JPS61197513A - Preparation of stabilized liposome including major medicines - Google Patents

Abstract

PURPOSE:The major active ingredients are dispersed or dissolved in a solution of solubilized micelles prepared from phospholipid, the dispersion or solution is dialyzed in a dialysis membrane, the remaining solution is subjected to gel filtration, then, the liposome dispersion containing the major active ingredients are rapidly freeze-dried to give the titled substance. CONSTITUTION:A solubilized micelle solution is prepared from phospholipid and a cholesterol as a liposome membrane composition, and major active ingredients are added to the micelle solution under stirring. The resultant dispersion is dialyzed with water in a dialysis membrane, then, the remaining solution is subjected to gel filtration by means of column chromatography to separate into a fraction of the liposome including the active ingredients and the other fraction free from the active ingredients. Then, the former fraction is rapidly freeze-dried to give the objective liposome powder. EFFECT:When the product is suspended, again, the elution of the active ingredients from liposome is extremely suppressed.

Description

DETAILED DESCRIPTION OF THE INVENTION [A] Object of the invention: It relates to a method for preparing stable somes that are capable of incorporating fat nose moe, kuzumono, and the like.

[Industrial application field]

The formulation of the Nyoro liposome of the present invention is stable as it maintains the main drug in a well-encapsulated state, so it can be used, for example, as a therapeutic drug or a diagnostic agent, as well as cosmetics, foods, etc. It can also be used when processing products.

[Conventional technology]

Lysome preparations are being actively researched as a means of drug therapy that efficiently or directly transfers (absorbs) the main drug into in-vivo tissues. In other words, when it comes to research on the application of leesomes to pharmaceutical preparations, rihasome membranes act as a good barrier that is impermeable to water-soluble solutes such as enzymes, electrolytes, and sugars that exist in living organisms.
This increases the ability of the drug to migrate into the blood and liver, and in vivo, it becomes susceptible to phagocytosis especially by macrophages. It has been proven in in vivo animal experiments that the system is easily taken up intensively by the system, and based on these results, agents for strengthening systemic biological defenses, ~macrophage activation factors, antibodies, etc. Based on this, research is progressing into new medical fields with many prospects as a more selective dosage form.

However, there are currently very few examples of its application in actual medical settings, and the primary reason for this is that liposomes themselves are chemically and physically unstable;
Looking at the phospholipids that make up the rhinosome membrane substances, for example, lecithin has four ester bonds in its molecule, and these are quite unstable in aqueous solutions, and they are highly resistant to glitter. When one of the two fatty acid esters undergoes hydrolysis, lysolecithin is formed and this triggers the destruction of blood cells and in-vivo cell membranes, resulting in hemolysis and cell lysis.

Examples of publicly known publications regarding lysome formulation include the following.

(Known Publications) (1) Published Patent Publication No. 1982-82310 (2)
u No. 57-136514 [Problem to be solved by the invention] Why is the liposome itself the most important in its formulation? However, in the conventional method, it is in a state of suspension, and although it is stable and contains the main drug forever,
There was a drawback that the main drug gradually leaked out.

As mentioned above, the liposomes themselves are not physically stable, and the lipid bilayer membrane vesicles that make up the liposomes are unstable to heat and can aggregate with each other due to temperature changes. However, because of this, it often leaks out easily, and it is especially unstable in electrolytes with strong dehydration properties or aqueous electrolyte solutions that have strong binding properties with gosomes, and tends to form large aggregates.

Therefore, it has not been easy to formulate gosomes in a practical manner.

In other words, the points to be solved in liposome formulation are:
The main drug is encapsulated in liposomes composed of lipids such as lecithin and cholesterol, and this is made into an aqueous suspension (
Various methods have been devised to solve the problem of how to make it stable over a long period of time (suspension), that is, how to make it possible to preserve it for a long period of time.

For example, in the above-mentioned Publication (1), a method has been developed in which the main drug is encapsulated in rhinosomes, and a method is developed in which Nishiso is freeze-dried and used by returning it to the state of Sassinjon at the time of administration. ing.

Furthermore, in the above-mentioned publication (2), by encapsulating the main drug in lyxome and then dialysis, the suspension of the liposome within the dialysis membrane has a constant diameter of the lysome, compared to the conventional gluesome. It has been shown that it is easy to obtain and has good stability.

The present inventors tried each method again based on publicly known publications (1) and (2), and added further studies. As a result, freeze-drying as shown in the former (1) can certainly be a means of stably supplying bisome preparations. Stable for long periods in dry conditions. Unfortunately, however, the main drug encapsulated in the ligasomes was eluted during the freeze-drying process, resulting in most of it leaking out.

On the other hand, in the dialysis method shown in Publication (2), the encapsulation of the main drug into liposomes is good. However, in order to put it into practical use, there is a limit to preserving it as a suspension of linosomes containing the main drug for a long period of time. It was thought that one ideal method would be to use this in the form of a suspension again as needed.

However, based on the linosomes encapsulating a low-molecular-weight main drug obtained by the dialysis method described in Publication (2), this was freeze-dried and then dispersed and floated in an aqueous medium again, and the suspension was Unfortunately, when this form is used, the main drug leaks out of the soma, and the number of remaining normal ribosomes decreases to almost zero.

Therefore, in order to satisfy the long-term storage stability of liposome preparations, their supply, and the conditions for actual administration, the present inventors encapsulated the main protein drug in ribosomes at a high rate, and Next, the main drug in the lysome is
We began research into formulating stable liposomes that do not leak into aqueous media, and attempted to solve these problems using the methods described below.

[Example] Structure of the Invention The present invention resides in a liposome formulation method in which a lysome suspension containing a main drug is dialyzed and then freeze-dried to form a powder. When the somes obtained by this method are resuspended, leakage of the main drug from within the ribosomes is extremely small and the ribosomes are stable. Examples and experimental results are shown below, and will be specifically described.

[Example 1] Na0421 at pH 7.2 (Tris-HCl 1mM)
Add 17mM of soybean lecithin 17mM of sodium fluorate to a small 60mM solution - stir at room temperature day and night;
Prepare the solubilized micelle solution. Next, 6m of this solution! To,
For example, if the main drug is arbitrarily selected and ferritene is the main drug, 6 mg of it is dissolved, placed inside an automatic dialysis machine, and placed on the outside across the dialysis membrane with N2 at a pH of 7.2.
acj! A small 160mM solution was incubated for 24 hours at 25°C (2
, 2mj! /win) After dialysis, the inner solution is taken out and then subjected to column chromatography and gel filtration, which separates the suspension in which ribosomes are formed and the main drug from being encapsulated. The suspension in which somes had been formed was collected, and this solution was subjected to a rapid freeze dryer to obtain a dry powder.

The automatic dialysis device used here was Liederets i' (
Lipoprep) automatic dialysis device: Diachemer AG
Diachama AG Zuric
h) Manufactured by

[Example 2] In accordance with the procedure of Example 1, the proteins or enzymes shown in the following table (Table 1) were substituted for the substances used as crude drugs, and rapid freeze-dried lipsomes were obtained for each of them.

[Example 3] The procedure of Example 1, dialysis and rapid freeze-drying were exactly the same, but 30% of the moles of phospholipid (soybean lecithin), which is the membrane material of ribosomes, was replaced with cholesterol. As for the main drugs, lyochrome C1 and catalase were used to obtain rapid freeze-dried products of each lysome.

[Example 4] The procedure of Example 1, dialysis and rapid freeze-drying are exactly the same, but in preparing the solubilized micelle solution to form the liposome membrane, the concentrations of soybean lecithin and sodium cholate were changed. A rapid freeze-dried product of lilysomes was obtained by changing the amount by 2 to 3 times and using aldolase as the main drug. In addition, the dialysis time was also increased 2 to 3 times in response to changes in the concentrations of soybean lecithin and sodium cholate.

The sodium cholate used in each of the above-mentioned examples was passed through a dialysis membrane (cut off at a molecular weight of 10,000) at a rate of 2.2 mj/min! The external dialysis solution (pH 7) flows at a rate of
,2,NaCj! 160mM).

Further, the temperature was 25°C, and the dialysis time was 24 hours.

Furthermore, in the fractionation (separation) treatment after dialysis by column chromatography, the main drug substance in each example was a protein or enzyme, and the column was Sepharose 4B correspondingly.

Length 20cI11. The experiment was carried out at a radius of 0.5■, which allowed the resome suspension containing the main drug,
Proteins or enzymes that coexist in an eluted state without being encapsulated can be easily separated.

[Results of the evaluation test of rhinosomes according to the examples] The composition results regarding rhinosomes obtained based on the above-mentioned examples are as follows.

(Experimental composition) "Table 1" Stability of liposome inclusions according to Examples Regarding the stability of both liposomes, the one in the state of sassefushi 5 and the one in the state of sassinsigon after freeze-drying, the unique absorption wavelength of each inclusion was measured by absorbance and the encapsulation rate was determined. This is a comparison.

(Measurement conditions in Table 1) Column: Sepharose 4B, long 20G.

radius 0.50 Eluent Nidris-HCl2 1mM.

NaCj!　160mM.

Buffer (pH near, 2) As shown in Table 1, even after dialysis according to the present invention, the substances encapsulated in the dialysis membrane leak out even after being freeze-dried and returned to the suspension. Without a doubt,
It was revealed that 100% of the liposome remained in the liposome.

[Experimental results 2] Figure 1 shows the steps shown in Example 1, before and after freeze-drying the linosome suspension containing ferritin, and again after freeze-drying, and for both samples.
The relationship between the inclusion amount and elution volume was measured by gel chromatography based on absorbance and shown in a graph.

Elution volume 13mj! , and the peaks near 28 m2 are absorptions due to ferritin encapsulated in lysomes and free ferritin, respectively.

In other words, 19.7% was encapsulated in the leesome before freeze-drying, and 19.1% was still encapsulated even when it was freeze-dried and returned to the suspension. I understand.

In other words, conventional glue-some formulations had the disadvantage that most of the glue leaked out of the liposome membrane when it was freeze-dried and returned to the suspension, but according to the method of the present invention, it is possible to However, the main drug is stably retained within the somal membrane.

[Experimental results 3] In the present invention, as shown in each example, substances with different molecular weights are used as main drugs. For example, when soybean lecithin and cholesterol were present at 17 mM in a 7:30 molar ratio, 6 mg of the main drug was used in production with respect to the total amount of the solubilized micelle solution. When the encapsulation rate of the main drug was determined for the rhinosomes thus obtained, the results shown in Table 2 were obtained.

That is, as shown in Table 2, substances with large molecular weights have an increased inclusion rate.

"Table 2" The relationship between the molecular weight of the main drug and the encapsulation rate.
The number of moles) is replaced with cholesterol, and the other steps are as follows:
It was found that when lysomes were obtained in the same manner as in Example 1, the encapsulation rate increased slightly.

In addition, when preparing the solubilized micelle solution, the concentration of phosphorous substance (soybean lecithin) and sodium cholate is increased by 2 to 3 times, and the dialysis time is also increased by 2 to 3 times accordingly, thereby obtaining the following: For example, the encapsulation rate of linosomes containing aldolase as the main drug is 2, as shown in the following table (Table 3).
It was found that when the number is doubled, the inclusion rate also nearly doubles. However, above this point, the inclusion rate has reached a plateau.

1 Table 3 Relationship between increase/decrease in composition of solubilized micelle solution and encapsulation rate (6 mj of solubilized micelle solution Even if the main drug is encapsulated or the monosome is returned to the suspension form, a stable encapsulation state can be obtained.

Therefore, if necessary, for example, the lyophilized powdered rhinosomes can be stored in a vial or the like, and the dispersion liquid can be injected into the bottle to resuspend the main drug. According to the linosome formulation method of the present invention, for example, various enzymes conventionally used for therapeutic purposes can be easily digested. It is easily degraded by proteolytic enzymes in the tract and blood, and as a result, absorption from the digestive tract is extremely small.
According to the final method, active ingredients such as enzymes and fluorescent substances, or other active ingredients, are protected across the lysome membrane,
It is also expected that it will migrate to the focal area at a high rate.

In other words, research and development of liposome preparations has continued to date with the aim of transferring drugs to living tissues with high efficiency, but all of them lack long-term stability.

Furthermore, the advantage of the method for preparing Nyorori lsomes of the present invention is that the size of the phagosomes can be easily controlled to a constant size.

In the conventional method, for example, in the above-mentioned Publication (2), when preparing a solubilized micelle solution, the number of moles of cholic acid is determined from the quantitative relationship of lecithin and cholesterol, and if it is made the same value as cholic acid, the obtained The diameter (size) of the liposomes produced is approximately 1000, and the cholesterol content is 30%.
However, according to the method of the present invention, in order to obtain liposomes of uniform size, as shown in Example 1,
By adopting automatic dialysis equipment, lecithin,
By simply changing the composition of cholesterol and sodium cholate, rhinosomes of a desired size can be obtained without any particular restrictions.

In other words, in the conventional formulation of liposomes, lysasomes of the required size were collected by obtaining a coarse non-membrane and passing it through a Milliamenplan filter, etc. Other liposomes with non-standard sizes remained, causing a significant loss in terms of yield and efficiency, and this could not be said to be a production (formulation) method that could contribute to the medical field in a very practical manner. According to the final method, it has become possible to easily produce large quantities of uniform liposomes.

Until about 10 years ago, the formulation technology for Rihasomes was focused on how to prepare a solubilized micelle solution.
Various improvements have been made in terms of how to remove various dispersants and organic solvents that are inconvenient for administration from this solubilized micelle solution. And now,
The next step is to research how to formulate liposome formulations that are stable over a long period of time.

Under these circumstances, the liposome formulation method of the present invention allows the encapsulated main drug to be obtained in a stable state even if it is freeze-dried. However, during the freeze-drying process, most of the main drug that was not encapsulated leaks out, or when the main drug that has been encapsulated is returned to the suspension form after freeze-drying, most of it is The fact that the drawbacks such as leakage from inside the Ritz-7° can be solved is considered to be a sufficient contribution to the establishment of a new administration method.

[Brief explanation of drawings]

FIG. 1 shows a sample containing ferritin obtained in Example 1.
This is a graph showing the state before freeze-drying and the elution no.P turn when the suspension was used again after freeze-drying using a liposome suspension after dialysis. A is before freeze-drying. The mouth is after freeze-drying.

Claims (1)

[Claims] As a liposome membrane composition, a solubilized micelle solution is prepared based on phospholipids or phospholipids and cholesterol,
Next, add the main drug to the solubilized micelle solution and disperse and dissolve it while stirring.Next, use a dialysis membrane, inject the main drug solution into the membrane, seal it, and dialyze under running water. After performing this, the solution inside the dialysis membrane is taken out, put into column chromatography, and subjected to gel filtration.This separates the solution into a dispersion solution of liposomes encapsulating the main drug and a liquid in which the main drug is not encapsulated but in a free state. 1. A method for preparing stable liposomes encapsulating a main drug, which comprises fractionating, separating a dispersed solution of liposomes encapsulating the main drug, and rapidly freeze-drying the same.