JPH01197431A - Liposome including antibiotic - Google Patents

Abstract

PURPOSE:To obtain liposome including an antibiotic which is used in treatment for intractable infectious diseases, by allowing the liposome to include an antibiotic and coating the liposome with a polysaccharide derivative occurring in nature on its surface. CONSTITUTION:An antibiotic, preferably ampicillin or minocycline is included inside the liposome whose membrane is composed of a phospholipid or a combination thereof with cholesterol. Then, the liposome is coated with a natural polysaccharide derivative such as mannan or amylopectin on its surface. The polysaccharide derivative is substituted 0.5-5 primary alcohol groups per 100 units of the monosaccharides with cholesteryloxycarbonyl- aminoethylaminocarbonylmethyl groups. Natural release of the antibiotic included inside is controlled and the efficiency is improved in the transfer of liposome into cells.

Description

[Detailed Description of the Invention] (Industrial Application Classification) The present invention relates to a liposome in which an antibiotic is encapsulated inside the liposome, and specifically, an antibiotic such as ampicillin or minocycline is encapsulated inside the liposome, and the liposome is This invention relates to a liposome whose surface is coated with a naturally occurring polysaccharide derivative.

More specifically, the present invention specifically improves the bioavailability of antibiotics encapsulated inside liposomes by coating them with specific naturally occurring polysaccharide derivatives. , to suppress the natural outflow of antibiotics encapsulated inside.
This invention relates to a liposome with improved cellular transfer efficiency.

(Conventional technology and its problems) Liposomes, which are formed when naturally derived lipids are redispersed in water, have a very high approximation as an artificial cell model with a cell membrane structure, and can be used as tissue-directed drug carriers (drug carriers). It has good biocompatibility as a pharmaceutical material such as carriers), artificial red blood cells, cell modifiers, and enzyme immobilization bases, and its potential for use in a wide range of fields of medicine and pharmacology has been proposed. There is.

However, even when conventional liposomes are applied to the above-mentioned purposes, no results have been obtained that are suitable for actual use. The reason for this is firstly that conventional liposomes themselves are essentially an assembly of natural lipids through non-covalent interactions, so even if they have good biocompatibility, they do not meet the requirements for practical use. lack of structural stability of the liposome itself;
The second point is that specific target cells or target tissue tropism, which is most important as a drug carrier, is hardly exhibited.

Therefore, the present inventors have conducted studies to improve the above-mentioned drawbacks, and have improved the mechanical strength of liposomes under physiological conditions by coating the surface of liposomes with polysaccharide derivatives. We have discovered that when liposomes treated with the above are administered to living bodies, they exhibit the ability to selectively target target organs, and we have already completed patent applications for these points [e.g., Japanese Patent Application Laid-Open No. 61-69801 No. (Patent Application No. 59-189746).

In this way, the technology of coating the surface of liposomes with a certain type of polysaccharide derivative is thought to bring great light to the field of liposome utilization, which had been considered difficult to put into practical use. development was strongly desired.

By the way, the recent development of antibiotics has been remarkable, including penicillin antibiotics and cephalosporin antibiotics, which are being used to treat various diseases caused by pathogenic bacteria that were previously considered difficult to treat. It's starting to look like this. However, with the development of these antibiotics, there are some diseases for which antibiotics are almost completely ineffective. For example, it has been confirmed that conventional oral administration or intravenous injection of antibiotics themselves does not effectively act against pathogenic bacteria that proliferate within cells.

In particular, Listeria monocutogenes
Listeriosis, caused by Listeriosis (Ria monocu to enes), is a disease caused by bacteria that proliferate in phagocytes (macrophages), causing pneumonia, meningitis, and sepsis (Listeria sepsis), with a high mortality rate. It is quite expensive and is one of the intractable diseases.

If we were to consider an effective treatment for listeriosis, we would be able to kill or bacteriostatic the pathogenic bacteria on the spot by directly acting on the bacteria that proliferate within phagocytic cells, such as ``notanm'' and ``nes''. Although this is a good idea, the current situation is that no means of effectively administering antibiotics only to phagocytes has been established.

Therefore, the present inventors conducted studies to provide a therapeutic means for this type of intractable disease, and found that when the surface of the liposomes described above was coated with a certain type of polysaccharide derivative, the liposomes had a specific cellular migration property. We focused on specific technology that improves

In other words, if an antibiotic is encapsulated inside the coated liposome, the liposome becomes a drug carrier with specific cell tropism derived from the terminal sugar chain structure of the polysaccharide used for coating, and is taken up into phagocytes. Then, the liposome collapses on the spot, and the antibiotic encapsulated inside is released, allowing it to directly attack pathogenic bacteria within the phagocytic cells, while causing almost no harm to other normal cells. They thought that this method could provide an effective means of treating intractable diseases, and as a result of their extensive research, they completed the present invention.

That is, the present invention encapsulates an antibiotic inside a liposome and coats the surface of the liposome with a polysaccharide that has a high affinity for phagocytes, thereby stabilizing the structure as a drug carrier and improving cell migration.
Furthermore, the toxicity of the antibiotic encapsulated inside the liposome can be reduced, and the antibiotic itself, which has not been studied much so far, can be applied as a missile therapy and can be effectively applied to the treatment of intractable diseases.

(Structure of the Invention) The present invention to achieve the above-mentioned object specifically provides: A liposome characterized in that an antibiotic is encapsulated inside the liposome and the surface of the liposome is coated with a naturally-derived polysaccharide derivative. Regarding.

The configuration of the present invention will be explained in more detail below.
The liposome itself provided by the present invention can be produced by a conventionally known method, and conventionally known liposomes whose liposome membranes are composed of phospholipids or phospholipids and cholesterol can be used. Examples of such phospholipids include egg yolk phospholipids such as egg yolk lecithin, soybean phospholipids such as soybean lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, dicetyl phosphate, stearylamine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol, etc. These can be used alone or in a mixture of two or more.

The liposome of the present invention is composed of at least the above-mentioned phospholipids, but may also contain cholesterol or glycoprotein depending on the type of antibiotic encapsulated inside and the type of naturally-derived polysaccharide derivative coated, which will be described later. .

The naturally occurring polysaccharide derivatives that coat the surface of the liposome of the present invention are, for example, derivatives of water-soluble naturally occurring polysaccharides such as pullulan, amylopectin, amylose, dextran, and mannan. Among these, mannan and amylopectin are preferably used when considering the specific interaction with phagocytes that is the object of the present invention.

In the present invention, such a water-soluble naturally occurring polysaccharide is used as a specific derivative, and the surface of the liposome is coated with the derivative. Examples of such a derivative include the following derivatives. That is, in order to enhance the coating effect of the above-mentioned water-soluble polysaccharide through non-covalent interactions on the liposome surface, and to achieve structural reinforcement and phagocytosis of the liposome, the monosaccharide constituting the polysaccharide must be Approximately 0.5 to 5 primary alcohol groups per 100 units of (glucose) have the following formula: %
Polysaccharide derivatives substituted with groups of the formula % (representing a carbonyl group) are preferred.

The reason why a cholesteryloxycarbonyl group is preferable as R in the above substituent in this case is because it has been demonstrated that when a liposome is coated, the cholesterol group is oriented in a wedge shape in the lipid layer of the liposome (Biochem
, Biophys, Acta, 857265-2
70 (1986)), such substituents are believed to be particularly suitable.

In addition, the above-mentioned specific naturally occurring polysaccharide derivatives basically contain 0.5 to 5 per 100 units of monosaccharide constituting the polysaccharide.
It is obtained by reacting chloroformic acid corestyryl ester with a substance in which a certain amount of primary alcohol groups are substituted with groups represented by the following formula: % formula %.

Various antibiotics can be encapsulated inside the liposome of the present invention constructed as described above, depending on the intended use of the specific liposome of the present invention, including penicillin antibiotics and cephalosporin antibiotics. substances, tetracycline antibiotics, etc. can be used. However, in order to treat listeriosis, which is an intractable disease mentioned above, ampicillin, a penicillin antibiotic,
Liposomes in which minocycline, a tetracycline antibiotic, is encapsulated inside the liposome are preferred.

(Particularly preferred embodiments of the present invention) The present invention relates to antibiotic-encapsulated liposomes, and a particularly preferred embodiment is a liposome in which minocycline or ampicillin is encapsulated inside the liposome, and the surface of the liposome is coated with cholesterol group-substituted amylopectin. .

(Effects, etc.) The present invention will be described in more detail below along with its manufacturing means, effects, etc.

In the present invention, liposomes refer to phospholipids such as the above-mentioned egg yolk phospholipids, soybean phospholipids, phosphatidylcholine, etc. alone, or unilamellar liposomes and multilayer liposomes composed of these phospholipids and cholesterol. This is a multilayered liposome, and was obtained by a conventionally known manufacturing method for liposomes. For example, one method for producing such liposomes is to place egg yolk lecithin or soybean lecithin together with a suitable solvent in an eggplant-shaped flask, distill off the solvent under reduced pressure, and form a thin film of phospholipids on the flask wall. are peeled off by adding glass beads and an appropriate buffer (e.g., 0.01M phosphate buffer containing pH 7, 4, 0, 1M NaCl: PBS buffer) to form a liposome membrane, and after ultrasonication. This includes liposomes obtained by collecting liposome fractions through a Sephadex or Sepharose column and distilling off the solvent.

In addition, for example, freeze-drying means can be applied to the solvent distillation, and the liposome can be taken out in the form of a powder by such means.

In the liposome of the present invention, a desired antibiotic is encapsulated inside the liposome, and the means for encapsulating the antibiotic may be, for example, a membrane component of the liposome (phospholipid alone or An antibiotic-encapsulated liposome can be obtained by coexisting a desired antibiotic with phospholipids and cholesterol and treating in the same manner.

For example, in a preferred embodiment of the present invention, ampicillin or minocycline is placed together with egg yolk phospholipid or soybean phospholipid in an eggplant-shaped flask, chloroform is added to dissolve it, and a thin film is formed by distilling off the solvent under reduced pressure using a rotary evaporator. After forming and drying, PBS
After swelling with a buffer solution, it can be stirred with glass beads to form liposomes.

2. Ta
The naturally occurring polysaccharide derivative used in the present invention is one that coats the surface of the antibiotic-encapsulated liposome selected in the previous section. About 0.5 to 5 primary alcohol groups are esterified with a cholesterol derivative.

Such esterification can be carried out roughly as follows.

That is, a naturally occurring polysaccharide such as pullulan, amylopectin, amylose, dextran, mannan, etc. is reacted with sodium monochloroacetate to obtain a sodium salt of carboxymethyl polysaccharide. Next, this was reacted with ethylenediamine hydrochloride to obtain aminoethylaminocarbonylmethyl polysaccharide hydrochloride, which was dissolved in anhydrous dimethylformamide. To this solution was added a solution of cholesteryl chloroformate in anhydrous dimethylformamide, and pyridine was added dropwise. By performing the reaction, the naturally occurring polysaccharide derivative used in the present invention can be obtained.

The naturally occurring polysaccharide derivative thus obtained can be identified by infrared absorption (IR) spectrum, nuclear magnetic resonance (NMR) spectrum, etc., and the number of introduced cholesterol groups can also be determined by elemental analysis and IH-NMR. It can be measured by the ratio of the number of protons in the polysaccharide moiety to the number of protons in the cholesterol moiety.

3. In the liposome of the present invention, which is a +posome, the antibiotic-encapsulated liposome produced in the previous section 1 is coated with the naturally occurring polysaccharide derivative obtained in the section 2. The coating means can be carried out as follows.

That is, by adding the derivative-containing aqueous solution obtained in Section 2 to the aqueous solution in which the antibiotic-encapsulated liposomes obtained as described in Section 1 are formed and stirring, the desired coating treatment can be carried out. can be administered.

In addition, the naturally occurring polysaccharide derivatives used in the coating treatment may be used in combination of two or more types in addition to using a single derivative.

The coated liposome can be obtained in powder form by freeze-drying.

(Example) Examples of preparing liposomes of the present invention and pharmacological activity tests using the liposomes will be described below.

Amylopectin (average molecular weight: 50,000) 1.0 g (6,17X1
0-3 mol sugar unit) was dissolved in a 1.35 M aqueous solution of sodium monochloroacetate 18,5+++1, and the mixture was stirred with l
Add 5.0 ml of 0N aqueous sodium hydroxide solution, dilute with distilled water to a total fi of 50 ml, and react at 25° C. for 7 hours. Then IM-sodium dihydrogen phosphate solution 5m
The reaction was stopped by adjusting the pH to 7 with a 5N aqueous hydrochloric acid solution, and then using a dialysis tube (Visking).
After dialysis (4 days against a saturated aqueous solution of toluene and 1 day against distilled water), the solution was concentrated under reduced pressure to about 10 ml and used as it was in the next reaction.

In addition, absorption of carbonyl groups was confirmed in the IR spectrum of the freeze-dried product.

Next, the concentrate obtained as above was diluted with 15 ml of distilled water.
Ethylenediamine dihydrochloride 0.74 diluted with stirring
g (5,56X 10-3 mol) and 1-ethyl-3-(3-dimethylamino)propylcarbodiimide hydrochloride 0.21 g (1,10X10X10-3)
was added, and the pH was further adjusted to 4.7 with IN-hydrochloric acid aqueous solution and IN-sodium hydroxide aqueous solution. This solution was stirred at 25°C for 7 hours, and after the reaction, it was dialyzed (4 days against 0.2M sodium chloride and 1 day against distilled water), freeze-dried, and the aminoethylaminocarbonylmethyl (1,4 )-0.75 g of amylopectin was obtained.

Freeze-dried product obtained as above 0.54 g (3,3
3X 10-3 mol sugar units) was added to 14 ml of anhydrous dimethyl sulfoxide and dissolved by heating at 70-80°C in an oil bath.

Then, after adding 3 ml of anhydrous pyridine to this solution, 0.60 g of cholesteryl chloroformate (1,33X
10-3 mol) of anhydrous dimethyl sulfoxide 4 ml
The solution was added and the reaction was carried out at the same temperature for 7 hours. After the reaction is complete, add ethanol and collect the resulting polysaccharide derivative by filtration.
After thorough washing with ethanol and ether, it is dissolved in a small amount of water and freeze-dried to obtain the target cholesterol group-substituted (degree of substitution 1.33) -amylopectin with 0.
I got 7g.

Incidentally, a reaction was carried out using mannan and dextran instead of amylopectin in the same manner as above, and cholesterol group-substituted mannan and cholesterol group-substituted dextran could be obtained.

(a) Take 30 mg of egg yolk lecithin in an eggplant-shaped flask, dissolve it in 3 ml of ethyl ether, and add 100 ml of minocycline to this solution.
g and 1 ml of physiological saline were added thereto, and ultrasonic waves were irradiated at 0° C. and 28 KHz for 10 minutes using a bath-type sonicator to obtain a liposome dispersion. After removing ethyl ether from the obtained dispersion using a rotary evaporator at 200 rpm under a reduced pressure of 350 mmHg, 3 ml of physiological saline was added and peeled off by shaking using a Portex mixer, and further 200 r under a reduced pressure of 700 mm Hg.
The ether is completely removed using a pm rotary evaporator for 1 to 2 hours. The thus obtained liposome dispersion was centrifuged at 20,000 rpm (1 hour) at 0°C, further washed with physiological saline, and subjected to the same centrifugation twice to obtain a large monomer containing minocycline. A lamellar liposome (LUV) was obtained.

(b) Next, 30 mg of cholesterol group-substituted amylopectin obtained in step I above was added to the liposome prepared as described above, and minocycline, which is the object of the present invention, was encapsulated by incubating at 20°C for 30 minutes. Amylopectin derivative-coated liposomes were obtained.

In addition, ampicillin-encapsulated amylopectin derivative-coated liposomes could be obtained by performing the same treatment using the same amount of ampicillin instead of minocycline.

Furthermore, mannan was used as the polysaccharide derivative in place of the amylopectin derivative used in step (b) above to obtain a corresponding polysaccharide-coated liposome.

As mentioned above, the antibiotic-encapsulated liposome of the present invention is obtained by encapsulating an antibiotic inside the liposome and coating the surface of the liposome with a naturally-derived polysaccharide derivative. Basic experiments show that the liposome has high tropism toward phagocytic cells, becomes a drug carrier, is taken up by the phagocytic cell, and the liposome undergoes metabolic collapse on the spot, releasing the encapsulated antibiotic and acting against pathogenic bacteria. has been proven.

Therefore, it can be said that the antibiotic-encapsulated liposome of the present invention is effective in treating Listeria sepsis caused by bacteria that proliferate in phagocytes. Therefore, we experimentally caused Listeria infection in mice and observed the therapeutic effect of the antibiotic-encapsulated liposome of the present invention.

1.1 Steps: (1) Li5teria monoctoenes isolated from a septic patient associated with malignant lymphoma was used as a bacterial strain, passed through the abdominal cavity of a mouse five times, and then cultured on a blood agar medium for 24 hours. It was suspended on the order of 109 CFU/ml in phosphate buffer (PBS) at pH 7.4, divided into 1.0 ml portions and stored at -80°C, and the required amount was used after 7 days or more (maintaining on the order of 108 CFU). .

(2) Listeria infection model was created using ddY strain SPF mice (weight 18-20 g, male) by inoculating 0.2 ml of the following bacteria into the tail vein.

LD50 for mice of L, monoc to enes is 5.7 X106 CFU/m1 (1,4X
105 CFU/mouse) was confirmed in advance, and approximately 10 times this fi (106 CFU/mouse) was used.

2, 1 (1) Amount and method of use of antibiotics A group administered with antibiotics alone was subjected to the experiment as follows, and the amount of antibiotics used was determined based on its therapeutic effect.

10 times the amount of glue of LD5o, monoc to en
es was injected intravenously into the tail, and from 12 hours onward, ampicillin (-daily doses of 40 mg/kg and 200 mg
/kg) and minocycline (-day dose, 4 mg
/kg and 20 mg/kg) was administered via the caudal vein for 7 days in two divided doses at 9:00 am and 7:00 pm.

Figure 1 shows the change in survival rate as a therapeutic effect. As is clear from the results in Figure 1, ampicillin 200m
g/kg and minocycline 20 mg/kg administration group (
(10 animals in each group) had a survival rate of 30%.

(2) Intravenous administration of antibiotic-encapsulated liposomes (1) above
Judging from the results, it was decided that treatment with antibiotic-encapsulated liposomes would be carried out at a daily dose equivalent to 40 mg/kg of ampicillin and 4 mg/kg of minocycline.

Liposomes containing various amounts of these antibiotics were administered twice a day through the tail vein of mice for 5 days, and changes in body weight and survival rates of infected mice were examined.

-1-i-Izumi The results are shown in Figure 2.

As is clear from Figure 2, in the ampicillin treatment group, the survival rate was 70% in the ampicillin alone group, and the survival rate in the liposome-encapsulated group was 70%.
00%, and no significant difference was observed between the two,
The treatment results of the liposome-encapsulated group tended to be superior overall.

On the other hand, in the minocycline treatment group, the survival rate was 20% for the minocycline alone group and 80% for the liposome-encapsulated group, with a significant difference (p<0,05) between the two, indicating that the liposome-encapsulated group had a particularly excellent therapeutic effect. It turns out that the results are as follows. As another indicator, changes in the body weight of infected mice were recorded, and the ampicillin-encapsulated liposome group showed an increase in body weight over time, and on the 6th day of treatment, the average weight at the time of infection was 20 g, but it was 24.5 g. In other groups, those with significant weight loss died early, and those that survived until day 6 were treated with ampicillin alone (8 animals).
Minocytalif encapsulated liposome group (10 animals) averaged 20
g, and the average body weight of surviving mice (3 mice) treated with minocycline alone decreased to 18.5 g. Up to this point, all control groups (no treatment group, liposome only treatment group) had 100% mortality.

Comprehensive judgment of the above results reveals that the antibiotic-encapsulated liposome of the present invention is extremely effective against refractory infections.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the therapeutic effects of various antibiotics on mice infected with Listeria, and FIG. 2 is a diagram showing the therapeutic effects of the antibiotic-encapsulated liposome of the present invention on mice infected with Listeria.

Claims (5)

[Claims] (1) An antibiotic-encapsulated liposome characterized in that an antibiotic is encapsulated inside the liposome and the surface of the liposome is coated with a naturally-derived polysaccharide derivative. (2) The liposome according to claim 1, wherein the antibiotic is ampicillin or minocycline. (3) The liposome according to claim 1, wherein the liposome membrane is composed of phospholipids, phospholipids, cholesterol, and polysaccharide derivatives. (4) The liposome according to claim 1, wherein the naturally occurring polysaccharide is at least one selected from the group consisting of pullulan, amylopectin, amylose, dextran, and mannan. (5) A patent in which the naturally occurring polysaccharide derivative is one in which 0.5 to 5 primary alcohol groups per 100 units of glucose constituting the naturally occurring polysaccharide are substituted with cholesteryloxycarbonyl-aminoethylaminocarbonylmethyl groups. Liposome according to claim 1.