JPH0579644B2 - - Google Patents

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to liposomes.

(Prior art) Conventionally, egg yolk lecithin, egg yolk phosphatidylcholine, soybean oil lecithin, their constituent phospholipids, and even synthetic lecithin have been mixed in water.
Closed endoplasmic reticulum (vesicles) with a size of 250 nm to 5 μm,
That is, it is known that liposomes, which are structures having a lipid bilayer membrane structure similar to living organisms, are formed. These liposomes are used as ultra-microcapsule materials that encapsulate drugs and enzymes, and various water-soluble and oil-soluble drugs such as insulin, heparin, vitamin K, corticosteroids, and various anticancer drugs can be stored in the internal water of the liposomes. Research is underway to develop sustained-release or more effective therapeutic agents by encapsulating them in phase or hydrophobic wall membranes.

In the field of cosmetics, vitamin E, ascorbic acid, etc., which are unstable when stored at room temperature, are formulated into liposomes in order to stabilize their shelf life over a long period of time.

In addition, liposomes made of lecithin tend to accumulate in the liver, spleen, lymph nodes, etc., but liposomes that are permeable to the blood-brain barrier by adding sulfatide have also been developed. It is known that D-glucose oxidase encapsulated therein is effective in treating congenital enzyme deficiencies. Artificial red blood cells in which hemoglobin and iron porphyrin are encapsulated in liposomes have also been proposed. A new drug-type missile therapy that binds monoclonal antibodies that react with cancer cells to the surface of liposomes and retains anticancer drugs inside them to target cancer cells is also being researched. Furthermore, the application of liposomes to immunology has been studied, and attempts have been made to use liposomes to measure antibody titers in serum and as diagnostic agents.

(Problems to be Solved by the Invention) Various drugs such as pharmaceuticals and cosmetics encapsulated in liposomes are usually administered intravenously, intramuscularly, subcutaneously, orally.
The stability of water-dispersed liposomes during storage is important, as they are used by methods such as application to the skin. Moreover, there is currently a strong desire to develop liposomes with intelligent functions such that once used, the contents are quickly released at a predetermined site in the human body. Furthermore, it is naturally essential that the material for forming liposomes be non-toxic. The functional liposomes that the present inventors are trying to develop will stably keep the drug contained inside when the pH is close to neutral and acidic, but when the pH is 6.2 or higher,
In particular, it has high functionality such that drug release increases when it becomes slightly alkaline (7.2 or 8.2).

In other words, the pH of the stomach in the human body is 1 to 6, in most cases 1 to 4, and the pH of the intestine is increased by secreting bile etc.
It will be 7.0 to 7.2. Also, the pH of the skin is 6.0-6.5, and the pH of the tear fluid is 8.2.
The PH of the blood is said to be around 7.2 to 7.4.
The production and development of liposomes that make it possible to control the release of drugs in response to these PH changes has not yet been developed, and there is a particular need for the development of such liposomes.

JP-A-63-2921 discloses a liposome based on N-palmitoylserine, but this liposome releases the drug contained therein only under alkaline conditions with a pH of about 8 or higher. , it does not have the function of releasing the encapsulated drug at a release rate corresponding to the pH condition of about 6 to 8.

(Means for solving the problem) In view of the above circumstances, the present inventors conducted intensive research to develop a new liposome wall material that overcomes the conventional drawbacks, and as a result, they found that N-acyl homoserine and ( Or) By using N-acyl homoserine lactone together with phospholipids, which are other common liposome wall membrane components, stable liposomes can be formed, and drugs encapsulated in these liposomes can be The inventors have discovered that the release rate of the internal drug can be controlled by changing the amount of the drug, leading to the completion of the present invention.

That is, in the present invention, the wall film is made of N having 8 to 22 carbon atoms.
The present invention provides a liposome characterized by containing N-acyl homoserine and/or N-acyl homoserine lactone having a -acyl group.

N-acyl homoserine and N- used in the present invention
As the acyl group in the acyl-homoserine lactone, one having 8 to 22 carbon atoms is used, preferably an acyl group derived from a saturated or unsaturated higher fatty acid having 10 to 18 carbon atoms.

The method for forming liposomes in the present invention is not particularly limited, but conventionally known methods can be employed. For details of such known methods, see, for example, Hiroshi Kikuchi and Keizo Inoue, "Cell Engineering" 2 , 1136.
(1983), Fumiyoshi Ishii, Ichiro Sasaki, Fragrance Journal 91 , 100 (1988), Shunichi Noro, Fumiyoshi Ishii, Pharmacy, 35 , 1193; 1329 (1984), etc.

The phospholipid wall material of the liposome in the present invention is a two-chain surfactant that forms lamellar liquid crystals in water, and can stably produce a closed endoplasmic reticulum with a bilayer membrane structure depending on the conditions, such as a phospholipid. molecule and N-acyl homoserine and/or N-
A mixture with acylhomoserine lactone is used.
Cholesterol can also be added to this mixture to reinforce the bilayer membrane. Furthermore, dicetyl phosphate, stearylamine, etc. may be added as a charged substance. As the phospholipid, commonly used phospholipids can be used alone or in the form of a mixture. The amount of N-acyl homoserine and/or N-acyl homoserine lactone used is 5 to 30%, preferably 10 to 20% in mole % based on the total wall membrane.

Next, to specifically explain the method for producing liposomes of the present invention, the molar ratio of egg yolk lecithin: cholesterol: N-acyl homoserine is 70:20:10 or 50::40:10 in a 100 ml eggplant-shaped flask. and/or N-acylhomoserine lactone. Although any good solvent with a low boiling point can be used, it is usually dissolved using chloroform, dichloromethane, ether, methanol, ethanol, etc., or a mixed solvent thereof. When incorporating an oil-soluble drug, the drug is added and dissolved at this stage. Thereafter, the solvent is removed using an evaporator and the mixture is dried under reduced pressure. Add an appropriate amount of distilled water or a buffer solution with a pH of 5.2 or lower to this solution, apply vibration using vortex mixing method or ultrasonic method, and create an aqueous dispersion of liposomes. It traps oil-soluble substances that can be used as drugs or cosmetics. Untrapped drugs are removed by gel filtration, dialysis, centrifugation, etc. If a water-soluble drug is to be encapsulated in liposomes, egg yolk lecithin: cholesterol: N-acyl homoserine and/or N-acyl homoserine lactone are weighed, dissolved, and dried in the same manner as above, and then mixed with distilled water or PH5. Dissolve the drug in a buffer solution of 2 or less and add it, form liposomes using the same method, and perform treatments such as gel filtration, centrifugation, dialysis, etc. to remove the drug present in the external aqueous phase. . This results in
Liposomes containing the drug in the internal aqueous phase are obtained. The weight of the liposome wall material is 0.1 to 3% by weight based on water.
Since the liposomes of the present invention are PH sensitive, PH6
Even in the above neutral and weak alkaline conditions, the encapsulated drug will flow out during gel filtration, centrifugation, etc. due to liposome collapse, so it is recommended to use a buffer solution with a pH of 5.2 or lower when producing liposomes. desirable.

In our experiments, the fluorescent substance calcein was used as a drug substitute so that the outflow rate of the drug encapsulated in liposomes could be measured very precisely. The liposome of the present invention has the function of stably retaining the encapsulated drug in an environment with a pH of 6 or lower, but starts releasing the drug when the pH reaches 6 or higher.

Next, to explain the synthesis of N-acyl homoserine, homoserine, which is a raw material, has a hydroxyl group and an amino group as active groups that react with fatty acids, and also has a carboxyl group that reacts with fatty acid chloride. A special method is required to react. That is, 1,3-
N-acylthiazolidene-
2-thione is obtained, and then this substance is reacted with homoserine to obtain the target product, N-acyl homoserine. Since this reaction all proceeds at room temperature, there are few side reactions and the yield is very good. N-acyl homoserine lactone is obtained by dehydration of N-acyl homoserine. N-acylhomoserine lactone is prepared by dissolving it in ethyl alcohol and adding a buffer solution.
When the pH is changed from 4 to 10, the chemical structure of the molecule changes, with the acidic side becoming a lactone and the alkaline side becoming an alkali metal salt of a carboxyl group.

The main reason for the formation of PH-sensitive liposomes according to the present invention is that N-acyl homoserine becomes a homoserine lactone type even at acidic PH, becoming a bulky molecule that is almost insoluble in water; This is thought to be due to the fact that the group becomes an alkali metal salt, which is considerably soluble in water and has surface activity.

The phospholipids used in the present invention include:
For example, egg yolk lecithin, egg yolk phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, dicetyl phosphate, phosphatidylglycerol, phosphatidic acid, azolectin, L-α-dipalmitoylphosphatidylcholine, Mention may be made of phosphatidylinositol and mixtures thereof.
In short, any phospholipid that can form liposomes can be used, and natural and artificial vesicle-forming materials such as sucrose esters, rhamnolipids, spiclesporic acid alkylamine salts, and dioctadecyldimethylammonium bromide can also be used. be able to.

(Effects of the Invention) The liposomes of the present invention have a wall made of naturally derived and/or artificial substances, have high biostability, and when stored at pH 6 or below, they can be easily removed from ordinary standard materials such as egg yolk lecithin. Like liposomes, they are stable, the drugs they contain are not released, and they are chemically and colloidally stable for long periods of time.

On the other hand, in the liposome of the present invention, when the environment of the external aqueous phase of the liposome becomes PH6 or higher, the drug encapsulated in the liposome is gradually released, and the rate of release increases as the PH value becomes 7 to 8. Both oil-soluble and water-soluble drugs can be used as drugs to be encapsulated in liposomes, and the liposomes of the present invention are suitable as sustained-release, functional new drug-type liposomes encapsulating drugs and the like.

The liposome of the present invention stably retains the drug at a pH of 6 or lower in digestive juices, and gradually releases the drug at a pH of 6 or higher, so it is suitable for stable protection in the stomach.
Furthermore, since it has the function of starting to release the drug when it reaches the intestine and the pH reaches 6.2 to 7, it is suitable for oral administration and transdermal administration. Examples of liposomes for cosmetics include those containing vitamins C, E, and the like.

N-acyl homoserine and/or N-acyl homoserine lactone used in the present invention is obtained by an acylation reaction using non-toxic amino acids and fatty acids derived from living organisms, and is a highly stable and safe substance. be. Furthermore, the liposome of the present invention is also highly safe.

(Examples) Next, the present invention will be described in more detail based on Examples.

Reference Example 1 N-acyl homoserine and N-acyl homoserine lactone were synthesized by the following procedure.

26 g of 1,3-thiazolidine-2-thione, 44.12 g of triethylamine in a 1 liter three-neck round bottom flask,
Weigh out 500ml of dichloromethane. While stirring the reaction system, 65.91 g of palmitoyl chloride was added dropwise from the separating funnel. The reaction system was sealed with a calcium chloride tube to prevent moisture from entering, and the reaction was carried out at room temperature for 3 days. During the reaction process, a small amount of sample was taken from the reaction system, and the reactants and unreacted substances were checked using thin layer chromatography to check the reaction rate. After the reaction is complete, triethylamine and
The hydrochloride was filtered off, the filtrate was washed with an alkaline aqueous solution, and N-palmitoyl 1,3-thiazoliden-2-thione dissolved in the dichloromethane layer was obtained by evaporation of the solvent and recrystallization with acetone. yield
65g, melting point 60.5-61.0℃, yield 83%.

Next, 0.916 g of homoserine, 1 N-palmitoyl,
2.5 g of 3-thiazoliden-2-thione, 1.06 g of triethylamine, 1:1 of tetrahydrofuran and water.
1 was dissolved in 300 ml of a mixed solvent, and stirred and reacted at room temperature for 48 hours. After the reaction, the solvent was removed, a weak aqueous hydrochloric acid solution was added to make the system acidic, and ethyl acetate was added to obtain cloudy N-palmitoylhomoserine. This was recrystallized from ethanol. Yield 1.37g, yield 54.8g, melting point 126-127°C.

By the same method as above, the fatty acid chloride was changed to lauroyl chloride and capryloyl chloride and the corresponding N-lauroyl homoserine and N-capryloyl homoserine were obtained.

Each N-acyl homoserine lactone was obtained in 100% yield by dissolving N-acyl homoserine in xylene and removing water and xylene with an evaporator while dehydrating at 95°C.

Example 1 Egg yolk lecithin 26.1 mg, cholesterol 3.9 mg,
N-palmitoylhomoserine synthesized in Reference Example 1
1.79 mg (molar ratio 7:2:1) was weighed into a 50 ml eggplant-shaped flask, and 5 ml of dichloromethane/methanol mixed solvent (volume ratio 3:2) was added to dissolve it.

Next, this eggplant-shaped flask was connected to a rotary evaporator to remove the solvent, a thin film was applied to the inner wall of the flask, and the flask was placed in a desiccator and dried under reduced pressure for 1 hour. After this, 5 ml of a solution of calcein (fluorescent paint) dissolved in a pH 5.2 buffer to a concentration of 4 x 10 ^-4 mol/l was added, and the mixture was shaken for 20 minutes using a vortex mixer to give a somewhat cloudy yellow color. A suspension of was obtained. This solution was gel-filtered using a PH5.2 buffer through a Sephadex G-50 column (diameter 20 mm, height 250 mm), fractionated into a fraction collector, and the fluorescence intensity of each fraction was measured. First, the calcein-containing liposomes flowing out and the calcein in the external aqueous phase were completely separated, and the fluorescence intensity was separated into two peaks. The peak portion of the first fraction that flowed out was calcein-encapsulating liposomes, which were completely separated from unencapsulated calcein and flowed out as a subsequent fraction. Calcein-containing liposome is Coulter N
When the particle size distribution was measured using a -4 submicron particle analyzer using laser light, the average particle size was 293 nm. Fluorescence measurement
Using 480 nm as excitation light, the intensity at the maximum emission wavelength of calcein at 520 nm was measured. To confirm that the calcein fluorescent dye was encapsulated in the aqueous phase inside the liposome, take 3.5 ml of the liposome dispersion and add 10% Triton X-100 (polyoxyethylene isooctyl phenyl ether) aqueous solution.
The fluorescence intensity of the sample to which 100 μl was added was more than twice that of the liposome solution made to the same volume by adding water. This fact confirmed that the fluorescent dye was encapsulated within the liposome. The principle of this experiment is that the fluorescent dye encapsulated in the aqueous phase of the liposome is in a concentrated state from the beginning.
The characteristic absorption of calcein at 520 nm is only slightly observed due to the property of concentration quenching, whereas the liposomes are destroyed by Triton This method takes advantage of high-concentration self-quenching, in which the fluorescence of the molecule is observed to increase in intensity.

Furthermore, the liposome suspension was left at room temperature and the fluorescence intensity was measured twice a day, but no change in intensity over time was observed for 3 weeks.

In addition, the liposome suspension was separated into a water portion and a liposome portion using a centrifuge, and only the liposome was dried under reduced pressure and its infrared absorption spectrum was measured. In the absorption spectrum, a carbonyl absorption characteristic of the lactone ring was present at 1780 cm ^-1 . This demonstrates that N-palmitoyl homoserine easily generates lactone by drying under reduced pressure and is converted to N-palmitoyl homoserine lactone, and that N-palmitoyl homoserine is incorporated into the liposome wall. It is.

Example 2 A liposome (A) aqueous dispersion containing a calcein fluorescent dye at a concentration of 4 x 10 ^-4 mol/l and having a composition of 10 mol% N-palmitoylhomoserine, 70 mol% egg yolk lecithin, and 20 mol% cholesterol was prepared in Example 1. A pattern was prepared. In this case, both the internal and external aqueous phases have a pH of
5.2 buffer was used. At the same time, a concentrated calcein solution (4×10 ^-4 mol/ml) containing no N-palmitoyl homoserine and consisting of 1.93 mg of dicetyl phosphate (DCP), 26.1 mg of egg yolk lecithin, and 3.9 mg of cholesterol (molar ratio 1:7:2) was added. A liposome (B) containing 1) was prepared by the method of Example 1. For these liposomes (A) and (B), the pH of the external aqueous phase can be widened by dropping sodium hydroxide and hydrochloric acid (PH
4-10) The efflux rate of calcein was observed every 30 minutes by measuring fluorescence intensity. In the liposome (A) aqueous dispersion, when the outer aqueous phase pH is 5.2 and below, calcein does not leak out for more than a week, and when the pH is 6.2, the fluorescent dye gradually leaks out, and within 5 hours, the calcein trapped in the inner aqueous phase is released. 12% of the time, 28% in 10 hours
leaked into the external aqueous phase. In the case of PH7.2, 80% in 5 hours,
100% leaked out in 10 hours. In the case of pH 8.2, 94% leaked out in 5 hours and 100% leaked out in 8 hours. In this same experiment, standard liposomes (B) had a pH between 2.2 and 8.2.
No leakage of calcein into the external aqueous phase was observed even during one week of measurement observation.

Example 3 In Example 1, the combination of egg yolk lecithin-cholesterol-N-palmitoylhomoserine was changed to 6:
Calcein-containing liposomes prepared in the same manner except for a 3:1 molar ratio exhibited a PH sensitivity similar to that shown in Example 2.

Example 4 A liposome prepared by using 10 mol% of N-palmitoyl homoserine lactone in place of the N-palmitoyl homoserine used in Example 1 showed the same tendency of PH sensitivity as that shown in Example 2.

Example 5 A liposome containing a fluorescent dye was obtained by using 21.6 mg of dipalmitoylphosphatidylcholine in place of the egg yolk lecithin used in Example 1, and using the same other ingredients as in Example 1. Liposomes were prepared by using an ultrasonic generator instead of vibrating with a vortex mixer in Example 1 and applying ultrasonic vibration for 10 minutes while keeping the temperature around 50°C. This is because the phase transition temperature of dipalmitoyl phosphatidylcholine is as high as 45° C., so the liposome preparation temperature also needs to be higher than the phase transition temperature. The obtained liposomes exhibited PH sensitivity similar to that shown in Example 2. The particle size distribution measured by Coulter N-4 Submicron Particle Analyzer is based on the average particle size.
It was 380nm.

Example 6 Egg yolk phosphatidylcholine 20.4 mg, cholesterol 4.4 mg, N-lauroylhomoserine lactone
1.1 mg was weighed into a 50 ml eggplant flask, and a liposome encapsulating a concentrated calcein solution was prepared in the same manner as in Example 1. By passing the liposome water dispersion through a microfluidizer and filtering it under pressure, the particle size distribution is narrow, with 95% of the particles having a central particle size of 178 nm.
Liposomes with a standard deviation value of 53 nm were obtained. This liposome also exhibited the PH sensitivity shown in Example 2.

Claims (1)

[Scope of Claims] 1. A liposome characterized in that the wall membrane contains N-acyl homoserine and/or N-acyl homoserine lactone having an N-acyl group having 8 to 22 carbon atoms. 2. The liposome according to claim 1, which contains a drug.