JPH0214736A - Working substance system for lipoid exchange with target structure - Google Patents

Abstract

PURPOSE: To rapidly transfer lipid with different target structures by preparing an active ingredient system which transfers lipophilic and amphiphilic components to the target structures by incorporating >=1 kind of lipid constituting elements and >=1 kind of transfer protein into the system. CONSTITUTION: The active ingredient for transferring the lipophilic and/or amphiphilic components to the target structures or to transfer these components to the active ingredient as well as to exchange these components with the target structures is prepd. by compounding at least one kind of lipid constitution element and at least one kind of lipid transfer protein. More specifically, the lipid, the lipid transfer protein in an aq. soln. and additive components are emulsified to prepare an aerosol. As another method of the specific example, there is a method for preparing an emulsion by adding additives into the lipid transfer protein in the aq. soln. during the emulsification of the lipid.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application and Prior Art] The present invention provides methods for transferring lipophilic and/or amphipathic components to target structures or for transferring these components to agent systems. With regard to agent systems for the purpose of transporting lipophilic and/or amphipathic components, as well as for exchanging those components with the target structure, in particular for optionally transporting lipophilic and/or amphiphilic components, appropriately altering the composition of the target structure and thus presenting its properties. It concerns the modification of the active substance system according to the specified requirements. Here we will use the concept "transfer" for transport in one direction and the concept "exchange°" for transport in both directions. Target structures can be, for example, emulsions, micelles, liposomes, aerosols, liquids or Engineering systems such as monolayers, oligolayers or multilayers related to solids, or for example riboproteins,
vesicles, organelles, biological systems such as bacteria, fungi, viruses, parasites, or also e.g. tumor cells,
Pathological structures such as deposits in tissues, old pigments, etc.

Traditionally, lipid exchange between different structures has been carried out by vesicular transport, collision, fusion, or monomeric components contained in the vehicle. However, such processes proceed very slowly with half-lives that require many hours, so that effective and appropriate lipid structure transformations occurring on such bases are generally not possible. Alongside slow motion, the fact that the use of, for example, organic solvents, detergents, high temperatures, etc. often compromises the integrity of the target structure, lacks specificity, and sometimes produces undesirable side effects is also important. Even based on the fusion of relatively large lipid aggregates, no technically feasible system for rapidly and appropriately deforming lipid structures has been known so far. Publication number WO85104880 (
7) PCT/US 85100621 describes fusion. Fusion, in the technical terminology of membrane biophysics, is understood as the fusion of two membranes.

As the fusion progresses, countless molecules are transferred to the existing membrane, which precludes appropriate deformation of the target structure at the molecular level. Using the fusion published in PCT/US 85100621, it is not possible to transfer molecules that cannot form membranes, such as lipid molecules, for example. Furthermore, the membrane proteins in these cited examples are not basically transfer proteins, but this is directly clear from the fact that membrane proteins exist within a single membrane, as their name suggests. Furthermore, as described in the cited example, this type of membrane protein is basically incapable of performing a lipid transfer function because it is hydrophobic. In the fusion system in the cited example above, only the membrane itself can be treated as a lipid component. Also, it is absolutely impossible to extract individual lipid molecules by fusion.

[Problem to be solved by the invention]

The aim of the present invention is to propose a rapid, suitable, specific and effective lipid exchange system with different target structures and use it to modify the composition, properties or functions of the target structures.

[Means to solve the problem]

The purpose is to transfer lipophilic and/or amphiphilic components to the target structure or to transfer these components to the agent system and to exchange these components with the target structure. This is achieved by providing an agent system comprising at least one lipid component and at least one transfer protein.

[Specific explanation]

In the active substance system according to the invention, we deal with translocation, which is a fundamental process completely different from fusion in the field of membrane biophysics. In the active substance system according to the invention, appropriate modification of the target structure at the molecular level is possible by transfer of individual single molecules, for example lipid transfer of lipid molecules. Lipid molecules of this type, for example those which are not capable of forming membranes, cannot be transferred at all by the aforementioned fusion, but this is possible using the active substance system according to the invention. The transfer of various lipid species to the target structure in the present invention is
Rather than following the laws of chance, where the small number of individual lipid molecules in the aqueous medium results in long or short transfers, the transfer protein is facilitated by the lipid transfer system according to the invention. .

Such proteins are components of the translocation system and are thus proteins capable of catalyzing translocation. Lipid transfer proteins contained in an aqueous medium and having a lipid component therein transport said lipid component in order to transfer the aqueous medium to a target structure.

In transfer systems, the lipid stock associated with transfer proteins can be prepared as an emulsion, whereas in fusion systems only the membrane itself is considered as the lipid component. Furthermore,
Transfer proteins are unsuitable components for the fusion process The transfer system according to the invention is incompatible with fusion systems not only on the lipid side but also on the protein side. Lipid transfer systems also allow extraction of individual lipid molecules.

Transfer or exchange with the target structure may include, for example, lecithin, sphingomyelin, cholesterin, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, gangliosides, cerebrosides, fat-soluble vitamins and their derivatives, triglycerides, etc. Lipophilic or amphipathic components are used, which together with one or several lipid transfer proteins form an active substance system.

The lipid component can then be provided in various configurations, for example as liposomes, micelles, aerosols, emulsions, etc. For transfer or exchange of lipophilic or amphiphilic moieties, the respective agent system is brought into contact with the target structure.

The advantages that can be achieved by the present invention include the complex association of lipophilic and amphiphilic components such as membranes, tissues, micelles, emulsions, cells, organocytes, bacteria, viruses, parasites, deposits in tissues, etc. The aim is to appropriately change the structure and function of The origin of the lipophilic or amphiphilic components that the transition also specified for exchange is known to the expert. They can be processed on the basis of isolation, consisting of natural sources, such as vegetable or animal origin, or on the basis of chemical synthesis, or on the basis of biotechnological methods, consisting of microbial origin. be.

The origin of the proteins that support the exchange or transfer of lipid components is known to experts. Proteins with various specificities are
For example, isolation can be obtained by isolation from natural sources, such as animal tissue, or synthesis by genetic engineering. For example, isolation is carried out by the following methods: , Obtaining 100,000 g of supernatant fluid 4 Precipitation of associated proteins with acid, Precipitation with ammonium sulfate 5 Dialysis 6 Gel filtration 7 Ion exchange chromatography 8 Chromatofocusing Depending on the purpose of use and site of action, lipid components and The protein components synthesize active substance systems with different configurations, with specificity arising from the lipids used and the transferred proteins introduced, as well as from the direction of lipid transfer.

The table below shows possible configurations of lipid association: emulsion skin, blood skin structure, nutrition supplement lipid transfer system to obtain as an aerosol,
The lipid, lipid transfer protein in aqueous solution, and additional components are emulsified and sprayed into an aerosol. To obtain the lipid transfer system as an emulsion, additives are added to the lipid transfer protein during lipid emulsification.

To obtain the lipid transfer system as micelles, lipids as pure substances are added to an aqueous solution of the lipid transfer protein.

To obtain a lipid transfer system as a liposome,
Detergent dialysis or extrusion or microemulsification as well as high pressure homogenization or ultrasound methods can be applied. In the case of detergent dialysis, a dry lipid/detergent mixture is taken up with an aqueous solution of lipid transfer protein and the detergent is immediately removed by dialysis.

In the extrusion method, the lipids are extracted as pure substances with an aqueous solution of the lipid transfer protein, and the mixture is pressed through a membrane of decreasing pore size.

In the case of the microemulsification method, lipids are extracted as pure substances using an aqueous solution of lipid transfer proteins, and while being recirculated under high pressure,
Pass through the interaction chamber.

The active substance system according to the invention can be obtained as follows: Lipid transfer proteins with suitable specificity for phospholipids are isolated from tissues such as, for example, bovine brain. The aqueous solution of lipid transfer protein is mixed with a suitable lipid component, such as a 5-fold molar excess of phosphatidyl-choline. The mixture is converted into an emulsion by suitable techniques such as high pressure homogenization, microfluidization, etc. Aliphotos of such emulsions (relating to protein moieties) are added to suspended aqueous solutions of liposomes in the form of single flakes, and translocation activity is determined by suitable methods, such as fluorescence techniques or radioactivity.

Thus, a standardized transfer system becomes applicable.

The function of the lipid transfer system is that the lipid transfer protein system uses lipid components as donors for transfer to various target structures. At that time, by combining a specific lipid transfer protein with an appropriate lipid component, lipids can be extracted from the target structure and transferred to the lipid component.

Lipid transfer protein systems facilitate rapid lipid exchange between lipid components and target structures in both cases.

This type of lipid transfer system can be used in membranes. Therefore, the asymmetric distribution of lipids in the bilayer can be identified by comparative analysis of the lipid part exchanged by the lipid transfer protein and the entire lipid of the lipid membrane. For membrane labeling, a lipid transfer system is used to label radioactive lipids, fluorescent lipids, and ESR or MMR.
- Incorporating the labeled lipid into the membrane structure.

By using lipid transfer systems, asymmetric distribution of lipids can be achieved in natural or artificial membrane bilayer structures. By extraction, unique structures of lipids can be achieved from natural or artificial membranes. In the case of stabilization, lipid transfer systems can stabilize natural and artificial cells, organelles or membrane structures such as e.g. liposomes. This is because membrane defects can be removed in such systems by incorporating lipids.

The agent system can be used in techniques for the retention or formation of molar layers on materials such as slide films, for direct water temperature protection, or for increasing the biocompatibility of interfaces.

In cosmetology and dermatology, such active substance systems serve for the reorganization of membranous structures (skin) that have exhibited dysfunction or aging. In medicine, active substance systems as drug carriers can be used to stabilize liposomes relative to blood components. Another use in medicine is to use agent systems to create asymmetric posomes for precise drug delivery (drug targeting). The use of extracting cholesterol in the treatment of arteriosclerosis is particularly significant. For this reason, liposomes as cholesterol-free lipid components are used in combination with lipid transfer proteins.

Yet another application of the agent system is the treatment of lipid metabolic disorders. Thus, for example, lipid transfer systems are used to regulate bile acid synthesis.

Active substance systems are included in various engineering, medical or cosmetic products. For example, ointments, creams, gels or sprays may have active substance systems. The above explanation is
It merely shows the essential features of the embodiments of the invention. Therefore, insofar as features are made clear in the description and are not listed in the claims, they also serve, if necessary, to specify the subject matter of the invention.

Claims (1)

[Claims] 1. For the transfer of lipophilic and/or amphiphilic components to the target structure or for the transfer of these components to the agent system and for the exchange of these components with the target structure. An active substance system comprising at least one lipid component and at least one transfer protein. 2. A method for producing the active substance system according to item 1 as an aerosol, which comprises emulsifying the lipid, the lipid transfer protein in an aqueous solution, and the additional components and spraying the emulsified mixture into the aerosol. 3. Process for producing the active substance system according to item 1 as an emulsion, characterized in that during emulsification of the lipids, additives are added to the lipid transfer protein in aqueous solution. 4. A method for producing the active substance system according to item 1 as a micelle, characterized in that the lipid as a pure substance is added to an aqueous solution of the lipid transfer protein. 5. Process for producing the active substance system according to paragraph 1 as liposomes, characterized in that a dry mixture of lipids/detergents is introduced into an aqueous solution of lipid transfer proteins and the detergents are then removed by dialysis. Method. 6. A method for producing the active substance system according to item 1 as liposomes, which comprises introducing the lipid as a pure substance into an aqueous solution of the lipid transfer protein and pressing such a mixture through a membrane of decreasing pore size. A method characterized by: 7. A method for producing the active substance system described in item 1 as a liposome, which comprises adding lipid as a pure substance to an aqueous solution of a lipid transfer protein, passing it through an interaction chamber while recirculating it under high pressure, and A method characterized by homogenization with , or treatment using ultrasonic waves. 8. Use of the active substance system according to claim 1, characterized in that the active substance system serves to hold or build up monolayers or multi-monolayers on the material. 9. Use of the active substance system according to claim 1, characterized in that it serves as a dermatological or cosmetic application in the reorganization of membrane structures (skin) that have shown aging or dysfunction. 10. Use of the active substance system according to item 1, characterized in that it serves to stabilize liposomes as drug carriers for blood components. 11. Use of the agent system according to paragraph 1, characterized in that it serves to create asymmetric liposomes for use in drug targeting. 12. Use of the active substance system according to claim 1, characterized in that it serves for the extraction of cholesterol for the purpose of treating arteriosclerosis. 13. Use of the active substance system according to item 1, characterized in that it is useful for the treatment of lipid metabolic disorders. 14. Products characterized in that they contain the active substance system according to claim 1.