=200mum comprises (A) a lipo-soluble platinum complex of the formula [R1, R2 are (organic group-substituted) ammine which may be linked to each other through a divalent organic group; R3 is (un)saturated higher fatty acid residue], (B) a phospholipid, preferably purified soybean lecithin or purified york lecithin, and (C) an unsaturated fatty acid such as a 12-20C fatty acid containing one to four double bonds, and/or a fatty acid ester such as the ester of a 12-20C fatty acid containing one to four double bonds. In order to stably store the liposome, the liposome preparation is preferably lyophilized, or substituted with nitrogen gas when prepared, and further controlled to an initial pH of 6-7.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a liposome preparation, and more particularly to a liposome preparation containing a fat-soluble platinum complex and having excellent anticancer activity.

[Conventional technology]

Cisplatin is an excellent anti-tumor tumor drug that has already been marketed and is known to exhibit great anti-tumor effects, but it has the disadvantage that the dosage is limited due to side effects such as nephrotoxicity and neurotoxicity. have.

In order to overcome the drawbacks, some cisplatin derivatives have been synthesized, for example, in JP-A-62-96, the following general formula is shown. represents ammine (provided that these two ammines may be linked via a divalent organic group), and Ro represents a saturated or unsaturated higher fatty acid residue. ) describes the lipophilic platinum complex and its antitumor activity ′, J
). However, since this compound has extremely low solubility in water, it is difficult to formulate it into aqueous preparations such as intravenous injection or drip preparations.

Liposomes are vesicles consisting of a lipid bilayer, and are morphologically classified into: ■ Unilamellar liposomes with a small diameter (SUV) ■ Unilamellar liposomes with a large diameter (LUV) ■ Multilamellar liposomes (MLV) . Liposomes contain biologically derived phospholipids and have been used as T1 drugs since the 1970s. Since liposomes have not only a water compartment but also a hydrophobic compartment, it is possible to retain hydrophobic drugs and substances. It is also known that by holding a drug in liposomes, it is possible to achieve sustained release of the drug and change its distribution in the body, thereby reducing the toxicity and side effects of the drug. For example, it has been found that retaining cisplatin in MLV reduces sageness.

[Problem to be solved by the invention]

Phospholipids that make up liposomes include phospholipids produced in huang, soybeans, and other animals and plants, and their hydrogenated products.
Synthetic phospholipids with uniform fatty chains are also known. Liposomes composed of synthetic phospholipids or hydrogenated phospholipids have much higher chemical stability of the phospholipids themselves and physical stability of the liposomes than liposomes composed of natural phospholipids. itself is expensive and impractical.

Therefore, it is desirable to produce stable liposome preparations using inexpensive natural phospholipids. Particle size is 200
The size exceeded nanometers. The liposome size is 2
If the particle size exceeds 00 nm, approximately 50% or more will be trapped in the liver and pancreas, so it takes 2Q to develop liposome formulations.
It is necessary to create Onm liposomes. Saroni 200
Sub-nm liposomes also have the advantage that they can be passed through a 220 nm filter for sterilization.

Therefore, an object of the present invention is to provide a liposome preparation that is composed of natural phospholipids, retains the platinum complex of formula (2) above, and has an average particle size of 200 nm or less.

Means for Solving the Problem] In the liposome preparation consisting of the platinum complex and natural phospholipid of the above formula, the average particle size of the liposomes can be reduced by adding an unsaturated fatty acid or fatty acid ester as a particle size reducing agent. The thickness can be reduced to 200 nm or less. That is, the present invention provides a fat-soluble platinum complex represented by the above formula CI,
The present invention relates to a liposome preparation characterized by consisting of (1) a phospholipid, and (2) an unsaturated fatty acid or a fatty acid ester.

The organic group in the organic group-substituted ammine represented by R1 and R7 in the platinum complex of the above formula [■], and these 2
2 when two ammines are linked via a divalent organic group
As a valent organic group, diammineplatinum (II) has traditionally been used.
Examples include those used as a substituent for the ammine moiety (RNH2) in complex anticancer drugs. That is, as an organic group, for example, a carbon atom group such as an isopropyl group has 1 to 1 carbon atoms.
5 alkyl groups and cycloalkyl groups having 3 to 7 carbon atoms, such as cyclopropyl groups and cyclohexyl groups. On the other hand, examples of divalent organic groups include 1.
Chloalkylene groups such as 2-chlorohexylene groups and, for example, 2.2-pentanoethylene-trimethylene groups: 1.2-tetramethylene (trimethylene) groups: 1.2-
Alkylene groups having 2 to 3 carbon atoms which may have substituents such as alkyl having 1 to 5 carbon atoms, alkyl having 2 to 6 carbon atoms, kylene or phenyl groups, such as diphenylethylene group; For example, 1f1.2-phenylene group, etc.
Examples thereof include alkyl having 1 to 5 carbon atoms, alkoxy having 1 to 5 carbon atoms, and 1,2-phenylene group which may have a substituent such as a 10-gen atom. In the case of divalent organic groups (e.g., tlfl, 2-cyclohexylene groups, etc.), isomers, i.e., cis and trans forms, exist; however, the platinum complex in the present invention may be any of these isomers. or a mixture thereof.

The saturated or unsaturated higher fatty acid residue represented by R3 includes fatty acid residues having 10 to 24 carbon atoms, and more specifically, for example, capric acid, lauric acid, myristic acid, valmitic acid, and stearic acid. , quidic acid,
Residues of saturated fatty acids such as behenic acid and lignoceric acid, and carbon atoms of 16 and more, such as oleic acid and liluic acid.
20 unsaturated fatty acid residues, etc.

Examples of natural phospholipids include lecithins derived from animals and plants, such as egg yolk lecithin and soybean lecithin, but highly purified lecithin with a high phosphatidylcholine content is preferred. As such highly purified lecithin, commercially available products such as purified egg yolk lecithin and purified soybean lentin can be used.

Examples of unsaturated fatty acids include fatty acids having 1 to 4 double bonds and 12 to 20 carbon atoms, such as myristoleic acid, palmitoleic acid, oleic acid, and lylunic acid. There are fatty acid esters and unsaturated fatty acid esters. The fatty acid moiety of the saturated fatty acid ester includes fatty acid residues having 12 to 20 carbon atoms, such as stearic acid and myristic acid. Regarding the fatty acid moiety of unsaturated fatty acids, the same examples as those for the above-mentioned unsaturated fatty acids can be cited.

Examples of the ester include alkyl esters having 5 or less carbon atoms, such as methyl ester and ethyl ester. More specifically, examples of fatty acid esters include ethyl myristate, ethyl oleate, ethyl stearate, and ethyl lylunate.

These fatty acids and unsaturated fatty acid esters alone
Alternatively, they can be used in combination, and the blending amount is 5 wt% or more of the phospholipid, preferably 10 wt%.
% or more, and in the case of fatty acid addition, the concentration at which phospholipids do not form micelles, that is, 35 wt% or less of phospholipids,
Preferably it is 20 wt% or less. When a fatty acid or a fatty acid ester is contained, an excellent effect is exhibited in reducing the particle size, which is the objective of the present invention. Furthermore, by retaining the negative charge of fatty acids on the surface of the liposome, it is expected that the rate of disappearance from the circulation will be slowed down.

In order to increase the strength of the membrane, sterols such as cholesterol and tocopherol are usually added to the liposomes of the present invention. Its blending amount is 0 or more in molar ratio to phospholipids,
Preferably it is 0.3 or more and 1°0 or less. Furthermore, the liposome of the present invention can contain additives normally used in injections, such as isotonic agents, pH regulators, preservatives, and the like.

Although the liposomes of the present invention can be produced according to conventional general methods, large-scale production is possible by using a high-pressure injection emulsification method. As a high-pressure injection emulsifying device, for example, Microfluidizer (registered trademark: Microfluidisc Co., Ltd.) is known. Microfluidizer ■ is capable of continuous production of liposomes by forcibly passing a dispersion containing drugs and lipids under high pressure through a reaction chamber maintained at a constant temperature.
Large-scale industrial production is also possible using the 510-type microfluidizer (2).

This formulation and production method made it possible for the first time to produce large-scale production of sushi-soluble platinum complex-containing liposomes with an average particle size of 200 nm or less.

In order to stably store the liposomes prepared in this way, it is recommended to freeze-dry them using a conventional method to obtain a lyophilized preparation, or to perform nitrogen substitution during liposome preparation and to set the initial pH to 6 to 7. preferable.

The liposome preparation of the present invention can be administered orally as an oral preparation, intravenously, intramuscularly or locally intraarterially as an injection, or rectally as a suppository. The dosage and frequency of administration vary depending on symptoms, age, body weight, administration form, etc., but it is usually administered to adults in the range of 1 m (g to 3 g, preferably 10 to 500 mg) per day as the active ingredient. can.

The following examples illustrate preferred embodiments of the invention in more detail without restricting the invention thereby.

Example 1 3 g of purified soybean phospholipid, cyclohexane-1°2-diammineplatinum (It) similistoate (formula [I
In E, R3 together with R2 forms fuclohexane-
1,2-diammine, R3 represents myristoate)
80 mg of cholesterol and 550 μl of cholesterol were weighed out and dissolved in 80 ml of chloroform in an eggplant flask (volume 500 ml). Next, chloroform was removed using a rotary evaporator, and a membrane component mixture was completely dried using a vacuum dryer. When a 5% aqueous glucose solution (100%) was added to this mixture and the mixture was shaken at 0°C, the dried material was immediately dispersed to form MLV. Furthermore, oleic acid 4
50 tng was added to obtain a white liquid. A transparent SUV or LUV was obtained by passing this liquid through a microfluidizer (11,000 ps rx for 10 minutes). The particle size distribution was measured using a dynamic light scattering device.

Furthermore, the fat-soluble platinum complexes incorporated into the liposomes and the fat-soluble platinum complexes that were not incorporated into the liposomes were separated by gel permeation chromatography, and the rate of incorporation into the liposomes was measured.

Example 2 The same operation as in Example 1 was performed to obtain MLV. 450 mg of ethyl stearate was added thereto to obtain a white liquid. Transfer this liquid using a microfluidizer ■ (11,000 psi)
x 10 minutes) to obtain a transparent SUV or LUV. The particle size distribution was measured using a dynamic light scattering device. Furthermore, the fat-soluble platinum complexes incorporated into the liposomes and the fat-soluble platinum complexes that were not incorporated into the liposomes were separated by gel permeation chromatography, and the rate of incorporation into the liposomes was measured.

Comparative Example The same operation as in Example 1 was performed to obtain MLV. Transfer this MLV to a microfluidizer @ (11000 ps) x
Transparent SUV or L
I got UV. The particle size distribution was measured using a dynamic light scattering device. Furthermore, the fat-soluble platinum complexes incorporated into the liposomes and the fat-soluble platinum complexes that were not incorporated into the liposomes were separated by gel permeation chromatography, and the rate of incorporation into the liposomes was measured.

The average particle diameter of the liposomes of Example 1.2 and Comparative Example and the uptake rate of the fat-soluble platinum complex are summarized in a table.

In this way, by adding fatty acids and fatty acid esters, the average particle size of the fat-soluble platinum complex is 200 nm.
It has become possible to produce the following liposomes on a large scale.

Claims (5)

[Claims] (1) B) The following general formula ▲ Numerical formula, chemical formula, table, etc. ▼ (In the above formula, R_1 and R_2 represent organic group-substituted or unsubstituted ammine;
R_3 represents a saturated or unsaturated higher fatty acid residue. ) A liposome preparation comprising a fat-soluble platinum complex represented by (b) a phospholipid and (c) an unsaturated fatty acid and/or a fatty acid ester. (2) The liposome preparation according to claim 1, wherein the phospholipid is purified soybean lecithin or purified egg yolk lecithin. (3) The liposome preparation according to claim 1, wherein the unsaturated fatty acid is a fatty acid having 12 to 20 carbon atoms and 1 to 4 double bonds. (4) The liposome preparation according to claim 1, wherein the fatty acid ester is a fatty acid ester formed from a fatty acid having 12 to 20 carbon atoms and 0 to 4 double bonds. (5) The liposome preparation according to claim 1, which is a freeze-dried type.