JPS5874619A - Liposome and its preparation - Google Patents

Abstract

PURPOSE:To obtain a liposome for the oral administration, by incorporating a steryl glucoside and steryl glucoside monopalmitate as active constituents with a phospholipid, etc. CONSTITUTION:One or two or more types of steryl glucoside, e.g. beta-sitosteryl-beta- D-glucoside, and steryl glucoside monopalmitate, as main drugs are added to chloroform containing a phospholipid, e.g. lecithin or sphingolipid phosphoglyceride, dissolved therein, and if necessary a membrane stabilizer, e.g. cholesterol, or a charging agent is added thereto, and dissolved therein. The chlorofrom is then distilled away, and a buffering agent, etc. is then added to give a liposome. The ratio between the main drugs and the lipid component as follows: based on one pts.wt. main drugs is 1-10pts.wt. phospholipid, 0.1-5pts.wt. sterol and 0.05-0.5pts.wt. charging agent. EFFECT:Intravenously administrable and easily sterilized under heating. USE:An injection adaptable to the remedy in the region of the hemostasis, reinforcing the blood vessel and antishock.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to steryl glucosides or steryl dulcosides. Steryl glunide and steryl glucoside novaluntate contain nitrogen in plants, so soybean 1
m yi.

Various natural ingredients such as koma beans and grapefruit dregs.

For example, the method of T. Kiribucbi et al.
ulturs+I Biologlcs+1ch*m1
try Σ Volume 8, fyo page ~] page 8.

(1966), mainly β=sitosteryl”-p
-D-Gluknight, stigmasteryl β-D-
Gluconide.

A mixture of campesteryl glutunides and fatty acid esters of these mixtures are extracted and separated. In order to obtain steryl gluconide from plant materials, a method is used in which the mixture obtained by the above method is hydrolyzed with an alkali. In addition, steryl gluconide peak nopaltate is
A method is used in which the steryl glucoside obtained by the above hydrolysis is used as a raw material to synthesize pallic acid.

Table 1 shows the composition ratio of steryl glucoside extracted and separated from plants for some materials.

Table-1 On the other hand? IJ ruglucoside can be synthesized using known synthetic methods (for example, Chemlcs +
I BrIchta>Volume 105, 10117-1131
These gluconides can be synthesized using the above methods (page), and monopalmitate can also be synthesized using these steryl glucosides as raw materials.i Steryl glucosides are soluble in pyridine and slightly soluble in dioxane. However, it is soluble in alliums and ketones ((.

It is almost insoluble in general organic solvents such as surface hydrogen and halogen-containing solvents, and almost insoluble in water. There are almost no differences in the physical and chemical properties of steryl gluconides, even if the sterol moieties are different, or even if they are mixtures extracted from plants. Tate is slightly soluble in nonpolar solvents, alcohols, but almost insoluble in water. There are almost no differences in the physical and chemical properties of steryl glucoside monopalmitate, even if the steryl moiety is different or if it is a mixture extracted from plants.

Steryl glucoside or steryl glucoside monopalmitate is disclosed in 41kai No. 854-11849, 41
Kaisho 53-1099! ! As shown in Newsletter No. 4, -Has hemostasis, blood vessel reinforcing, and anti-shirotic effects;
I [It is an important compound as a medicine. These compounds are desired to be made into injections because of their drug layer action, but as mentioned above, they are insoluble in water, so it has been impossible to dissolve them in an aqueous solvent to make injections. Therefore, in order to make these compounds into injectables, we tried dissolving them in non-aqueous solvents or making them into suspensions, but in the former method, we tried to dissolve them in non-aqueous solvents or suspend them. The latter has low solubility and the desired efficacy cannot be obtained, and although it is possible to prepare the latter, when it is administered to a living body, the elution of the compound from the administration site is slow, so a certain level of medicinal efficacy cannot be expected. It was also unsuitable as an injection. in this way.

With the conventional method of making injectables from poorly soluble compounds, it was impossible to provide injectables of steryl glucoside or sterylglu; nide monopalmitate, and special technology was required. . In response to such requests, the present inventors have already responded to the 41st meeting II! $3-3121 (1
Issue Publication # *' in 1981-201! As revealed in the @7th newsletter, etc., we have successfully developed a method to make these compounds water-soluble using a hydrophilic solvent and a solubilizing agent. However, even with these technologies, still...

1) Steryl glucoside and steryl glucoside monopal electrotate have extremely low affinity for water, so a relatively large amount of solvent or external activator is required to make a certain amount of these compounds water-soluble. shall be.

2) The surfactant precipitates and adheres to the ampoule during heat sterilization. 3) When injected intravenously, the efficacy of the compound is affected by the solubilizing agent used. However, there were still some issues to be improved, such as the lack of medicinal efficacy with the exception of some solubilizers such as ethylene (604-numbered castor oil). Moreover, it has a composition that can be solubilized to a high degree of purity and can be applied to areas such as vascular reinforcement and anti-ciliary therapy that require high dosages.In addition, it is an injection solution that shows medicinal efficacy instantly even when administered intravenously. Developed (as a result of intensive research,
The inventors have discovered that these objectives can be achieved all at once by including the Todoroki compound in liposomes, and have completed the present invention. That is, the present invention is a liposome, which is a novel composition of steryl glucoside and steryl glubunide monopal ζtate, and a method for producing the same.

In recent years, liposomes have been used as a drug in the literature, and their structure, composition, manufacturing method, etc. are explained in some reviews.

For example, Tyrell, D. H., et al., Blochlm.
icm at BlophyslcmActs MR4
B? , P, 2! Ito3G! (1976), Pls.
Arrived at der JH et al.

LIf@8cIence20(7)'Xh, 1109
~1020 (II??) > - Ironically, liposomes are made by taking the phospholipid chloroform S* in an eggplant I17 tube, distilling off the chloroform to form a thin film of the lipid in the container, and adding it to the container. The aqueous drug solution is contained in small spheres (vesicles) that are formed when the aqueous drug solution is added together with a buffer solution and then removed from the container by stirring. Therefore, in this case, it was necessary to separate the drug not incorporated into the liposomes by gel filtration or ultracentrifugation. Furthermore, as long as the liposome takes the form described above, even if it is separated and purified from the free drug, the aqueous drug solution inside the liposome will diffuse into the outer aqueous layer within a short period of time, making it difficult to put it into practical use as a pharmaceutical preparation. It had However, through intensive research, the present inventors accidentally discovered that steryl glucoside or steryl glubunide monopal ζtate has a strong affinity with the lipids that make up liposomes. 111 It is possible to incorporate these compounds into liposomal lipids.
Completed the present invention. That is, in the present invention, phospholipids are dissolved in chlorotetraform, steryl gluconide or steryl gluconide monopaltate is added thereto, and if necessary, a membrane stabilizer such as cholesterol or a charging agent is added. After dissolving, the aa form is distilled off, and physiological saline or a buffer solution is added thereto, followed by stirring or ultrasonic irradiation. A liposome composition containing steryl glucoside or steryl glubunide monopal ζtate in a lipid, and a method for producing the same.

The phospholipids used in the present invention include natural phospholipids such as lecithin, sphingolipid phosphoglyceride, and ganglioside, or di-2ristoyl, di/(
Thin toy roux. Distearyl-, dioleyl-, phos-7
Synthetic lecithins such as T-tidylcholine are mentioned, among which natural or synthetic lecithins are mentioned.As liposome membrane stabilizers, prestyrol, β-cytosteel, and stigmasterol are mentioned.

Examples include campesterol or a mixture of sterol extracted from plant materials, and the charging agents used include stearyl avy, which provides a positive charge, and 7-os71 tidic acid, diaceteryl, which provides a negative charge. Shun can be given.

On the other hand, the steryl daltunide targeted by the present invention is as follows.

β-p) st4lyl-p-D-/runite, stidamasteryl β-D-gelcosa 1do, cambesteryl β-
D-/lucoside, cholesteryl β-D-glucoside, and the above-mentioned steryl glucoside extracted from plant materials. 6-! of the above steryl gluconide! Monopal ζtate is used. The ratio of crude drug to lipid component is 1 part (weight) of crude drug to 1 to 10 parts of phospholipid, preferably 3 to B parts, sterols, 0.1 to S parts, preferably O, S to 3 parts, charged Depending on the strength and time of stirring or ultrasonic irradiation, multilamellar liposomes or ennilamellar liposomes can be obtained.

The liposome containing steryl glucoside or steryl glubunide monopal ζtate obtained in the present invention can be used for parenteral administration and has the following advantages.

1) As low as 3 to 5 parts (by weight) for 1 part (by weight) of the main drug 3) With the above ratio, it is possible to prepare an injection solution with a crude drug content of several 70%, which requires not only hemostasis but also a high dosage. It is possible to provide an injection solution that can be used to treat areas such as blood vessel reinforcement and anti-sickness.

3) The composition of the present invention exhibits reliable drug layer action even when administered intravenously. 4) Heat sterilization can be carried out easily.

In addition to the above-mentioned advantages, if 0 liposomes are filled into an ampoule with N1 gas and stored under direct light, they have the excellent property of being stable in terms of appearance and content for at least 2 years at room temperature.

The present invention will be explained in a different manner using typical examples below.

Example 1 Add egg yolk lecithin zoq to S.- eggplant m7 rasp,
Taroroform! m&:@understood. This was followed by β-sitosteryl-pD-dalbunide swI/1. After dissolving 3 volumes of Restyrol, chloroform was distilled off using a rotary evaporator on a SO' water bath. After spraying the residue with N and a scalpel for 10 minutes, it was dried in a vacuum dryer for 6 hours, and then saline was added to it for 10 minutes. - Added to the top part - enemy wave ho genaizu - (ultrasonic technology 11R2S K
Hz, I SOW) rNselftjXkiRlower85kna
*qgenize V, a pale yellow, almost clear case was obtained. When this was sterilized twice using a Poffilter 08 type, 99.6% of the β-sitosteryl 1-D glucoside before filtration was detected in the P solution. Fill a 991 ampoule with N and gas, and place it in an autoclave for 1 hour.
Sterilization was performed at 20v for 20 minutes. No precipitation of the active ingredient or lipids was observed during sterilization. This injection also contains 11
When stored under light, no precipitation of foreign substances or decrease in the content of the main drug was observed for more than 2 years at room temperature. This injection solution and a blank injection solution obtained by removing the crude drug from the composition of this injection solution were added to
The method of Motohashi et al. (Journal of Tokyo University of Medical Sciences) S(5) 106111<
ill! ! As shown in Table 2, the average hemostasis time in the blank group was 14.7±0682 minutes, while the average hemostasis time in the blank group was 14.7±0682 minutes. In the 2q/# administration group, bleeding stopped in 12.3±0.27 minutes, and a significant difference was observed at the P<0.01 level, proving the effectiveness of intravenous administration of one injection.

Implementation-2″ dipalmitoyl lecithin 2 in s〇- eggplant l17 lasco
01IP.

β-F steryl, β-D-gluside, Novatal tate 1019, Restyrol 519, chloroform 3
-I understand.

The chloroform was dissolved in water at 30°C and distilled off under reduced pressure using a rotary evaporator. This residue was blown with ash gas for 10 minutes and further dried in a vacuum dryer for 6 hours. 0 to pH 6.3 with phosphate buffer Im (trisodium phosphate 1
．． 11L Sodium dihydrogen phosphate 64F and salt 5. IP dissolved in distilled water for injection (1/1) S
- was added and homogenized for 3 minutes using an ultrasonic polymerizer to obtain a slightly cloudy liposome solution. When this was filtered with a Millipore filter - HA type, F
1 contained 19.24 β-sitosteryl 1-D-glucosoid peak nopal ζtate. P-Kan is N5t
Fill ampoules with ice and place in autoclay.

High-pressure steam sterilization was performed for 120 minutes at 110 tons. The pharmacological effects of intravenous injection of this injection are shown in Table 3, Section 2.

Example 3! ! Take 4 q of dioleylphostudecidylcholine and 4 q of dipalitoyl sulfatedylpurine in an eggplant-sealed flask of 011j, dissolve them in 2-chloroform, and add 3 q of cholesteryl β-D-gluconide and 1 q of restyrol. After dissolving, the nitroform was distilled off under reduced pressure on a Set+ water bath using a Tully evaporator. Cholesteryl β-D was prepared in the same manner as in Example 1.
- Liposome liquid 91 containing gluconide 3 capes was obtained.

The pharmacological activity of this injection solution is shown in Tables 2 and 13.

Example 4 11 dipalmitoyl lecithin in 50 eggplant 11 flask
519 was dissolved in chloroform 2-, and stigmasteryl β-D-glucoside swg and stigmasterol 6-stearyl urethane 111 II were added and dissolved therein, and the chloroform was distilled off using an evaporator on a 30°C water bath. did.

Thereafter, by the same operation as in Example 2, a positively charged liposome solution 1sllj containing stigmasteryl β-D-gluconide 5wI/I was obtained. Table 2, 1 shows the pharmacological activity of this injection.
4.

Example S A negatively charged liposome solution 5d containing 1 part of stigmasteryl β-gluconide was obtained in the same manner as in Example 4 by adding 1119 dicetyl phosphate in place of 1 q of stearyl acene in Example 4. Table 2 shows the pharmacological activity of this injection.
Shown on Cliff S.

Example 6 Add egg yolk lecithin zeoq to 100-117 eggplants.

Couloform 511/ was added to this to dissolve it, and then steryl gluconide SOQ and COV extracted from soybeans were dissolved.
After adding and dissolving Stit-Ru 2swi, Set
Chloroform was distilled off under reduced pressure using a rotary evaporator on a + water bath.

This residue was sprayed with N gas for 10 minutes, and then dried in a vacuum dryer for 6 hours. This was combined with the phosphoric acid mild II used in Example 2! ! 111 was added to the mixture, and the mixture was homogenized using a probe ultrasonic generator for 5 minutes under a N and air stream to obtain a slightly cloudy ribosome solution. This is a Millipore filter
Filtered with HA type. At this time, F* contained 98.24 steryl gluconides. Thereafter, an sml ribosome solution containing steryl gluconide SOq was obtained in the same manner as in Example 1. Table 1 shows the medicinal properties of this injection.
Shown on Cliff 6.

Example 1 Add egg yolk lecithin 20019 to a 100-liter eggplant flask.

To this, add chloroform 5II7 and dissolve the steryl gluconide nopal hyotete extracted from II scales by adding 60 capes and cholesterol SO.
The following procedure is the same as in Example 6 to extract a11 steryl group: y
? Idomonopal Shoguchi! ! Liposome containing 019*
Obtained S-.

The am activity of this injection solution is shown in Tables 2 and 1. From the results in Table 2, it is clear that the composition of the example of the present invention exhibits drug layer activity when administered intravenously.

Claims (1)

[Claims] (11 Step 9 contains dalphnide and sterylglutz!id) j (Containing one or more types of Rumichii as an active ingredient (21 Single lame-shaped claim sgi)
* Liposome = (3) multi-lamellar IJ posome according to claim 1. (41 active ingredient is contained in the lipid. The liposome according to any one of claims 111 to 3) (5) The lipid is a natural or synthetic phosphatidylpurine. JI The liposome according to any one of Claims 118511 to 4; (6) The liposome according to Claim 1111!i, wherein the natural sulfantidylpurine is egg yolk lecithin. ()) Synthetic Claim 1, wherein the phosphatidyl phosphorus is at least one of didensudyl phosph tidyl purine, dibar hyalophosph t tidyl purine, distearyl phosph decyl coli y, and dioleyl phosph 1 tidyl purine. Liposome. (8) Contains steryl as a lposome stabilizer. 41. The liposome according to any one of Claims 1 to IIT. (9) The sterol contains at least one of cholesterol, β-sitosterol, stigmasterol, and campesterol, a substance that imparts a positive charge to the liposome as set forth in claim 8. Patent I! Riboso according to any one of Clauses 1 to 9 above, characterized in that Patent I! Riboso according to any one of Clauses 1 to 9. I Claims I! Clause 10 of Clause 1, wherein the substance imparting a positive charge is stearylamine Liposome I-▲. @Characterized by containing a substance imparting a negative charge. *Liposome according to any one of claims 1 to 9. The substance imparting a 113lk charge is phosphatid acid or It is dicetyl phosphate. Ribono ▲● steryl darconide described in the patent claim IIs 1z is β-sitosteryl β-D-gluconide, stigmasteryl β-D.
- Glupnide. It is a monovartate. 411. The liposome according to any one of claims 1 to 411. One or more of steryl glucoside and steryl dal sf nopal ζ tate are dissolved in a chloroform solution containing the tetralipid and optionally a sterol and a charging agent. Then, the chloroform was distilled off to form a lipid film in the container. It is characterized by adding a medium to this and stirring or irradiating it with ultrasonic waves. Method for producing liposomes.