JPS62501631A - Liposome retention method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Liposome retention method Industrial applications The invention relates generally to liposomes and more particularly.

This invention relates to a method for retaining liposomes containing biologically active molecules. The method of the present invention In vivo drug administration and diagnostic agents It can be applied to the retention of ENTs).

This invention is a joint venture of the National Science Foundation and the University of California under license number CM82-17538. Achieved with government assistance. The Government has certain rights in this invention.

light skin row swelling Liposomes are unilamellar or multilamellar that encapsulate a fluid space (5 spaces). They are lamellar lipid vesicles. The vesicle wall has a polar head and a non-polar tail. formed by a bilayer of lipid components on top. In aqueous (or polar) solutions -m The polar head of is directed outward and diffuses into the surrounding medium. And the non-polar type of lipid The core parts meet each other.

Generates a polar surface and a nonpolar core in the vesicle wall. Unilamellar liposomes include the above There is a single bilayer, whereas multilamellar liposomes generally have substantially concentric rings. There are multiple bilayers of .

Encapsulation of drugs and other therapeutic agents and lipolysis of these drugs in vivo It is known that somesomes are useful. For example, Rahman et al. No. 3, 9!33, 75 issued on November 23, 1976 for Issue 4 includes improved chemistry for encapsulating and injecting antitumor agents in liposomes. Therapy is described. April 21, 1981 against Apple, etc. U.S. Pat. No. 4,263,428, issued in By binding the drug in somesomes, it is possible to target selected cellular sites (s) in mammalian tissues. elective cell 5ite) to enable Attack 91 (delivere d) antitumor agents have been described. Liposomes reduce the toxicity of drug administration 1 tissue distribution is altered, increasing the efficacy of the drug and further improving the therapeutic index.

Furthermore, liposomes can carry various chemicals, biochemicals, genetic materials, etc. in vitro ( useful for leading to viable cells (in vitro) and as a carrier for therapeutic drugs. It is used effectively.

Many methods are known for making liposomes.

Many of them are from Szoka and Papahad. jopoulos) by Ann, Rev, 7 Lμ匪L9:467-50 8 (1980). In addition, several liposome encapsulation methods are patented. In the literature, in particular, U.S. Pat. , 235,871 and U.S. Pat. No. 4,016, issued April 5, 1977, to Suzuki et al. , No. 100.

Encapsulation of therapeutic agents and conventionally active compounds in liposomes has significant commercial potential. However, there are significant difficulties in commercially using liposome encapsulation. The stability is the stability of the liposome over a long period of time. Under certain storage conditions, Although the structure of the posome is fully preserved, such conditions are usually inconvenient and prohibitive. It cannot be used.

One invention has been made to solve this problem.

Natsume Matashiri Therefore, one object of the present invention is to provide a commercially viable method for retaining liposomes. It is.

Another object of the present invention is to produce commercially viable liposomes by freeze-drying. By providing a retention method, the liposomes are able to restore their original encapsulation upon rehydration. Retains nearly 100% of the substance.

Yet another object of the present invention is to provide carbohydrates that can preserve structure and function in biofilms. , to provide a method for retaining liposomes.

Yet another object of the present invention is to use the present invention as an encapsulation material inside liposomes or as Retaining agents, such as trehalose, present in the external solution during drying or both Than.

An object of the present invention is to provide a liposome retention method.

Yet another object of the invention is to retain up to 100% of the original encapsulated material upon rehydration. It is an object of the present invention to provide a lyophilized compound that

Other objects and advantages of the present invention can be found in the following description, claims and claims. It will become clear from the attached drawings.

According to one aspect of the present invention, as a method for retaining liposomes, the structure and mechanism in biofilms are There is a method of freeze-drying liposomes in the presence of a retention agent that can preserve their potency. . Preferred retention agents include carbohydrates having at least two monosaccharide units, especially A preferred compound is the disaccharide sucrose.

Maltose and trehalose.

According to another aspect of the invention, the method also contains a biologically active molecule or therapeutic agent. Liposomes are freeze-dried with a retention agent such as trehalose contained inside them. It is characterized by In preferred embodiments of the invention, suitable compounds, e.g. Trehalose exists both inside and outside lipid membranes. of total retention agents for lipids. The preferred weight ratio is about 0.1:1 to 3.0:1. Especially preferred weight The ratio is approximately 1.0:1.0.

The present invention further provides that when reconstituted by rehydration, liposomes retain their original encapsulation. A heart-warming day that retains virtually all of its quality The present invention retains liposomes containing biologically active molecules through the use of retention agents. Regarding the method. The method of the invention freeze-dries liposomes in the presence of a retention agent. The method of It includes a method of drying or a combination of both methods. favorable retention The agent is a carbohydrate having at least two monosaccharide units attached to a gudicosidic linkage . Particularly preferred retention agents include sucrose, maltose and trehalose. Among the above, trehalose is the most preferred for use in the method of the present invention. It was found to be an effective retention agent.

Trehalose is present in high concentrations in tissues and is involved in survival dehydration. It is a natural sugar capable of hydration. Trehalose plays a role in the dry biofilm structure. It is particularly effective in preserving structure and function. Freeze-dried in the presence of trehalose Liposomes that also contain the trehalose to be encapsulated are particularly well encapsulated. hold the psel. i.e. both internal and external during freeze-drying of liposomes. is exposed to trehalose, upon rehydration it loses 100% of the original encapsulated material. Nearly % is retained. Freeze-dried liposomes without any retention agents This is in sharp contrast to the loss of liposomes and their components, which Combine extensively.

Examples of phospholipids used to form liposomes that can be used in the method of the invention include phosphatide. Zirchloraine, phosphatidylserine, phosphatidic acid and mixtures thereof It is. Both natural and synthetic phospholipids are useful.

Preferably, the biologically active substance or therapeutic encapsulation material is water soluble. Main departure An example of an appropriate therapeutic drug for which the retention method by light can be effectively carried out is sympathetic nervous stimulation. agent.

e.g. atropine or scopalamine; bronchodilators such as inprotanol ; vasodilators, such as dilciazene; hormones, such as insulin; and anti-inflammatory agents; There are neoplastic drugs, such as adriamycin. Suitable biologically active molecules include e.g. Examples include RNA, DNA, enzymes and immunoglobulins.

Small unilamellar vesicles (SUVs) serve as starting material before encapsulation of trehalose. Preferably, it can be produced by any method. A suitable method is to remove lipids in an organic solvent with water. A method of injecting into a French pressure cell There are extrusion methods and sonication methods. Small unilamellar particles that trap substances It can be added at any stage of vesicle production, but in practice large unilamellar vesicle production Immediately before mixing an aqueous solution of the substance to be trapped with small unilamellar vesicles, It is most convenient to The preferred weight ratio of encapsulating material to lipid is about 1. 0: 1.0.

Large unilamellar vesicles (LUVs) with high trapping efficiency are then freeze-thawed or is produced by rotary evaporation.

An example of a rotary evaporation method and a particularly effective method is given by G. Gregoria Zois. riadis) 4g of liposome technology (1984) Nakanori, W., De. Deamer, D.W., “Encapsulation of macromolecules in liposomes” A novel method for This method involves first liposomes and capsules. The invention is characterized by producing a polar solution containing a certain amount of a fluorinated substance. Substantially dissolved After removing all the liquid, the liposomes are recovered by hydration of the concentrated mixture. This person No. 493, filed May 12, 1983, with Diemer et al. as inventors. , No. 952. Then filtration, centrifugation or gel permeation The vesicles produced by chromatography are further homogenized.

Trehalose may be added at any stage during large unilamellar production, but phosphorus The presence of trehalose on both sides of the lipid bilayer significantly improves retention efficiency. . Therefore, it is preferable to add trehalose before manufacturing large unilamella; As a result, trehalose is trapped inside. Preference of total amount of trehalose to lipids Preferred weight ratios range from about 0.1:1 to about 3.0:1, particularly preferred. The range is approximately 1.0:1.0. Next, freeze large unilamellae in liquid nitrogen. ,dry. Under certain conditions, for lipids that are susceptible to loss due to the presence of oxygen, Preferably produced dry in vacuum conditions, rewatering simply adds water to the dry mixture. This is done by adding.

In a preferred embodiment of the invention, the liposomes are exposed to trehalose; Various holding agents can be used in place of the loin, e.g. at least two units of monosaccharide. There are carbohydrates that contain In particular, sucrose and maltose are preferred examples. be.

The following examples show specific examples of the present invention, but these do not fall within the scope of the claims. It is not intended to limit the scope of the invention described.

X-seed, one spot, one spot. Dipalmitoyl phosphatidyl chlorine and phosphatidyl chlorine in a molar ratio of 95=5 A phospholipid mixture containing approximately 40 mg of dinic acid was made optically clear in a radiofrequency decomposition bath. High frequency decomposition was performed. Ink citric acid as encapsulating compound in 50mM aqueous solution was produced by freezing and thawing Kasha lamellar vesicles. Excess ink citric acid is removed by dialysis. I left. Trehalose (trehalose by weight) either after or before freezing and thawing Niphospholipid = 2. O: 1.0) was added to train only the outside of the lamellar vesicle. Those containing halose, and those containing trehalose both externally and internally. Mela vesicles were produced. Broad (Plaut) et al. The method described in Morology, Volume 5 (New York: Academic Press), By adding incitrate dehydrogenase and NADP to the outside of the cell, isocitrate was measured. isocitrate outside the vesicles by incitate dehydrogenase. oxidation, resulting in the reduction of NADP to NADPH. The speed and amount of fireflash Recorded by photometry. After measuring the total amount of ink citric acid in the vesicles, Triton -100 (octylphenoxypolyethoxyethanol, Philadelphia, USA) A cleaning agent and emulsifier manufactured by Rohm & Barr of Philadelphia. triton (trade name of Rohm & Haas) was added. To this Triton X-100 More trapped isocitrate is released into the surrounding medium.

Ink citric acid trapped in vesicles both before and after lyophilization and rehydration. The efficiency of trapped isocitrate was measured and evaluated. As shown in Table 1, If trehalose is present both inside and outside the vesicle.

More than 60% of the trapped isocitrate was retained upon lyophilization of the vesicles.

Retention efficiency was also significantly increased when trehalose was present only externally, but The extent was smaller compared to the case where treharth was present on both sides of the plasma membrane. When frozen When the lipid concentration of the There was no significant impact.

(■/mQ) Outside inside FT umbrella io, 8 o --.

FT 11.1 0.08 -+ O FT 10.8 1.78 + -42 FT 11.1 1.78 + + 6 1 umbrella FT = freeze-thaw MeL-1shi-"ji-jiζ High frequency decomposition of a solution containing 43μ of egg phosphatidylchlorine in 4mQ of water Car-heavy lamellar vesicles were produced by. Trehalose 32■ and isocitric acid 13 (2) The phospholipids were spin-dried in the presence of 2 to produce lamellar vesicles. Phosphorus fat n The weight ratio of nidrevalose:isocitric acid was approximately 4:3:1. distilled water Dialysis was performed to remove excess incitric acid and trehalose.

The enzymatic assay method described in Example 1 was then used to detect isoqueous molecules trapped in the vesicles. The amount of phosphoric acid was measured. Trehalose is added to the liposomes after dialysis to form the final The weight ratio of the phospholipid nidrevalose was set to 1.0:1.4, and the sample was freeze-dried. Next, the sample was rehydrated with distilled water, and the remaining ions in the liposomes were determined by enzymatic assay. The amount of citric acid was measured. The lyophilized vesicles retained 75% of their original content.

Next-JIL-1 Balmitoyl oleoyl phosphatidyl chloraine (90%) and phosphatyl A phospholipid mixture of dirserine (10%) was hydrated to 10×1011Q and then Car-heavy lamellar vesicles were produced by radiofrequency decomposition. Ingredients that act as encapsulating molecules Fire lamellar vesicles were prepared by spin drying in the presence of citric acid. Example 1 and Methods essentially similar to those described in Sections 1 and 2 were used. Lyophilized and rehydrated Record the retention efficiency of ink citric acid, first in the presence and absence of trehalose. The efficiency of freeze-dried lamellar vesicles was also recorded. As shown in Table 2, Kasha lame When la vesicles were lyophilized and rehydrated under the conditions described above, incitric acid was It is clear from the results that 0% was maintained. As the example above shows, external The presence of trehalose both inside and outside is favorable for retention of the encapsulate.

LEFT-】L””-TEXT One possible cause of loss that occurs during freeze-drying is that the lamellar vesicles are close to each other. Too much contact results in melting and destruction of the vesicle contents. Both Melting was measured by sound energy transfer. This fluorescence method uses an excitation probe (donor probe). based on the energy transfer from the nib lobe (acceptor probe) to the second nib lobe (acceptor probe). Ku. For measuring melting, the acceptor probe emits fluorescence when energy transfer occurs. can use a mixture of probes. - The manufacturing method uses a donor probe, while the other used acceptor probes to produce large unilamellar vesicles, but before lyophilization Mixed. Following lyophilization and rehydration, probe intermixing was determined. result are shown in Table 3 below.

This result shows that as the concentration of trehalose increases, the intermixing of the probes decreases. That is clear. Furthermore.

If trehalose is only present inside the liposome.

Probe mixing is significantly reduced. Therefore, the use of trehalose reduces vesicle melting. Make it less.

external internal RD umbrella 0.06 - + O RD 3.2 + +100 RD 0.19 ++49 RD 0.33 ++69 RD 0063 ++76 RD　O,91++86 RD 1.76 ++99 Umbrella RD = Rotary drying external internal R1>0.05-+72 RD　O,15++39 RD 0.25 ++29 RD 0.50 ++12 RD 0.95 ++8 FT$-0,--93,0 FT 0.4 + + 79.0 FT O, 8+ + 59.0 FT1.2+　10　NU, O FT 1.6 + + 38.0 FT 2.0 ++15.0 Umbrella RD = Rotary drying Umbrella FT = Freeze-thaw Balmitoyl oleoyl phosphatidyl chlorine vs. phosphatidyl serine ( 90:10) Melting of lamellar vesicles, resonance energy transfer between fluorescent probes measurement 迭YOSH-Two Kingdoms, Belly Further experiments were carried out in a manner similar to that described in Example 3, but with the exception of using mulch as a retention agent. Tose and sucrose were used. The results are shown in Tables 4 and 5 below. this According to their results, maltose and sucrose also improve the encapsulated material after freeze-drying. It is clear that it holds well.

RD　0.05　-+3 RD　O,15++41 RD 0.25 ++88 RD 0.49 ++95 RD O, 64+ + 100 external old part RD 0.07 -+20 RD O, 35++57 RD 0.49 ++89 RD　O,83+　+　86 RD 1:15 ++91 Submission of translation of written amendment (Article 184-7 of the Patent Law) 1. Indication of patent application PCT/US 8610 OO16 2. Name of the invention Liposome retention method 3. Patent applicant Address Addison, Berkeley, California 94720, United States 2199 Street Name: The Regents of the University ・of california 4. Agent 6. List of attached documents M-jL"υ" [-thief 1. In the liposome retention method.

Providing an initial liposome with a lipid membrane that the liposome encapsulates inside. a first reservoir containing an initial amount of a water-soluble substance, said substance containing at least two monosaccharide units; Contains a preservative, The initial liposome is contacted with a second retention agent containing at least two monosaccharide units in an aqueous solution. and lyophilize the initial liposomes in the presence of the second retention agent. A method for holding liposomes characterized by forming a substance.

2. By mixing the freeze-dried product with an aqueous solution, the produced liposomes can be obtained from the freeze-dried product. It also includes a step to recover the encapsulated material, so that the resulting liposomes contain a small initial amount of encapsulated material. The method of claim 1, wherein at least about 25% by weight is encapsulated.

3. The first retention agent is trehalose, and the generated liposome is the encapsulating material. The method of claim 2, wherein at least about 60% of the initial amount is encapsulated. .

4. The combined weight ratio of the first and second retention agents to the lipid of the lipid membrane is about 0.1. : 1 to about 3.0 : The person according to claim 1 or 2, which is in the range of 1. Law.

5. The method of claim 4, wherein the weight ratio is about 1.0:1.0.

6. The first retention agent contains at least two monosaccharide units linked with a glycoxide bond. A method according to claim 1 or 2.

7. The first retention agent is selected from the group consisting of trehalose, maltose and sucrose. A method according to claim 1 or 2, which is selected.

8. The second retention agent contains at least two monosaccharide units bonded to a glycoxide bond. The method according to claim 1 or 2.

9. The second retention agent is selected from the group consisting of trehalose, maltose, and sucrose. 9. The method of claim 8, wherein the method is selected.

10. A lyophilized compound useful for storage of encapsulated materials.

The initial amount of material that supplies the initial liposome and that the liposome encapsulates inside. and the substance contains a certain amount of trehalose.

contacting the initial liposome with an amount of trehalose in an aqueous solution; Lyophilize the initial liposomes in the presence of trehalose to form a lyophilizate. A lyophilized compound produced by

11. Claims in which the encapsulating material is a water-soluble therapeutic agent or biologically active compound A compound according to item 10.

12. The compound according to claim 11, wherein the encapsulating material is a macromolecule.

13. The encapsulated substance is a sympathomimetic agent, a bronchodilator, a vasodilator, an antineoplastic agent. drug, RNA, DNA, enzyme or 14, the first retention agent and the second retention agent are 1ip The lipid membrane of the initial liposome during hilosate formation and recovery of the produced liposomes. 3. The method of claim 2, wherein the amount is effective to retain

15. The method according to claim 14, wherein the first and second retention agents are trehalose. Method.

16. A retention compound comprising: Contains lifilosate with lipid component, trisaccharide component and initial amount of encapsulated component The trisaccharide component is trehalose, and the weight ratio of the trisaccharide component to the lipid component is about 0.1: 1 to about 3.0: 1, the lipid component limits the lipid membrane on the inside and outside, and the Sugar components are present in the phyllosade both inside and outside the lipid membrane, and At least most of the initial amount of encapsulated material in the glue phyllosede is present inside the Compounds that exist.

17. The trisaccharide component trehalose forms glue phyllosade both inside and outside the lipid membrane. encapsulated substances inside the lipid membrane during the reconstitution of lifilosate. The compound according to claim 16, which is effective to retain most of the initial amount of Compound.

18. The encapsulating material is a water-soluble therapeutic agent, biologically active compound, or diagnostic agent; By mixing Ficerot with an aqueous solution, at least the initial Most of the amount of lifilosate can be recovered as encapsulated liposomes. A compound according to claim 16 or 17.

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Claims (13)

[Claims] 1. In the liposome retention method, The initial liposomes are supplied with a water-soluble substance that the liposomes encapsulate inside. the substance contains a first retention agent; contacting the initial liposome with a second retention agent in an aqueous solution, and Lyophilizing the initial liposome in the presence of the second retention agent to form a lyophilizate. A liposome retention method characterized by: 2. By mixing the lyophilizate with an aqueous solution, the lyophilizate is produced from the lipolyte. It also includes a step to recover the encapsulated material, so that the resulting liposomes contain a small initial amount of encapsulated material. The method of claim 1, wherein at least about 25% by weight is encapsulated. 3. The first retention agent is trehalose, and the resulting liposome is The method of claim 2, wherein at least about 60% of the initial amount is encapsulated. . 4. The combined weight ratio of the first and second retention agents to lipid is about 0.1:1 to about 3. ．． 3. A method according to claim 1 or 2, wherein the ratio is 0:1. 5. 5. The method of claim 4, wherein the weight ratio is about 1.0:1.0. 6. Claim 1 or 2, wherein the first retention agent contains at least two monosaccharide units. The method described in. 7. The first retention agent is selected from the group consisting of trehalose, maltose and sucrose. 7. The method of claim 6, wherein the method is selected. 8. Claim 1 or 2, wherein the second retention agent contains at least two monosaccharide units. The method described in section. 9. The second retention agent is selected from the group consisting of trehalose, maltose and sucrose. 9. The method of claim 8, wherein the method is selected. 10. A lyophilized compound useful for storage of encapsulated materials, comprising: The initial amount of material that supplies the initial liposome and that the liposome encapsulates inside. , the substance contains a certain amount of trehalose, contacting the initial liposome with an amount of trehalose in an aqueous solution; Lyophilize the initial liposomes in the presence of trehalose to form a lyophilizate. A lyophilized compound produced by 11. Claims in which the encapsulating material is a water-soluble therapeutic agent or biologically active compound A compound according to item 10. 12. 12. A compound according to claim 11, wherein the encapsulating material is a macromolecule. 13. The encapsulated substance is a sympathomimetic agent, a bronchodilator, a vasodilator, an anti-neoplastic agent. The drug according to claim 12, which is a drug, RNA, DNA, enzyme or immunoglobulin. Compounds listed.