RU2211027C2 - Method for designing, controlling and applying macromolecule associates and complex aggregates with increased useful loading and controlled association/dissociation degree - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves preparing substance combination two of which show amphipathetic properties and build expanded surfaces, mainly membrane-like surfaces, when making contact with liquid medium. The first and the second substances showing amphipathetic properties are different in their solubility in the fluid medium. The substances possessing higher solubility build surfaces of smaller length when compared to the second one. The substance possessing higher solubility is to be selected from lipids or lipid-like materials. The second one is to be selected from surfactants. The third substance has one or several chain molecules. EFFECT: optimized drug delivery. 59 cl, 12 dwg, 4 tbl

Description

 The invention relates to combinations of substances that exhibit amphipathic properties and can form extended surfaces, in particular membrane-like surfaces in contact with a liquid medium. In particular, the invention relates to the association at the molecular level of other amphipathic substances with such surfaces, these other amphipathic surface-associated substances usually being larger molecules with repeating subunits, such as oligomers or polymers, and often belong to the class of biologically active agents.

 In addition, the invention relates to methods for creating such surfaces and to associate these larger molecules with such surfaces, as well as to various methods of using such surfaces and associates.

 Amphipathic chain molecules and related macromolecules, such as proteins, are adsorbed on any type of surface, but to different degrees and very often in different conformations. The present invention describes the state of the art and provides a new rationale for optimizing and controlling macromolecular associations with soft complex surfaces. It can be used in biology, biotechnology, pharmaceuticals, therapy and diagnostics.

(Macro) molecular adsorption / binding to an adsorbing surface (adsorbent / adsorbate association) is a multi-stage process:
i) the first stage involves the redistribution, preferably accumulation, of the adsorbate at the adsorbent / solution interface. This stage usually proceeds quickly and depends on the speed of diffusion.

 ii) in the second stage, the adsorbate molecules are hydrophobically associated with a soft (membrane) surface. This process involves several steps, such as partial molecular binding and sequential rearrangement (s), with at least some of them proceeding slowly.

 It has been proven (Cevc G., Strohmaier L., Berkholz J., Blume G. Stud. Biophys. 1990, 138: 57ff) that the possibility of specific binding of a large molecule to a surface-attached ligand embedded in a “soft” lipid membrane decreases due to the proximity of the interface. This is probably due to the same non-Coulomb force, depending on hydration, which also prevents colloidal fusion of neighboring lipid membranes with one another. The total resulting force decreases with a decrease in hydrophilicity and stiffness of the solution-lipid interface (Cevc G., Hauser M., Kornyshev A.A. Langmuir 1995, 11: 3103-3110).

 It has also been previously suggested that the degree of non-specific adsorption of proteins on a lipid bilayer (Cevc, et al., Op. Cit: 1990) is proportional to the availability of protein hydrophobic binding sites in the membrane. It was found that the creation of defects in lipid bilayers by mechanical means (for example, by means of sonication) or by inducing lipid phase transitions increases the amount of membrane-bound protein.

 As a rule, it is believed that the more hydrophobic the surface, the higher the degree of adsorption of amphipathic macromolecules. For example, K. Prime and G.M. Whitesides (Science, 1991, 252: (164-1167), which used self-organizing monolayers of long chain alkanes with terminal groups of different hydrophobicities to systematically alter protein adsorption by binding hydrophobic amino acids, have confirmed this “rule” or “principle.” Currently The “hydrophobic attraction” moment is thus considered to be the predominant force in protein adsorption.

 On the other hand, it is widely recognized that in the total macroscopic interaction between a hydrophilic macromolecule, such as a protein, and a hydrophilic surface, such as glass or montmorillonite clay, immersed in an aqueous solution at neutral pH, strong repulsion prevails. Thus, under conditions for which the Van der Waals rules, Lewis acid bases and double layer electrical interactions are applicable on a macroscopic scale, the adsorption of hydrophilic proteins on hydrophilic inorganic surfaces is usually weak (H. Quiquampoix et al., Mechanisms and Consequences of Protein Absorption on Soil Mineral Surfaces, Chapter 23 in Proteins at Interfaces (PAI), TA Horbett and JL Brash, eds., ASC Symposium Series 602, 1995, New York: 321-333). However, some hydrophilic proteins are indeed adsorbed on glass from a solution, although more rarely than they would adsorb on a hydrophobic surface; such proteins are also adsorbed on surfaces of montmorillonite clay. To explain this unusual phenomenon, it was suggested and confirmed by experimental data that proteins can bind to an equally (e.g., negatively) charged hydrophilic inorganic surface immersed in an aqueous medium through multiply charged counterions (e.g., calcium) associated with (negatively) charged hydrophilic proteins . Other subtle effects of charges include the formation of hydrogen bonds, the formation of salts in proteins, and the binding of counterions. For example, it was suggested that “structural rearrangements in the protein molecule, dehydration of the sorbent surface, redistribution of charged groups and surface polarity of the protein” - all these factors can affect protein adsorption (Haynes C.A et al., Colloids Surface B: Biointerfaces, 2, 1994: 517-566). Accordingly, although Coulomb interactions are important, they are generally not predominant in the adsorption of proteins on solid surfaces, as in the case of strong adsorption of a-LA (alpha-lactalbumin) on PS (polystyrene) under conditions when the protein carries a significant total negative charge. Another recent review recognized that “there is currently no clear agreement on the extent to which charges affect protein adsorption” (Reversibility and the Mechanism of Protein Adsorption, W. Norde and C. Haynes, Chapter 2 in (PAI), op . cit .: 26-40).

 Currently, there is a point of view that in the case of soft surfaces, such as membranes, at least the first stages of protein adsorption occur due to electrostatics and / or charge interactions predominate on them (see, for example: Deber CM; Hughes DW; Frasez PE; Pawagi A.V .; Moscarello MA Arch. Biochem. Biophys. 1986, 245: 455-463; Zimmerman, RM, Schmidt, CF, Gaub, NHEJ Colloid Int. Sci. 1990, 139: 268-280; Hernandez-Caseldis T .; Villalaain J .; Gomes-Fernandez JC Mol. Cell. Biochem. 1993, 120: 119-126.) Leading experts also believe that electrostatic forces are decisive when binding secretory phospholipases to various idnymi aggregates (Scott D.L .; Mandel A.M .; Sigler RV .; Honig B. Biophys J. 1994. 67: 493-504).

 Until now, experts believed that the main determining factor in the final adsorption of proteins is hydrophobic attraction, despite the fact that ionic interactions play a role, combined with an increase in entropy caused by conformational changes in the protein during its adsorption.

 Typically, proteins are strongly adsorbed to surfaces that are oppositely charged, and not to surfaces that carry the same charges. The dependence of protein adsorption on pH reflects this fact. The effect of charges can be confused with “hidden” factors, such as small multivalent counterions, which can form bridges between the protein and surface areas with the same charge, from which repulsion would normally be expected.

 The final conformation of the adsorbed protein is rarely identical to that at the beginning. This is the reason that in most models of protein adsorption, a transition occurs from a state of reversible adsorption to a state of stronger retention, which arises as a result of molecular restructuring or relaxation of the protein on the surface. Macromolecular rearrangement during adsorption is often catastrophic and ends with protein denaturation. Based on the fact that enzymes and antibodies retain at least part of their biological activity in an adsorbed state, and biological activity solely depends on the preservation of the native structure, it can be concluded that changes in the conformation of the adsorbed protein are nevertheless often limited in time and in time volume. Protein coagulation is most strongly affected by hydrophobic interactions. Both phenomena, protein binding and conformational changes, are sensitive to the presence of certain amphiphilic substances, such as surfactants and phospholipids. It was believed that protein adsorption decreases or becomes reversible with the addition of such molecules.

 Thus, proteins are more often mixed than not mixed with surfactants during their isolation in order to minimize non-specific adsorption and protein loss. In one study, protein adsorption decreased to an insignificant level as the surface concentration of a grafted surfactant like Pluronic increased. The number of ethylene glycol (EG) units in the monomeric side chain of the surfactant was 4, 9, and 24, and the monomer with the smallest number of EG units (4) was the most "inert" with respect to blood components (Analysis of the Prevention of Protein Adsorption by Steric Repulsion Theory TB McPherson et al., Chapter 28 in PAI, op. Cit .: 395-404).

 It was shown that short polymers covalently bonded to the surface and increasing the thickness and hydrophilicity of the interface and, thus, reducing the availability of hydrophobic binding sites under it, reduce the possibility of protein binding to modified surfaces, as well as protein denaturation.

 The fact that surfactants, which also often contain short polymer segments at one end, tend to inhibit the binding of proteins to different surfaces or even make it partially reversible, consistent with the above conclusions. This phenomenon may include solubilization or substitution of the protein, depending on the relative strength of the interactions of the surfactant with the surface and the binding of the surfactant to the protein; usually both of these factors play a role.

In another experiment, the addition of a non-ionic surfactant of the Brij series (alkyl polyoxyethylene ether) to the aqueous phase at pH 7.0 in the concentration range of about 10 ^-4 wt.% Caused a significant displacement of the protein from the air-water interface (T. Arnebrant et al, op. cit.).

The removal of pre-adsorbed proteins using surfactants has been extensively studied (Protein-Surfactant Interaction at Solid Surfaces, T. Arnebrant et al. Chapter 17 in PAI, op. Cit .: 240-254). There are three types of interactions:
i) Binding of a surfactant via electrostatic or hydrophobic interactions to specific regions of a protein, such as alpha-lactoglobulin or serum albumin;
ii) Cooperative adsorption of a surfactant on a protein without significant conformational changes;
iii) Cooperative binding of a surfactant to a protein, accompanied by conformational changes;
For example, the removal of a protein from a methylated (hydrophobic) surface from silicon dioxide is the same for different surfactants, which indicates that the proteins are removed by their substitution due to the higher surface activity of the surfactant. It can be concluded that the effects of the head group of the surfactant are most pronounced on hydrophilic surfaces and less important on hydrophobic surfaces (Protein-Surfactant Interaction at Solid Surfaces, T. Arnebrant et al. Chapter 17 in PAI, op. Cit. : 240-254).

 The same conclusions apply to other lipids. The amount of plasma proteins adsorbed on a plastic surface decreases when it is pretreated with a suspension of DPPC liposomes; adsorption of insulin on the surfaces of the catheter demonstrates the same pattern.

 The authors of the present invention have found that amphipathic substances, in particular macromolecules, are adsorbed on soft surfaces, including a mixture of lipids and surfactants, more efficiently than on lipid aggregates that do not contain surface-active molecules. In general, a mixture of molecules forming a stable membrane - usually, but not necessarily in the form of lipid vesicles (liposomes) - and at least one highly amphipathic, i.e. relatively water-soluble, destabilizing bilayer component (often a surfactant), for example such a mixture can be a mixture of phospholipids and surfactants, more prone to bind amphipathic substances such as proteins than pure phospholipid surfaces, especially vesicles or liposomes, which consist only of phospholipids or also include at least one lipid stabilizing bilayer, such as cholesterol. It was found that the relative number of bound amphipathic macromolecules (proteins) was unexpectedly higher for surfaces that had total charges of the same sign as the total charge of the adsorbed particle. This clearly contradicts the published information, from which it follows that in order for electrostatic bonding to be strong, opposite charges on interacting particles are required.

The authors suggest that one of the requirements of the above improvement of supramolecular (for example, for drug carriers) association is the general adaptability of the adsorbing surface. This ability to improve adsorption allows adsorbed macromolecules to:
i) firstly, to accumulate near the adsorbing surface due to charge-charging and other interactions creating local attraction;
ii) secondly, to optimize non-electrostatic interactions / binding with the absorbent surface. (The latter process usually requires the presence of hydrophobic and hydrogen bonding sites that form or become accessible due to the flexibility of the surface and / or its adaptability.)
(Macromolecular) drug / drug carrier combinations that fully satisfy these requirements and allow control of their implementation are best suited for practical use.

 The authors further suggest that each stage involved in the process of protein adsorption on a soft (membrane) surface depends to varying degrees on the proximity and number of hydrophobic binding sites in / at the membrane-solution interface. The kinetics of the hydrophobic association between the macromolecules and the binding surface, therefore, should be sensitive to the number of sites available for binding, which in turn increases due to the presence of surface-active ingredients in the membrane and the softness of the membrane.

The rate at which adsorbed (macro) molecules can conformationally adapt to many binding sites is also important. For example, in the case of uncharged flexible (Transfersome ^® ) membranes, hydrophobic interaction is the main reason for the association of insulin with the surface. However, the multistage binding underlying it requires substantial rearrangements in the system and, therefore, a long adsorption time to complete it. As a result, the optimal incubation times for the formation of the Transfersome ^® -incyline complexes can be quite large.

 The adsorption flow diagram described above is consistent with the main adsorption flow scenario described in the specialized literature. Despite this, several differences and even controversial issues clearly distinguish these developments from well-known knowledge that is currently disclosed.

 Surprisingly, the addition of charged surfactants to a surface in accordance with the invention accelerates the binding of the protein to the surface and provides a means of controlling the degree and rate of association of the macromolecule with the membrane. This contradicts the aforementioned widespread belief that surfactants inhibit protein binding. On the other hand, at least partial removal of the surfactant from such a surface accelerates the process of macromolecular desorption and releases some molecules. It also directly contradicts published information.

 Surprisingly, we found that macromolecular adsorption on a soft deformable surface in accordance with the invention, in particular on a corresponding membrane, is stronger than on a less deformable surface. Since it is known from the prior art that soft membranes are more hydrophilic and mutually repulsive than membranes of a less adaptable type, such a discovery directly contradicts expectations.

 Therefore, one of the objectives of the present invention is to determine the conditions that provide maximum association between large, often macromolecular, amphipathic molecules, such as proteins, or any other type of suitable chain molecules and a complex adsorbing surface.

 Another objective of the present invention is to determine the primary factors that control the rate of macromolecular adsorption to or, respectively, the rate of desorption from the complex surface.

 Another objective of the present invention is to provide methods for producing compositions suitable for (bio) technological and medical applications.

 Another objective of the present invention is to describe methods that are particularly suitable for the practical use of the obtained compositions, including, but not limited to, the use of the obtained adsorbates in diagnostics, separation technology and (bio) technology, bioengineering, genetic manipulation, for stabilization, concentration or delivery of the agent, for example in medicine or veterinary medicine.

 DEFINITIONS

 An “associate,” as defined in this application, is a complex of two or more different molecules, at least one of which forms aggregates with one or more well-defined surfaces, regardless of the reason for the formation of the complex, but excluding covalent binding. The association between different types of molecules can be based on encapsulation (e.g., insertion into a vesicle containing the molecule (s) forming the surface (s)), insertion (e.g., incorporation into the aggregate layer at and below the surface) or adsorption (on the surface of the aggregate) ; combinations of two or more of these principles are also possible.

 The terms “adsorbate”, “adsorbed (macro) molecule”, “binding (macro) molecule”, “associating (macro) molecule”, etc. are used interchangeably herein to describe associations between molecules that do not form extended surfaces under selected conditions and an “adsorbent” or “binding surface”, etc. in the above conditions.

 "Carrier" means an aggregate, irrespective of nature and source of its origin, that is capable of being associated with one or more macromolecules, used for practical purposes, such as application to or delivery to a human or animal body.

"Lipid" in accordance with the meaning of the present invention is any substance whose properties are the same as in fats. As a rule, the molecules of this series contain an extended non-polar segment (chain, X) and in most cases also contain a water-soluble, polar, hydrophilic group, the so-called main group (Y). The basic structural formula 1 for such compounds looks like
XY _n (1)
where n is greater than or equal to zero. Lipids with n = 0 are called nonpolar lipids, with ni 1 - polar lipids. In the context of the present description, all amphiphilic substances, such as glycerides, glycerophospholipids, glycerophosphinolipids, glycerophosphonolipids, sulfolipids, sphingolipids, isoprenoidlipids, steroids, sterols or sterols, etc. and all lipids containing carbohydrate residues are simply called lipids. For a more detailed definition, PCT / EP91 / 01596 is proposed by reference.

 "Border-active" substance or "surfactant" in this application means any substance that increases the tendency of the system to form edges, protrusions or other strongly curved structures and segments with a large number of defects. In addition to conventional surfactants, this category includes auxiliary surfactants and other molecules that promote lipid solubilization in the presence of more commonly used surfactants; as well as molecules that induce or contribute to the formation of (at least partially hydrophobic) defects in adsorbing (hetero) aggregates. The direct surface-active action or indirect catalysis of the (partial) separation of the mixture of molecules, or also the conformational changes of the corresponding molecules caused by the surfactant, are often the cause of this effect. Accordingly, many solvents, as well as asymmetric, and therefore amphipathic, molecules and polymers, such as numerous oligo- and polysaccharides, oligo- and polypeptides, oligo- and polynucleotides and / or their derivatives belong to the above category in addition to the more common commonly used surfactants. A fairly detailed list of the most well-known standard surfactants, some suitable solvents (otherwise called auxiliary surfactants) and many other suitable surfactants can be found in the description of PCT / EP91 / 01596. A more complete list can be found in Handbook of industrial surfactants; Michael Ash, Irene Ash, eds., Gower Publishing, 1993.

 A "chain molecule", or "macromolecule", is a linear or branched chain molecule that contains group (s) of at least two types or types of structures with unequal affinity for the "adsorbing surface". Another requirement characteristic of the corresponding alternative (item 2) or combined (item 3) variants of the present invention is that at least one type of such group must be (partially) charged in the donor solution and / or at the adsorbing surface. The different surface affinities in individual groups are often explained by their different amphipathicity, that is, different hydrophilicity / hydrophobicity. Different groups can be arbitrarily distributed along the chain, but often several groups with similar physical properties (for example, several hydrophilic or more than one hydrophobic) are located on the same chain segment.

"Macromolecules", in accordance with the meaning of this application, among others, include:
Carbohydrates with the basic formula C _x (H ₂ O) _y , for example, sugar, starch, cellulose, etc., most often require modification in order to implement the present invention to give them an additional affinity for the bonding surface. This can be done, for example, by attaching hydrophobic residues to carbohydrates intended for association with a (partially) hydrophobic surface, or by introducing such groups that can participate in other non-Coulomb interactions (for example, in the formation of hydrogen bonds) with a more hydrophilic binding surface.

 Oligo- or polynucleotides, such homo- or hetero-chains of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) acid, as well as their chemical, biological or molecular biological (genetic) modifications (for a more detailed definition, you should consider the lists given in the PCT / EP91 / 01596).

 Oligopeptides or polypeptides include 3-250, often 4-100 and most often 10-50, identical or different amino acids that are naturally linked by amide bonds, but in the case of proteomimetics, they can be built on the basis of various polymerization schemes and can even be partially or fully cyclic ; Optically pure compounds or racemic mixtures may be used (see PCT / EP91 / 01596 for a more detailed and complete definition).

 Long polypeptide chains are usually called proteins, regardless of the detailed conformation and the exact degree of polymerization. Most, if not all, proteins are quite effectively associated with surfaces.

 For illustration purposes only, the following relevant classes are described.

 Enzymes that include oxidoreductases (including various dehydrogenases, (per) oxidases, (superoxide) dismutases, etc.), transferases (such as acyl transferases, phosphorylases and other kinases), transpeptidases (such as esterases, lipases, etc.) etc.), lyases (including decarboxylases, isomerases, etc.), various proteases, coenzymes, etc.

 Immunoglobulins of the IgA, IgG, IgE, IgD, IgM classes of all subtypes, their fragments, such as Fab or Fab2 fragments, single chain antibodies or parts thereof, such as variable or hypervariable regions, in their natural form or after chemical, biochemical or genetic manipulations may be useful for the present invention. These include, but are not limited to, IgG-gamma chains, IgG-F (ab ') 2 fragments, IgG-F (ab), IgG-Fc fragments, Ig-kappa chains, immunoglobulin light chains (e.g., kappa and lambda chains), and also include smaller fragments of immunoglobulins, such as variable or hypervariable regions of any of these substances or fragments.

 Immunologically active macromolecules other than antibodies (endotoxins, cytokines, lymphokines and other large immunomodulators or biological mediators) also belong to the class of heterologous chain molecules. They also include phytohemagglutinins, lectins, polyinosines, polycytidylic acid (poly I: C), erythropoietin, “granulocyte-macrophage colostimulating factor” (GM-CSF), interleukins 1 to 18, interferons (alpha, beta or gamma and their ( bio) synthetic modifications); tumor necrosis factors (TNFs); all sufficiently large and amphipathic extracts from tissues and plants, their chemical, biochemical or biological derivatives or substitutes, their parts, etc. All of these molecules, respectively, can suitably be efficiently associated with complex surfaces, as described herein.

 Other biologically similar examples include substances that affect local or general growth, such as major fibroblast growth factor (BFGF), endothelial cell growth factor (ECGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin, insulin-like growth factors (such as LGF I and LGF II), nerve tissue growth factors (such as NCP-beta, NGF 2.5s, NGF 7s, etc.), platelet derived growth factor (PDGF), and t .d.

 Derivatives particularly useful for the purposes of the present invention include modifications made by the (bio) chemical, biological or genetic route, in which the adsorbate molecules are replaced by several, often more than 3 non-polar (hydrophobic) residues, such as aryl, alkyl, alkenyl, alkenoyl, a hydroxyalkyl, alkenylhydroxyl or hydroxyacyl chain with 1-24 carbon atoms as suitable groups, or reactions that increase the ability to participate in other non-Coulomb interactions actions between the adsorbate and the adsorbent. When hydrophobizing macromolecules, side chains with a relatively small number (1-8, or even better 1-4) carbon atoms are preferred. Extensive information is known from the prior art on how to hydrophobize chain molecules for various purposes. For the purposes of the present invention, the strong adsorbent compound disclosed in other publications (Torchilin VP; Goldmacher VS; Smirnov VN Biochem. Biophys. Res. Comm. 1978, 85: 983-990) is excluded, not only because it is known from prior art, but also because it is likely to lead to difficult reversible associations.

 It is also known from the prior art that the addition of surfactants to a membrane constructed from an amphipathic substance changes the ability of the membrane to adapt. Moreover, it has already been suggested that this fact can be used to improve the transport of the agent through pores that are otherwise too narrow in the barrier by incorporating the agent into tiny droplets surrounded by appropriate membranes and suspended in a suitable liquid medium. This is described in more detail in earlier applications PCT / EP91 / 01596 and PCT / EP96 / 04526.

 The paths that should be chosen to optimize the indicated vesicles with membranes with high adaptability with the aim of penetrating through the pores in the barrier generally do not coincide with the steps that must be taken to ensure or control the degree or rate of association between the chain molecule with one side and such membranes on the other. In addition, the ability to three-dimensionally adapt such membrane surfaces that surround these vesicles (and hence the possibility of deformation of the vesicles themselves) does not necessarily matter, for example, in association processes when the indicated surface with which the specified macromolecule is associated is on a solid substrate and therefore should not have the ability to three-dimensional adaptation inherent in free membranes.

 In order to provide and / or control the processes of macromolecular association with the surface, on which the present invention is focused, two main effects can be used, which were mentioned above.

 The first important phenomenon is that amphipathic molecules, i.e. macromolecules or chain molecules already discussed are better associated with an extended surface, which includes at least one amphipathic substance, which in turn is prone to the formation of extended surfaces, and at least one more substance, which is more soluble in a suspending liquid medium, and also prone to the formation of less extended surfaces than the first amphipathic substance. In other words, the presence of a substance that tends to destabilize the surface gives the surface / solution interface a relatively greater attractiveness with respect to adsorbed macromolecules compared to the corresponding surfaces formed by only the less soluble substance forming the surface, in the absence of the previous, more soluble surface destabilizing second substance. A surface in the context of this document is considered extended if it can spread and / or develop cooperative surface excitation in two dimensions. The surface of the vesicles, for example, satisfies this criterion, as it contributes to irregularities or fluctuations; depending on the flexibility of the membrane, the average diameter of the vesicles needed for this is between 20 nm and several hundred nanometers. (Mixed) lipid micelles that do not reach this size in at least one of the directions do not satisfy this requirement; in this case, their surface is not considered as extended in accordance with the meaning of the present invention.

 The second, more soluble and destabilizing surface substance, as a rule, is a border-active substance or surfactant.

 The second recently described effect is that, contrary to expectations, electrically charged macromolecules or chain molecules are easier and better associated with a surface of the same charge (i.e., the charges are both negative or both positive), when the latter is complex and includes at least two amphipathic substances , one of which is more soluble than the other, and also tends to destabilize the surface formed by the less soluble substance. In other words, although, as a rule, it is true that the same charges repel each other, charged macromolecules or chain molecules can associate with the same charged surface better when either the associating substance and the surface of the substrate are negatively charged, or even when both components involved in the process Associations carry a total positive charge, provided that the surface complexity makes possible the necessary intra- and intermolecular rearrangements. Based on existing experience, one would expect that the association will proceed easier and stronger in the case when negatively charged macromolecules are associated with a positively charged surface, i.e. when it is promoted by electrostatic attraction, and vice versa.

 The two effects described in the previous paragraphs can be advantageously combined, as specifically defined in independent paragraph 3.

 The choice of amphipathic surface-forming substances can be determined in terms of the different solubilities of the substances involved, which together form the membrane or surface that the macromolecule or chain molecule is about to bind to and which most often takes the form of vesicles suspended in a liquid medium. As a rule, the effect of the invention is more noticeable, i.e., the attractiveness of the surface with respect to the binding macromolecules is greater, the greater the difference in solubility of the participating molecules. The more soluble membrane ingredient should be at least 10 times, but preferably at least 100 times more soluble, than the less soluble component involved in surface building. So, when an amphipathic surface-forming substance, such as a phospholipid, is combined with a second substance, for example a surfactant, in a suitable liquid medium, such as water, the use of a surfactant as a second component, which is more soluble in water than phospholipid (in the right amount).

 On the other hand, the choice to be made can also be determined in terms of the values of the curvature of the resulting surface. Using the above example of a phospholipid (as the main surface-forming substance) mixed with a surfactant (as the surface destabilizing more soluble second ingredient) in water (used as a liquid medium), the resulting vesicles acquire some characteristic surface curvature. The (average) curvature, as a rule, is defined as the reciprocal of the average radius of the space enclosed inside the surface under consideration. Typically, the addition of a surfactant will increase the surface curvature of the mixed lipid vesicles compared to the curvature of phospholipid vesicles not containing a surfactant. If there is a saturation concentration of a surfactant at which the stability of the curved surface is not at catastrophic risk, the optimal concentration of surfactant is usually chosen so that it is less than 99% of such a saturation concentration; more often a value between 1 and 80 mol% is selected, even more preferably between 10 and 60 mol% and most preferably between 20 and 50 mol% of the saturation concentration.

 On the other hand, if the saturation concentration in the system under consideration is unattainable due to the fact that after the surfactant is added, the surface is destroyed before saturation is achieved, the amount of surfactant used is usually less than 99% of the solubilizing concentration. Again, the optimal concentration of surfactant in the system is often between 1 and 80%, more often between 10 and 60%, and preferably between 20 and 50% of the concentration that limits the formation of the absorbent surface of solubilized mixed lipid aggregates, i.e. at a higher concentration, at which the extended surface is replaced by a significantly smaller average surface.

 A suitable mixture of substances used in practice can also be determined in terms of average values of the curvature of these surfaces. As indicated in paragraph 7, the surfaces have an average curvature (defined as the reciprocal of the average radius of the areas covered by the surfaces) corresponding to the average radius between 15 and 5000 nm, often between 30 and 1000 nm, more often between 40 and 300 nm, and most preferably between 50 and 150 nm. It should be noted, however, that the curvature of the absorbent surface is not necessarily due to the properties of the absorbent membrane. When surfaces are used on a solid substrate and they are constructed in accordance with the present invention from a selected mixture of amphipathic substances, the average curvature of these surfaces is usually determined by the curvature of the surface of the solid substrate.

In addition, the invention can be defined in terms of the relative concentration of charged components associated with the surface, at least when the association between the same charges is used. The relative concentration of such surface-bound charged components is between 5 and 100 mol%, more preferably between 10 and 80 mol%, and most preferably between 20 and 60 mol% of the concentration of all surface-forming amphipathic substances taken together. Determined from the point of view of the density of the total surface charge, the surface is characterized by density values between 0.05 and 0.5 C • m ^-2 (pendant per square meter), even better between 0.075 and 0.4 C • m ^-2 and best between 0.10 and 0.35 C • m ^-2 .

 It is preferable to select the concentration and composition of the background electrolyte, which preferably includes mono- and oligovalent ions, so as to achieve the maximum positive effect of charge-charge interactions on the desired association. Typically, the total ionic strength is maintained between I = 0.001 and I = 1, preferably between I = 0.02 and I = 0.5, and even more preferably between I = 0.1 and 1 = 0.3.

 Another useful definition of the invention focuses on absorbent surfaces in the form of a membrane surrounding a tiny droplet of liquid. Such membranes are often similar to a bilayer and include at least two types or types of (self) aggregating amphiphilic substances, the solubility of which in the (preferably aqueous) liquid medium used to suspend droplets varies at least 10 times, preferably at least 100 time. In these cases, the choice of substances that form the membrane can be dictated by the requirement that the average diameter of the homoaggregates of the more soluble substance or the diameter of the heteroaggregates comprising both substances be smaller than the average diameter of the homoaggregates containing only the less soluble substance.

 The total content in the system of all amphipathic substances that are capable of forming a surface, preferably has a value between 0.01 and 30 wt. %, especially between 0.1 and 15 wt.%, and most preferably between 1 and 10 wt. % of the total dry weight, in particular if the combination is used to obtain a composition for application to the body or to a person or animal, mainly for medical purposes.

 The substance involved in the construction of the surface, or the substance supporting the surface, i.e. a substance that is capable of forming extended surfaces can be advantageously selected from biocompatible polar or nonpolar lipids, especially if the absorbent surface must have a bilayer-like structure. Specifically, the lipid or lipoid from any suitable biological source or an appropriate synthetic lipid, or a modification of such a lipid, preferably glyceride, glycerophospholipid, isoprenoid lipid, sphingolipid, steroid, sterol, sulfur or carbohydrate lipid or any other lipid may be selected as the main surface-forming substance. capable of forming bilayers, in particular a half protonated liquid fatty acid, and preferably belonging to the class of phosphatidylcholines, phosphatidylethane laminates, phosphatidylglycerols, phosphatidylinositols, phosphatidic acids, phosphatidylserines, sphingomyelins or sphynophospholipids, glycosphingolipids (for example, cerebrosides, ceramidopolyhexosides, sulfatides, sphingoplasmolene, or diphenyl diphenolidene, or other , diarachidoyl, dilauroyl, dimyristoyl, dipalmitoyl, distearoyl or the corresponding sphingosine derivatives, glycolipids or diacyl, dialkenoyl or dialkyl lipids.

 Another destabilizing surface and a more soluble substance is predominantly a surfactant and can mainly belong to the class of nonionic, zwitterionic, anionic or cationic detergents; Particularly suitable for use are a long chain fatty acid or alcohol, an alkyl tri / di / methylammonium salt, an alkyl sulfate salt, a monovalent salt of cholate, deoxycholate, glycocholate, glycodeoxycholate, taurodeoxycholate or taurocholate, acyl or alkanoyl dimethyl dimethyl dimethyl alkyl, or alkanoyl-N-methylglucamide, N-alkyl-N, N-dimethylglycine, 3- (acyldimethylammonio) alkanesulfonate, N-acylsulfobetaine, polyethylene glycol octylphenyl ether, in particular nonethylen octylphenyl ether glycol, acyl-polyethylene ether, in particular non-ethylene dodecyl ether, isoacyl-polyethylene glycol ether, in particular octaethylene glycol isotridecyl ether, acyl-polyethylene ether, in particular octaethylenodecyl ether, acyl-sorbitan-polyethylene glycol ester (such as Twenty-glycol, 20) or polyethylene glycol-20-sorbitan monooleate (Tween 80), acyl-polyhydroxyethylene ether, in particular polyhydroxyethylene-lauryl, -myristoyl, -acetyl stearyl or-oleoyl ether, as in polyhydroxyethylene 4, or 6, or 8, or 10, or 12, etc., in lauryl ether (as in the Brij series) or in the corresponding ester, for example, a series of polyhydroxyethylene-8-stearate (Myrj 45), laurate or oleate, or polyhydroxylated castor oil (Chemophor EL), sorbitan monoalkylate (e.g. Arlacel or Span), especially sorbitan monolaurate (Arlacel-20, Span-20), acyl or alkanoyl-N-methylglucamide, especially in decanoyl- or dodecanoyl-N-methylglucamide, alkyl sulfate (salt), for example, lauryl or oleyl sulfate, sodium deoxycholate, sodium glycodeoxycholate, sodium oleate, t sodium urate, a salt of a fatty acid such as sodium elaidate, sodium linoleate, sodium laurate, a lysophospholipid such as n-octadecylene (= oleyl) -glycerophosphatidic acid, phosphorylglycerol or phosphorylserin, n-acyl-, for example, lauryl or oleyl-glycero -phosphatidic acid, -phosphorylglycerol or phosphoryl serine, n-tetradecyl-glycero-phosphatidic acid, -phosphorylglycerol or phosphoryl serine, the corresponding palmitoleyl, elaidoyl, vaccenyl-lysophospholipid, or the corresponding short-chain phospholipid or .

 The concentration of charged membrane components is often in the range of relative concentrations of 1-80 mol%, preferably 10-60 mol%, and most preferably between 30-50 mol%, calculated on the basis of the amount of all components involved in the construction of the membrane.

 Preferably, phosphatidylcholine and / or phosphatidylglycerol is selected as the surface-supporting substance, and a lysophospholipid, such as lysophosphatidic acid or methylphosphatidic acid, lysophosphatidylglycerol or lysophosphatidylcholine, partially N-methylated lysophosphatidyletholate glycololamine, chololate, glycololamine, glycololamine, cholenecholamine another sufficiently polar derivative of sterol, laurate, myristate, palmitate, oleate, palmitoleate, elaidate or any other whether the fatty acid and / or surfactant Tween-, Myrj- or Brij- type, or even Triton, - surface-active sulfonate or -sulfobetaine, -N-glucamide or sorbitan (Arlacel or Span) fatty acids are selected as the substance less capable of forming an extended surface.

 Advantageously, the average radius of the space enclosed by said extended surfaces is between 15 and 5000 nm, often between 30 and 1000 nm, more often between 40 and 300 nm, and most preferably between 50 and 150 nm.

 Typically, a substance of the third type, which is associated with an extended surface formed by a combination of two other substances (and, if required, a third, fourth, fifth, etc. substance), can include any molecule with repeating subunits, especially in the form of chain molecules. Thus, the third substance may be an oligomer or a polymer. In particular, it can be an amphipathic macromolecular substance with an average molecular weight of more than 800 daltons, preferably more than 1000 daltons and often even more than 1500 daltons. Typically, such substances are of biological origin or are analogues of biological substances and predominantly have biological activity, i.e. are bioagents.

 The third substance (a substance of the third type) is preferably associated with membrane-like extended surfaces according to the invention, in particular by incorporating into the boundary layer (or boundary layers) between the membrane and the liquid medium, and this boundary layer (s) are (are) integral part of these membranes.

 The content of the specified third substance (molecules) or the corresponding chain molecules, as a rule, has a value between 0.001 and 50 wt.% Based on the mass of the adsorbing surface. Often this content has a value between 0.1 and 35 wt.%, More preferably between 0.5 and 25 wt.% And mainly between 1 and 20 wt.% Using the same relative units, and it was found that a particular ratio often decreases with increasing molar mass of these chain molecules.

 It has been found that whether the adsorbed macromolecule or chain molecule is a protein or part of a protein, it can usually be associated with an absorbent surface in accordance with the meaning of the present invention, provided that such a particle includes at least three segments or functional groups capable of binding to absorbent surface.

 Macromolecules or chain molecules, which in accordance with the present invention are capable of association with an extended surface formed by these amphipathic substances, can belong to the class of polynucleotides, such DNA or RNA, or polysaccharides with at least partial ability to interact with the surface, whether they are in natural form or after some suitable chemical, biochemical or genetic modification.

 The chain molecules associated with an extended surface can have various physiological functions and act, for example, as adrenocorticostatic, b-adrenolytic, androgenic or antiandrogenic, antiparasitic, anabolic, anesthetic or analgesic, analeptic, anti-allergic, anti-arrhythmic, anti-atherosclerotic and anti-atherosclerotic and antibiotic, antidepressant and / or antipsychotic, antidiabetic agent, antidote, antiemetic, anti epileptic, antifibrinolytic, anticonvulsant, anticholinergic agent, enzyme, coenzyme or corresponding inhibitor, antihistamine, antihypertensive agent, biological inhibitor of drug activity, antihypotonic, anticoagulant, antifungal, anti-myasthenic agent, anti-Parkinson's disease, antipyretic, antipyretic, anti-inflammatory respiratory analeptic or respiratory stimulant, bronchodilator, car iotonic, chemotherapeutic agent, coronary dilator, cytostatic, diuretic agent, ganglioblocker, glucocorticoid, anti-influenza agent, hemostatic, hypnotic agent, immunoglobulin or its fragment or any other immunologically active substance, biologically active carbohydrate (derivative), contceptive, anti-migraine antimigraine agent, mineral , morphine antagonist, muscle relaxant, narcotic, neurotherapeutic, antipsychotic agent, neurotransmitter or one of its antagonists, peptide (derivative f), an agent for the treatment of eye diseases, (para) sympathomimetic or (para) sympatholytic agent, protein (derivative), anti-psoriasis / neurodermatitis drug, mydriatic, psychostimulating, rhinological agent, any sleep-causing agent or antagonist thereof , sedative agent, antispasmodic, tuberculostatic, urological, vasoconstrictor or vasodilator, virustatic agent or any agent for treating wounds, or any combination of the above agents.

 The invention can also be advantageously used when the third substance is a growth regulating agent.

 Other examples of preferred embodiments of the invention include a third substance selected from the class of immunoregulators, including antibodies, cytokines, lymphokines, chemokines and, respectively, active parts of plants, bacteria, viruses, pathogens or other immunogens, or parts or modifications of any of them; enzymes, coenzymes or any other type of biocatalysts; recognizing molecules, including but not limited to adhesins, antibodies, catenins, selectins, chaperones, or parts thereof; hormones, especially insulin.

 In the case of insulin, the combination according to the invention preferably contains from 1 to 500 int. units insulin / ml, in particular between 20 and 400 int. units insulin / ml and most preferably between 50 and 250 int. units insulin / ml as an active substance. Preferably, recombinant human insulin or the like human insulin is used as a medicine.

 Other preferred uses of the present invention include the use of a variety of cytokines, such as interleukins or interferons, said interleukins suitable for use in humans or animals and include IL-2, IL-4, IL-8, IL-10, IL-12, and these interferons are suitable for use in humans or animals and include, but are not limited to, IF alpha, beta and gamma.

 The specified combination contains between 0.01 and 20 mg of interleukin / ml, in particular between 0.1 and 15 mg and most preferably between 1 and 10 mg of interleukin / ml, if necessary after final dilution to achieve the practically required range of drug concentration.

 The specified combination contains up to 20 relative wt.% Interferon, in particular between 0.1 and 15 mg of interferon / ml and most preferably between 1 and 10 mg of interferon / ml, if necessary after final dilution, due to which the concentration of the drug is established in the range of practically preferred concentrations.

 In another embodiment, the present invention describes the introduction of nerve growth factor (NGF), associated as a (third) active substance with the surfaces of the invention. The preferred type of this agent is human recombinant NGF, the optimal concentration ranges for its use are up to 25 mg of nerve growth factor (NGF) / ml suspension or up to 25 relative wt.% NGF as an agent, in particular 0.1-15 rel. wt.% protein and most preferably between 1 and 10 Rel. wt.% NGF, if necessary diluted before use.

 It is possible to use the technology of the invention described herein for administering immunoglobulin (Ig) as intact antibodies, portions of antibodies, or any other biologically acceptable and active modifications thereof. Advantageously, the suspension contains up to 25 mg of immunoglobulin (Ig) / ml suspension or up to 25 wt.% Ig, relative to the total lipid content, preferably from 0.1 to 15 rel. wt.% protein and most preferably from 1 to 10 Rel. wt.% immunoglobulin.

 The invention describes methods for producing the combinations described above, in particular as compositions of an active agent, in particular a biologically, cosmetically and / or pharmaceutically active agent, as discussed above, and these methods include selecting at least two amphipathic substances that differ in their solubility in a suitable liquid medium and which, at least in combination, are capable of forming an extended surface, especially in the form of a membrane, in contact with said medium. It is recommended as a selection criterion for these methods to use an extended surface formed by a combination of substances that can attract an active agent and promote association with the specified surface, provided that the specified surface is more attractive for this agent than the surface formed only by that of two substances, which forms longer surfaces per se than another substance per se, and / or select at least two amphipathic substances that differ in their size solubility in a suitable liquid medium, provided that such substances, at least in combination, are capable of forming an extended surface, in particular a membrane-like surface in contact with said medium, and also provided that said surface including a combination of both substances is more attractive to the active agent and has a better ability to bind it than a surface formed by one of the two substances alone, which forms longer surfaces than the other ETS, and last but not less important condition is that in the case where both the surface and the agent bear total electric charge, and the surface, and the agent in the average are both negatively charged or both positively charged.

 Preferred methods for producing the extended surfaces of the invention include mechanical operations on a suitable mixture of substances, such as filtration, pressure change or mechanical homogenization, shaking, mixing, stirring, or the action of any other controlled mechanical fragmentation method in the presence of agent molecules that must be associated with the surface formed during such processing.

 Preferably, if the selected combination of surface-forming substances is allowed to adsorb on suitable surface (s), which is (them) a solid substrate, or in some other way, the selected combination is brought into constant contact with it (them) and then with a liquid medium by adding one substance after another or several at the same time, at least one of the last stages of surface formation is carried out in the presence of an agent, which is subsequently associated with the surface on a solid substrate.

 An advantageous option is to first obtain adsorbent surfaces or their precursors suspended in a liquid medium or on a solid substrate, using steps that may involve sequential mixing of the surface-forming molecules, and then associating molecules are added and allowed to associate with said surfaces, using shaking, stirring or incubation, provided that such treatment does not destroy the pre-formed surfaces.

 In accordance with the present invention, a preferred method for preparing compositions for the non-invasive use of various agents, in particular through intact human or animal or plant skin, is the creation of surfaces capable of associating with agent molecules in complexes comprising at least one amphiphilic substance, at least one hydrophilic liquid, at least one border-active or surfactant, and at least one agent. Together, these ingredients constitute a composition suitable for non-invasive use of the agent, and other generally accepted ingredients suitable and necessary to achieve the desired properties and stability of the final composition may also be added.

 During the implementation of the method, it is possible to advantageously mix the selected ingredients separately and, if required, co / dissolve the components to form a solution, then combine the resulting mixture (s) or solution (s) in order to finally cause the formation of particles or surfaces that bind to the agent, preferably under the action mechanical energy, as already discussed.

 Amphiphilic substances suitable for this purpose, as described in the present invention, can be used either as such, or dissolved in a physiologically compatible polar liquid, such as water, or a solvent miscible with it, or in a solvation promoting reagent, together with a polar solution that also includes at least one border-active substance or surfactant.

 One of the preferred ways of inducing the formation of attracting agent surfaces is to add the substance to the liquid phase. Alternatives include reverse phase evaporation, injection or dialysis, or mechanical exposure, such as shaking, mixing, vibration, homogenization, sonication (i.e., ultrasonic waves), shearing, freezing and thawing or filtering using a conventional suitable displacement pressure. When filtering is used, the filter material can be advantageously selected so that it has a pore size between 0.01 and 0.8 μm, preferably between 0.02 and 0.3 μm, and most preferably between 0.05 and 0.15 μm . You can use several filters in series or in parallel, which is more suitable to achieve the desired surface formation effect and maximize the simplicity and speed of the process.

 Advantageously, if said agents and carriers are associated, at least in part, after the formation of an absorbent surface.

 For practical purposes, it is possible to form associates between the molecules of the agent and the binding surfaces immediately before using the resulting composition. In this case, a concentrate or lyophilisate can be used as starting material.

 The invention describes the preparation of carriers for agents, in particular for the purpose of drug delivery, depot preparations, or for any other type of use in medicine or biology. Thus, the invention can also be used in the field of penetration through pores in a barrier; in this case, an associating surface is predominantly obtained in the form of a membrane formed by amphipathic molecules surrounding the smallest droplets, as is already known from the prior art, with agent molecules associated with the surface of these droplets, which are transferred using the indicated ultra-deformable droplets through the pores in the barrier, even if the average pore diameter in the barrier is less than the average diameter of droplets or vesicles. However, it may be necessary to coordinate the properties that provide the association, on the one hand, and the properties that provide the best ability of the membrane to adapt, on the other hand, since these two properties, as discussed above, do not necessarily coincide and most often actually differ from the optimal properties of the composition, determined by one ability of vesicular membranes to adapt for passage through pores.

 Other uses for the associates of the invention include use in bioengineering, genetic manipulation, as well as use in separation technology, for (bio) technology and diagnostic purposes. Here, as in other cases of using the invention, including enzymatic processes and catalysis, it may be useful that aspect of the invention according to which the associating surface can preferably be on a solid substrate than used as a membrane vesicle. This allows the surfaces of the invention to be fixed on a solid substrate, which is then suitably treated, attached, separated, concentrated, etc., for example, in order to fix, to the maximum extent possible, the catalytically active macromolecules associated with this type of surface.

 It is possible to stabilize surface-associated molecules, especially chain molecules, which are at least partially amphipathic, such as (modified) proteins, polypeptides, polynucleotides or polysaccharides, and / or in the catalytic processes in which these molecules are involved in surface condition. Therefore, the present invention can be used to obtain, say, columns filled with catalytically active, high affinity or selective macromolecules with other activities. One example of such use is chemical reactions that are carried out by passing suitable reactant (s), for example, in solution, through a column containing surfaces on a solid support with active molecules non-covalently attached to them, surrounding the surface where the reaction involving these active macromolecules when passing the solution through immobilized macromolecules.

 In another illustrative example, a solution with molecules, at least some of which must be separated from the solution, is passed through a column or brought into contact with a suspension of adsorbing surfaces on a solid substrate in order to first allow target molecules to associate with the surface of the substrate, and then separate the liquid and solid phase by any suitable method, including, but not limited to, centrifugation, precipitation, flotation (with or without centrifugation), electrical or magnetic separation of a part adsorbent, etc.

 Another possibility of using the present invention relates to the control of the kinetics and / or reversibility of the association or dissociation between the surface-associated molecules on the one hand and the complex adaptable surface formed in accordance with the present invention by a combination of suitable amphipathic substances, in which an increase in the charge density on the surface and / or an increase in the softness of the surface and / or the density of defects on the surface can be used to accelerate the association ui. A decrease in the corresponding parameters can be used to reduce the rate of association or else to induce partial or complete dissociation.

The temperature of preparation of the composition and its storage rarely goes beyond the range from 0 to 95 ^o C. Due to the sensitivity to temperature conditions of many of the ingredients we are interested in, especially many macromolecules, it is preferable that the temperature is below 70 ^o C and even below 45 ^o C. , cryo-and thermal stabilizers can allow you to work at different temperature ranges. The practical application of the invention is usually carried out at room or physiological temperature, but its use at various temperatures is also possible and may be required for specific compositions and applications. One of the possible reasons for this is to maintain the efficiency of adaptation (flexibility, sign of charge and / or charge density) of the adsorbing surface at elevated temperatures; another possible example is to keep the agents in active form at low temperatures.

The characteristics of the composition are reasonably determined by the most sensitive component of the system. Preferred may be storage in cold conditions (for example, at 4 ^o C), as well as in an inert atmosphere (for example, in a nitrogen atmosphere).

The described compositions can be created at the site of their application using techniques specific to the adsorbent or adsorbate, depending on which one is more important. (Examples of phospholipid-based adsorbents can be found in "Liposomes" (Gregoriadis G., ed., CRS Press,
Boca Raton, Fl. Vols 1-3, 1987); 'Liposomes as drug carriers' Gregoriadis G., ed., John Wiley & Sons, New York, 1988; 'Liposomes. A Practical Approach ', New, R., Oxford-Press, 1989). The composition may also be diluted or concentrated (for example, by ultracentrifugation or ultrafiltration).

 At the right time or before using the composition, additives can be added to it to improve the chemical or biological stability of the final composition, (macro) molecular association or change, kinetics of dis / association, ease of administration, compatibility, etc.

 The additives of interest to us include a variety of system-optimizing solvents (the concentration of which should not go beyond the limits determined by maintaining or achieving the required characteristics of the system), chemical stabilizers (for example, antioxidants and other absorbers), buffers, etc., adsorption enhancers, biologically active adjuvant molecules (e.g. bactericidal, virostatic agents), etc.

 Solvents suitable for the aforementioned purpose include, but are not limited to, unsubstituted or substituted, for example, halogenated, aliphatic, cycloaliphatic, aromatic or aromatic aliphatic hydrocarbons, such as benzene, toluene, methylene chloride, dichloromethane or chloroform, alcohols, such as methanol or ethanol, propanol, ethylene glycol, propanediol, glycerin, erythritol, short chain alkyl esters of carboxylic acids such as acetic acid, alkyl acid esters such as diethyl ether, dioxane or the like trahydrofuran, etc., or mixtures thereof.

 It may also be necessary to change the pH of the adsorbent / adsorbate mixture after preparation or immediately before use. This should prevent the destruction of individual system components and / or associates. It should also improve the biological activity or physiological compatibility of the resulting mixture. To neutralize a mixture for in vivo or in vitro biological use, biocompatible acids or bases are often used to obtain a pH between 3-12, often 5 to 9, and most often between 6 and 8, depending on the purpose or site of use. Physiologically acceptable acids are, for example, dilute aqueous solutions of inorganic acids, such as hydrochloric acid, sulfuric acid or phosphoric acid, and organic acids, such as carboxyalkanoic acids, for example, acetic acid. Physiologically acceptable bases are, for example, dilute sodium hydroxide, suitably ionized phosphoric acids, etc.

 All directly or indirectly mentioned lipids and surfactants are known. A review of lipids and phospholipids that form aggregates suitable for association with macromolecules is provided, for example, in the 'Phospholipids Handbook' (Cevc G., ed., Marcel Dekker, New York, 1993), 'An Introduction to the Chemistry and Biochemistry of Fatty acids and Their Glycerides' (Gunstone FD ed.). A review of commercial surfactants is provided in the catalogs of 'Mc Cutcheon's, Emulsifiers & Detergents', (Manufacturing Confectioner Publishing Co.) and other sources such as Handbook of Industrial Surfactants M. Ash & I. Ash, eds. Cower, 1993. A review of relevant active compounds is provided, for example, in 'Deutsches Arzneibuch', The British Pharmaceutical Guide, European Pharmacopoeia, Japanese Pharmacopoeia, The United States Pharmacopoeia, etc. Relevant macromolecules are described in industrial catalogs, in scientific periodicals on this topic and in specialized publications.

 This description discloses some relevant properties of the associates, demonstrated by the example of some selected mixtures of polypeptide / protein and phospholipid / surfactant. The validity of the general conclusions is not limited to the presented choice, as well as the associations obtained that are extremely useful in the field of medicine and veterinary medicine.

 The following examples should illustrate the invention without establishing or outlining its limitations. The temperature is given in all examples in degrees Celsius, the dimensions of the carriers in nanometers, the ratios and percentages are given in molar units. All units are given in SI, unless otherwise indicated.

Examples
The following experiments are carried out in order to determine the ability of insulin to bind to complex vesicles. Various compositions of vesicle compositions are used. Options include various surfactants and lipids for introducing a total charge on / into the vesicles, different lipid / detergent ratios, different total lipid contents and different types and concentrations of insulin.

 In the first series of experiments, complex lipid vesicles containing a phospholipid / biosurfactant mixture were combined with insulin at various protein / lipid ratios in order to find the maximum binding. Suitable, single component vesicles (liposomes) are used for comparison.

Examples 1-27 Ultradeformable and flexible vesicles (Transfersomes ^TM ).

Initial suspension
The total lipid (TL) content of 10 wt.%, Including:
874.4 mg of soya bean phosphatidylcholine;
125.6 mg sodium cholate;
9 ml of phosphate buffer, pH 7.1.

Final suspension A
TL content of 5 wt. %, including lipids listed above, and 0.1, 0.5, 1, 2, 3, 4 mg of insulin per 100 mg TL
To achieve the required dilutions, the stored insulin solution (4 mg / ml Actrapid ^TM Novo-Nordisk) is mixed with buffer (see table 1).

 Final suspensions A are prepared by mixing 2.5 ml of the original lipid suspension (10% TL) and 2.5 ml of a suitable insulin dilution.

The final suspension In
TL content from 5 to 0.25 wt.%, Including the lipids above,
and 4, 5, 6.67, 10, 20, 40, and 80 mg of insulin per 100 mg of TL
In order to obtain different ratios of insulin / lipid, the pipetting scheme shown in Table 2 is used.

 Final suspensions B are prepared by mixing 2.5 ml of Actrapid HM (4 mg / ml insulin) with 2.5 ml of an appropriately diluted lipid suspension.

Final suspension C
TL content from 2.5 to 0.125 wt.%, Including the lipids above, and
4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 80 and 160 mg of insulin per 100 mg of TL.

 To obtain the indicated ratios of insulin / lipid, use the pipetting scheme shown in table.3.

 For test series C, a 5% suspension of vesicles is prepared from a 10% stored suspension by diluting it 1: 1 volume: volume with buffer and repeating the filtering and freezing-thawing process as described below.

Preparation of the adsorbent / adsorbate mixture. The buffer is prepared using standard methods and filtered through a 0.2 micrometer sterile filter. (For further use, the solution is stored in a glass jar.) The lipid mixture is suspended in a buffer in a sterile glass jar, tightly closed, and stirred on a magnetic stirrer for 2 days at room temperature. The suspension is then extruded sequentially through etched-track polycarbonate membranes (Nucleopore type) with a nominal pore size of 400, 100 and 50 nm, respectively. Each time, three passes are made using a displacing pressure between 0.6 and 0.8 MPa. The resulting suspension of vesicles is frozen and thawed 5 times at an appropriate temperature from -70 to +50 ^o C. To obtain the desired final size of the vesicles, the suspension is again extruded 4 times through a 100 nm filter at 0.7 MPa. At the last stage, highly deformable vesicles are sterilized by filtration through a sterile syringe filter with 200 nm pores. Vesicles are stored in sterile polyethylene containers at 4 ^{° C.} until used.

 Each insulin molecule carries a negative total charge in the neutral pH region due to the excess of negatively charged amino acids with respect to positively charged amino acids above pH 5.4.

For many, including this association study, a commercial insulin solution (Actrapid ^™ from Novo-Nordisk) is used. Thus, the original protein solution contains 4 mg of insulin / ml and 3 mg of m-cresol / ml. Various nominal insulin / lipid ratios are obtained by adding the appropriate amount of such a solution to a suspension of adsorbing vesicles. The resulting carrier insulin mixtures were gently but thoroughly mixed and incubated for at least 2 hours, depending on the experiment, at room temperature.

 In Test Series A, the final suspension is prepared by diluting the initial suspension of Actrapid vesicles to obtain a final lipid concentration of 50 mg TL / ml and various protein / lipid ratios. In test series B, the final lipid concentration varies between 2.5 and 40 mg / ml depending on the ratio of insulin / TL. In experimental series C, the final lipid concentration is in the range from 1.25 to 25 mg / ml. For comparison, similar diluted series are prepared using a buffer instead of a lipid suspension.

Test measurements are carried out with 4 ml of each insulin / vesicle mixture. After 2 hours, the lipid vesicles are separated from the aqueous lower phase to determine how much insulin has been associated in any way with the lipid vesicles and how much remains unbound in the aqueous lower phase. For this purpose, CENTRISART I-ultracentrifuge tubes with a notch of 100,000 Da are used. Three tubes are used for each dilution with 1 ml of an insulin-containing suspension, and centrifuged at 2000 g for 3 hours (T = 10 ^{° C.} ). The insulin concentration in the formed optically transparent supernatant (acceptable containing only buffer, insulin and a certain amount of mixed lipid (phosphatidylcholine / cholate) micelles together with the dissolved detergent) is determined. Supernatants that are not optically transparent are discarded because they have been shown to be contaminated with lipid vesicles that have passed through defective CENTRISAT I filters. Use the standard HPLC process for all of the insulin definitions mentioned in this description. Measurements duplicate.

 Initial dilutions serve as a positive control. With a negative control, the value of non-specific adsorption of insulin on the test device is determined. After correction, taking into account such non-specific binding, the difference between the initial and final concentration of insulin in the supernatant is calculated. "Missing" insulin is defined as associated with vesicles and is expressed in absolute or relative units.

 The results of the above experiment are shown in Figure 1. They show that when the insulin / lipid ratio is below 6 mg / 100 mg TL, 80-90% of the added protein is associated (bound) with the vesicles. At higher insulin / lipid ratios, the relative efficiency of the protein-surface association decreases and reaches only 5% binding for 2/5 (40 mg / 100 mg) dilution. In other words, 2 mg out of every added 40 mg of insulin at high dilution and high protein / lipid ratio tends to associate (nominally) with 100 mg of lipid in the form of highly deformable vesicles.

 Prolonging the incubation time or, but to a lesser extent, increasing the concentration of the added suspension improves the situation (figures 2 and 3).

 Examples 28-45.

Standard vesicles (liposomes), initial suspension:
1 g of soy bean phosphatidylcholine;
9 ml of phosphate buffer, pH 7.1.

Final suspension A
TL content of 5 wt.%, Including the above lipids and
0.1, 0.5, 1, 2, 3, 4 mg insulin per 100 mg TL
(0.1, 0.5, 1, 2, 3, 4 rel. Wt.%).

The final suspension In
TL content from 5 to 0.25 wt.%, Including lipids above and
4, 5, 6, 7, 10, 20, 40, and 80 mg insulin per 100 mg TL.

 An initial lipid suspension is prepared as described in Examples 1-27. However, in order to obtain a sufficiently monodisperse preparation of rather small liposomes, 6 additional extrusions are carried out through 100 nm filters.

 The binding of insulin to test liposomes was very low. Only from 2 to 5% of the administered drug was bound to standard lipid vesicles in the range of dilutions from 4 to 100 mg / ml - dilutions (graphic data not shown).

 To determine and experimentally exclude the effects of suspension dilution on the composition of highly deformable complex vesicles, the following experiments are carried out.

 Examples 46-59.

The initial suspension:
874.4 mg of soya bean phosphatidylcholine;
125.6 mg of sodium cholate (giving 10 vol.% TL content);
9 ml of phosphate buffer, pH 7.1.

The final suspension:
The composition of the final suspensions was the same as in series B and C of examples 1-27, including decreasing final lipid concentrations.

The measured insulin / lipid ratios were as follows: 4, 8, 10, 20, 40, 80, 160 mg of insulin per 100 mg TL
The preparation of a suspension of vesicles corresponds to the description given in examples 1-27 for the established ratios of insulin / lipid, except that in this case, dilutions are carried out either using Actrapid containing 10 mM cholate and / or using a buffer containing from 5 to 20 mM cholate (for control and test samples). This is done in such a way that the final concentration of cholate in all samples is 5 mM, which is close to the CMC of this detergent, in order to prevent dissociation of cholate from the vesicle membrane after dilution.

 When preventing the laundering of cholate from vesicles, not only the original existing composition of the vesicles is preserved, but also the average charge density of the surface of the vesicles. These improvements affect binding.

 In the examples of these test runs, special care was taken to keep the nominal cholate concentration below 5 mM during the pipetting process, to prevent accidental solubilization of the vesicles, which is especially likely in the range of low total lipid concentrations.

 The results show that up to a protein / lipid mass ratio of 10%, between 80 and 90% of the added insulin binds to the surface of the lipid vesicle (Figure 4). This means that the adsorbent / adsorbate association is almost complete and the protein binding efficiency is very high. The percentage of protein associated with a lipid slowly decreases with increasing protein / lipid ratio and reaches 7% with 1.6 mg of insulin / 1 mg of lipid.

 The absolute amount of carrier-associated insulin peaks at about 0.4 mg of insulin per 1 mg of lipid, when 15.6 mg of 40 mg of added insulin is found to be associated with 100 mg of total lipids in the form of highly deformable vesicles. The best yield is obtained with a relative ratio of 0.2 mg of insulin per 1 mg of the total amount of lipids, however, 14 mg of the injected 20 mg were measured as associated with mixed lipid vesicles. Figure 4 illustrates this data.

 Similar results are obtained if cholate molecules are introduced into a suspension of mixed lipid vesicles with a buffer or insulin solution.

 Examples 60-71.

The original suspension (20% TL):
1099.7 mg phosphatidylcholine from soybeans;
900.3 mg Tween 80;
8 ml of phosphate buffer, pH 7.4.

The final suspension, including:
lipid mixture above
2, 4, 8, 10, 20, and 40 mg of insulin per 100 mg of TL.

The preparation of a suspension of vesicles is carried out essentially as described in examples 1-27, except that the mixing time is increased to 7 days. Actrapid ^TM (Novo-Nordisk) is the source of adsorbed insulin in all cases.

 In order to be able to use a fixed insulin concentration of 4 mg / ml, an insulin / lipid ratio is prepared with a varying final total lipid concentration between 8 and 100 mg / ml. For comparison (regarding the possible dilution effect), vesicles of a similar composition are used to prepare various insulin / lipid ratios, but with a fixed final total lipid concentration of 10 mg / ml (1 wt.%). The vesicle-protein association time is 3 hours.

 The centrifugation time used to separate the non-associated insulin from the protein bound to the vesicle is 6 hours (at 1000 g). All other details of the experiment were the same as described in the first test series (examples 1-27).

 Results. Despite the fact that the binding of insulin to membranes that contain a non-ionic surfactant (Tween-80) is generally lower than to charged (holate-containing) membranes, the quantitative characteristics of both adsorbing systems are the same (see examples 1-27).

 The association of insulin with membranes at a relative insulin / lipid ratio of 0.04 mg of insulin / 1 mg of lipid is approximately 50%. A relative concentration of 0.2 mg of insulin / 1 mg of maximum binding lipid corresponds to only 5.2 mg of bound protein of the total amount of added 20 mg of insulin. An absolute optimum, that is, the best yield in the test series, is achieved at a ratio of 0.04 mg of insulin / 1 mg of lipid.

 Examples 72-76.

An initial suspension (10% TL) including:
874.8 mg of soya bean phosphatidylcholine;
125.6 mg sodium cholate;
9 ml of phosphate buffer, pH 7.1 (-7.4; with these buffers, the pH of the initial suspension is in the range of 7.3-7.6.

 Since the required pH range is 7.3–7.4, all of the following test runs with cholate as a surfactant are carried out with a buffer where the pH is 7.1).

A solution of insulin A:
4, 8, 10, 20 mg / ml phosphate buffer, pH 7.4;
30 μl of Hcl (1 M) per ml of dissolved dry insulin, followed by 30 μl of 1 M NaOH per 1 ml of solution.

Insulin Solution B:
4 mg Actrapid / ml phosphate buffer, pH 7.4.

Mixtures of insulin vesicle
5 wt.% Total lipid concentration;
0.04, 0.08, 0.1 and 0.2 mg of dry insulin per 1 mg of total lipid;
(4, 8, 10, 20 rel. Wt.%).

 The preparation of a suspension of vesicles is carried out as described in examples 1-27, using a similar composition of the membranes. However, to achieve high insulin / lipid ratios, using moderately high final concentrations of total lipid, dry insulin is dissolved to bring its concentration to higher values than used in commercial solutions.

Lyophilized recombinant human insulin does not dissolve rapidly in phosphate buffer at pH 7.4. To prepare an insulin solution, a dry, lyophilized recombinant human insulin “powder” similar to Actrapid ^™ is thus first added to 2 ml of buffer and thoroughly mixed. After temporary acidification (achieved by adding 60 μl of HCl), which sufficiently increases the solubility of insulin, increasing the possibility of a clear solution, add 60 μl of NaOH in order to return the pH value back to 7.4, at which the insulin is stable (as hexamers) and is resistant to destruction / deamidation. An additional solution is prepared by directly dissolving 8 mg of insulin in 2 ml of buffer, pH 7.4.

 A suspension of vesicles (2 ml) and insulin A solution (A) (2 ml) were thoroughly mixed and incubated for 12 hours at the above nominal insulin / lipid ratios. The final total lipid concentration is 50 mg / ml in all cases. For comparison, use solution B. The rest of the experiments are carried out as described in examples 1-27.

 Results. The binding of insulin from a solution prepared from dry protein powder (which at least temporarily makes it possible to obtain a monomer solution) is comparable to that measured for Actrapid insulin in Examples 1-27 (Figure 5). This suggests that it is possible to associate a large amount of insulin with a suspension of lipid vesicles at a concentration of 50 mg / ml. The maximum insulin binding is in the region of the 1/5 protein / lipid mass ratio when approximately 16 mg of added insulin is associated with mixed lipid membranes.

 At similar protein concentrations, identical results were determined with dissolved ad hoc and commercial insulin solutions.

 In the following experimental series, insulin adsorption is compared to various charged and uncharged, liquid, mixed lipid membranes.

 Examples 77-92.

Conventional vesicles, SPC liposomes, neutral (TL = 10 wt.%):
there is no total charge, only zwitterionic phospholipids are contained;
1 g of soy bean phosphatidylcholine;
9 ml of phosphate buffer, pH 7.4.

Conventional vesicles charged with SPC / SPG liposomes (TL = 10 wt.%):
total negative charge of 25 mol.% anionic phosphatidylglycerol;
750 mg of soy bean phosphatylcholine;
250 mg of soy bean phosphatidylglycerol;
9 ml of phosphate buffer, pH 7.4.

Highly deformable neutral vesicles (TL = 10 wt.%):
there is no total charge; they contain zwitterionic phospholipids and nonionic surfactants;
550 mg of soya bean phosphatidylcholine;
450 mg T ween 80;
9 ml of phosphate buffer, pH 7.4.

Highly deformable charged vesicles A (TL = 10 wt.%):
total negative charge due to 25 mol.% anionic cholate;
874.4 mg of soya bean phosphatidylcholine;
125.6 mg sodium cholate;
9 ml of phosphate buffer, pH 7.1.

Highly deformable charged vesicles B (TL = 10 wt.%):
total negative charge due to 25 mol.% (relative to PC) of anionic phosphatidylglycerol;
284.3 mg of soya bean phosphatidylcholine;
94.8 mg of soya bean phosphatidylglycerol;
620.9 mg Tween 80;
9 ml of phosphate buffer, pH 7.4.

Mixtures of insulin vesicle, respectively
50, 25, 10, 5 mg of the total amount of lipid per ml of the final suspension;
0.04, 0.08, 0.1 and 0.2 mg of insulin per 1 mg of total lipid;
(4, 8, 10, 20 rel. Wt.% Protein).

All vesicles are prepared as described above. Tween-containing vesicles are mixed for 7 days. Vesicles and liposomes containing cholate are mixed for 2 days. Actrapid 100 HM ^TM (Novo-Nordisk) is a source of insulin. This led to a variation in the final protein and resulting final lipid concentrations (50, 25, 10 and 5 mg TL / ml, respectively). However, with SPC liposomes, only sample 4 rel. Was studied. wt.%.

 The experimental protocol is the same as described for examples 1-27. For all preparations, the incubation time is 3 hours, the centrifugation time is 6 hours (at 500 g) to facilitate the comparison process. The measurement results are shown in figure 6.

 The results clearly show that insulin, despite its total negative charge, is better associated with negatively charged surfaces. High membrane flexibility, which provides high deformability of the vesicles, is also an advantage.

 The relative binding capacity is 80-90% for highly flexible charged membranes. Such a very high degree of protein association with the membrane is observed at a mass ratio of 1/25 insulin / lipid for both types of studied mixtures of phospholipid-surfactant. Uncharged membranes containing phospholipids and non-ionic surfactants exhibit a 50% relative binding at comparable insulin / lipid ratios.

However, insulin added only at a concentration of between 2.5% (p. Experiments 28-45) is estimated to bind to uncharged, phosphatidylcholine liposomes. Protein binding to charged liposomes, which are associated with 10-20% of the added insulin at a protein / lipid mass ratio of 1/25, exceeds this worst result. Consequently, conventional charged lipid bilayers are an intermediate between an uncharged liposome membrane and more flexible, but neutral (Transfersome ^TM ) membranes.

 These results suggested that the total surface charge (originating from charged lipids or other charged membrane-associated components) should be combined with the softness of the membrane (which is facilitated by the presence of detergents and other related molecules in the adsorbent) in order to maximize surface association or association carrier insulin. This is in favor of the fact that the charges “draw” (parts) of the adsorbed molecules into the adsorbent, which, if it is “softened”, allows the protein to easily penetrate into the interface of the surface.

 Examples 93-95.

Conventional vesicles, SPC liposomes, neutral (TL = 10 wt.%):
no total charge containing only zwitterionic phospholipids;
1 g of soy bean phosphatidylcholine;
9 ml of phosphate buffer, pH 7.4.

Highly deformable charged vesicles A (TL = 10 wt.%):
total negative charge due to 25 mol.% anionic cholate;
874.4 mg of soya bean phosphatidylcholine;
125.10 mg sodium cholate;
9 ml phosphate buffer, pH 7.1.

Highly deformable charged vesicles B (TL = 10 wt.%):
total negative charge, due to 25 mol.% (rel. PC) of anionic phosphatidylglycerol;
284.3 mg of soya bean phosphatidylcholine;
94.8 mg of soya bean phosphatidylglycerol;
620.9 mg Tween 80;
9 ml of phosphate buffer, pH 7.4.

Mixtures of insulin-vusicula, respectively
50, 25, 10, 5 mg total lipids per ml of final suspension
0.04, 0.08, 0.1 and 0.2 mg of insulin per 1 mg of total lipid
(4, 8, 10, 20 rel. Wt.% Protein)
Cooking. To study the kinetics of insulin adsorption: time-dependent measurements were taken on phosphatidylcholine Tween 80 mixed membranes. Test vesicles are prepared as described in the corresponding previous examples. The first point data is determined 2 hours after mixing the lipid suspension with a protein solution. For neutral highly deformable membranes, the next reference point is chosen after 3 hours. Other samples for all suspensions are taken after 4 or after 5 days and after 5 or 6 weeks of incubation.

 Results. A clear time dependence of insulin adsorption on uncharged SPC / Tween mixed membranes was found (see FIG. 9 for some of the data presented). The binding efficiency observed initially in the association process increases from 30% after 2 hours to 50% after 3 hours when the nominal insulin / lipid weight ratio is 1/25. At t = 4 days, binding increases to 64%, but this difference may be insignificant, since after 5 weeks, binding is only 58%.

 As measurements show, the binding of insulin to simple phosphatidylcholine liposomes increases only from a minimum of 2.5% after 3 hours to 5% after 6 weeks.

 Insulin adsorption on charged SPC / SPG / Tween 80 mixtures occurs much faster and stronger than in the case of neutral membranes, as indicated by an increase in protein binding to such membranes from 64% after 2 hours to 76% after 6 weeks. A small second increase, compared with the association in the first hours, indicates a relatively fast binding kinetics.

 For charged SPC / cholate mixed membranes, the rate of insulin binding is even higher. Experiments carried out with such charged vesicles reveal a lack of dependence on the time of protein adsorption on the mixed lipid membrane. Within the experimental error, after 2 hours, binding was already completed in the same way as after 5 weeks of incubation. This suggests that insulin adsorption on charged, flexible membranes occurs much faster than on uncharged membranes. In conclusion, it can be assumed that unusual electrostatic interactions can also cause desorption of protein molecules. The very weak and / or very slow association of insulin with phosphatidylcholine membranes shows that hydrophobic binding, in itself, is not sufficient to achieve high payloads. This may be due to the limited ability of insulin molecules to find suitable binding sites on the lipid bilayer surface. Repulsion between several unsuccessfully adsorbed protein molecules and neighboring experimental adsorbates may also be important.

 Examples 96-100.

Suspensions of ultradeformable vesicles with different charge density (TL = 10 wt.%):
total negative charge, due to 25, 33, 50, 67, 75 mol.% phosphatidylglycerol;
137, 205, 274, 343, 411 mg of soy bean phosphatidylglycerol;
411, 343, 274, 205, 137 mg of soy bean phosphatidylcholine;
452 mg Tween 80;
9 ml phosphate buffer, pH 7.4;
2 mg insulin / ml of the final suspension.

 Lipid vesicles are prepared as described in Examples 93-95. The increase in the relative concentration of charged lipid in the membrane enhances the association of vesicle-insulin, as can be seen in figure 4, and moderately, but acceptable, increases the viscosity of the final suspension.

 Lipid suspensions at higher SPG / SPC molar ratios prepared as described in Examples 93-95 are rather viscous and difficult to handle. However, a higher relative concentration of the charged lipid component should increase the relative amount of insulin associated with the vesicles. This is illustrated in figure 7.

 A change in the content of a charged lipid affects the binding efficiency (insulin) of a protein in a uniform manner. First, the relative amount of insulin associated with vesicles rises. When the ratio of SPC / SPG close to 50, there is a maximum of relative binding. This suggests that a very high SPG content damages the effective binding of insulin, possibly due to the effect of interfacial displacement and / or the effect of a charged surface on the kinetics of protein adsorption. (The latter does not have to be very fast to allow macromolecular rearrangements to occur on the surface, which thus leads to maximum packing density).

 Examples 101-104.

Highly flexible charged membranes (TL = 10 wt.%) Mixed 1/1 with insulin:
874: 10 mg of soya bean phosphatidylcholine;
125.6 mg sodium cholate;
9 ml of phosphate buffer, pH 7.1;
4 mg insulin / ml in stock solution.

 Various methods are used to prepare the vesicles: in addition to extrusion and the freeze-thaw cycles described in Examples 1-27, a much simpler technique was also tested (in which the suspension was only extruded sequentially).

 No noticeable difference was found in the efficiency of protein adsorption on mixed lipid membranes (Figure 8). However, the form of the ability to adapt lipid vesicles, as assessed in the “pore-limiting penetration test”, is different for different groups: as it was found, the ability to deform the vesicles obtained in examples 1-27 is the highest.

 Examples 105-106.

Ultra-flexible charged membranes with various additives (final composition):
437 mg of soya bean phosphatidylcholine;
63 mg sodium cholate;
1 ml of phosphate buffer, pH 7.1;
2 mg of insulin / ml in the final suspension.

Additive A
m-cresol 1.5 mg / ml (final).

Additive B
benzyl alcohol 2.5 mg / ml (final).

Co-solvent addition to Transfersome ^® containing sodium cholate affects the resulting amount of membrane-associated insulin. The relative binding efficiency is 60% in the presence of m-cresol and 90% after the introduction of benzyl alcohol into the test suspension.

 The additives used in examples 103-104 may also act as preservatives.

 Examples 107-110.

Similar membranes with insulin from various sources:
437 mg of soy bean phosphacholine;
63 mg sodium cholate;
1 ml of phosphate buffer, pH 7.1;
2 mg insulin / ml;
from Actrapid 100 NM ^TM (Novo-Nordisk) originally dry, human recombinant (Novo-Nordisk)
initially dry pork (Sigma Chemical Industries);
from Lispro ^TM , insulin analogue (Pfizer Inc.).

 No significant changes were observed in the efficiency of adsorption of various proteins on similar membranes. However, this does not exclude the possibility of various de / adsorption rates.

In particular, dry insulin, when dissolved in an acidic buffer and transferred back to the neutral pH range, is adsorbed on mixed lipid membranes with the same efficiency as insulin from the ready-to-use Actrapid ^™ solution (Novo-Nordisk).

 Examples 111-118.

Soft uncharged membranes
The original suspension (10% TL):
1099.7 mg phosphatidylcholine from soybeans;
900.3 mg Tween 80;
19 ml phosphate buffer, pH 7.

The final suspension, including:
8.4 μl IF mixed with the lipid mixture above
when using from 1.84 mg TL / ml to 18.4 μg TL / ml to create increasing relative amounts of interferon, as shown in FIG. 10.

 Compositions containing protein / lipid mixtures with increasing molar ratios are prepared essentially as described in Examples 60-71. Tests are carried out as described in examples 1-27 with two modifications. The first involves the use of separation tubes (100 kDa notches) in the Centrisart process, which in these test runs are always pre-coated with albumin (from a solution containing 40 mg BSA / ml buffer) to reduce non-specific protein adsorption to below 15%. After incubation with BSA, the tubes are then washed twice with buffer and filled with interferon solution (obtained by diluting the stored solution in the same buffer) to achieve a suitable concentration. A commercial ELISA immunoassay for IF is used to evaluate the final protein concentration. To calculate the amount of interferon associated with the vesicle, use the same process as described in examples 1-18. The degree of protein binding is defined as “protein loss” from the supernatant, measured twice or thrice.

 The results are shown in FIG. 10. They reflect a picture qualitatively similar to that described for insulin binding.

 Examples 119-134.

 Highly flexible, charged membranes.

Initial suspension
The total lipid (TL) content of 10 wt.%, Including:
874.4 mg of soya bean phosphatidylcholine;
125.6 mg sodium cholate;
9 ml phosphate buffer, pH 7.1.

Final suspensions:
lipid / protein mixtures, as shown in FIG. 10;
(other data corresponding to these are given in examples 111-118).

The results of two different experimental series illustrated in FIG. 10 (filled diamonds and squares), reflect the desired effect of the negative charge of the membranes on the efficiency of interferon binding by highly deformable bilayers, despite the total negative charge of protein molecules
Examples 135-145.

The original suspension (10% TL):
Soft uncharged membranes;
SPC / Tw 80;
550 mg of soya bean phosphatidylcholine;
450 mg Tween 80;
9 ml of phosphate buffer, pH 6.5.

Soft charged membranes
SPC / NaChol;
874.4 mg of soya bean phosphatidylcholine;
125.6 mg sodium cholate;
9 ml of phosphate buffer, pH 7.1.

The final suspension, including:
lipids in the ratios above, and
10,000 IU (int. Unit) of interleukin-2 (IL-2)
This mixture of lipids and proteins is processed together. Then determine the degree of association. The separation is carried out essentially as described for examples 119-134, and the amount of IL-2 is determined using protein-dependent stimulation of in vitro growth of Renca cells, in comparison with the standard curve. The data obtained are shown in the following table. (Absolute IL-2 concentrations are given in IU (international units) and the relative amount of protein in%).

 Deviations between the initial and final (fully reduced protein) values are partly due to the loss of protein during separation of the vesicle / IL-2 and partly due to the modified activity of the protein due to the presence of lipids.

 The short-term association of interleukin and preformed lipid vesicles with different surface charge densities, as found, is less sensitive to the charging effect than is assumed based on the data in Table 4 (data not shown).

 Examples 146-148.

Conventional neutral vesicles (initial suspension):
1 g of soy bean phosphatidylcholine;
9 mM phosphate buffer, pH 6.5.

Highly deformable neutral vesicles (initial):
550 mg of soya bean phosphatidylcholine;
450 mg Tween 80;
9 ml of phosphate buffer, pH 6.5.

Highly deformable charged vesicles (initial):
874.4 mg of soya bean phosphatidylcholine;
1% 5.6 mg sodium cholate;
9 ml of phosphate buffer, pH 7.1.

Calcitonin (e.g. salmon) mixed with vesicles (final suspension) 100 mg of total lipid per ml of final suspension
1 mg protein per 100 mg of total lipids.

All lipid suspensions are prepared as described below. Protein (equipped with a small amount of ¹²⁵ I-labeled protein, purified shortly before use) is added to preformed vesicles and incubated for at least 24 hours; alternatively, a protein solution is added to the lipids and coextruded through a microporous filter during suspension preparation.

 To determine the relative binding efficiency of the polypeptide to the membranes, the protein / vesicle mixture is chromatographed using size exclusion gel chromatography on the size of molecules with sequential determination of radioactivity. This allows you to get two peaks containing a radioactively labeled protein, respectively associated with the vesicle and in solution. The areas under the curve are about 30 and 70% for ordinary vesicles, from 60-70% and 40-30% for neutral, soft membranes and> 80% and <20% for charged, highly flexible membranes, respectively.

 Examples 149-152.

Highly deformable neutral vesicles (initial):
550 mg of soya bean phosphatidylcholine;
450 mg Tween 80;
9 ml of phosphate buffer, pH 6.5.

Highly deformable charged vesicles (initial):
874.4 mg of soya bean phosphatidylcholine;
25.6 mg sodium cholate;
9 ml of phosphate buffer, pH 7.1.

Immunoglobulin G mixed with vesicles (final suspension):
100 mg total lipid per ml of final suspension;
0.5 and 1 mg of protein per 100 mg of total lipid.

 All lipid suspensions are prepared as described below. Immunoglobulin (monoclonal IgG directed against fluorescein) is included in the composition by adding a preformed suspension of vesicles. After separation of the amounts of vesicles associated with and free immunoglobulin, the relative contribution of the first of them was determined by using quenching of fluorescence in separated, initial and control solutions. This allows you to determine the final concentration of IgG in each gap.

 The efficiency of the association of IgG with a carrier membrane is estimated to be at least 85% in the case of charged, highly deformed vesicles and approximately 10% lower for neutral, soft membranes. The observed slight difference is probably due to the fact that Ig contains a large hydrophobic Fc region, which rapidly penetrates the lipid membrane even in the absence of softening membranes in the defective components.

 Examples 153-154.

Highly deformable charged vesicles. Type C:
130.5 mg of soya bean phosphatidylcholine;
19.5 mg cholate, sodium salt;
0.1 ml ethanol.

Highly deformable uncharged vesicles. Type T:
75 mg soy bean phosphatidylcholine;
75 mg Tween 80;
0.1 ml ethanol.

Human Insulin Recombinant
1.35 ml Actrapid ^TM 100 (Novo-Nordisk).

 Test composition. One of the lipid mixtures is introduced into alcohol until a uniform phospholipid solution is formed (note: Na cholate is not completely dissolved). The mixture is injected into an insulin solution and mixed thoroughly. After maturing for approximately 12 hours, the resulting crude vesicle suspension is filtered several times through a 0.2 micrometer filter (Sartorius, Gottingen) in order to facilitate and achieve good sample homogeneity. The final concentration of insulin is 80 units / ml.

Test. A healthy male volunteer (75 kg, age 42 years) fasts for 17 hours before the first determination of glucose concentration. In order to determine the change in the concentration of glucose in human blood depending on time, samples are taken in a volume of 2 ml to 4 ml every 10 minutes and up to 20 minutes through a soft intravenous catheter attached to one of his hands. After the initial test period, lasting 70 minutes, during which the average blood glucose concentration was 78.4, a suspension of Transfersulin ^® (45 IU, international units) was applied and spread evenly over a healthy skin surface on the inside of the other forearm (in several sequences) so as to cover the surface of the skin with an area of 56 cm ² . 30 minutes after applying the test suspension, the surface of the skin becomes macroscopically dry; 30 minutes later, only weak traces of suspension are visible.

 To determine the concentration of glucose in the blood, a standard gluco-dehydrogenase test (Merck, Gluc-DH) is used. Each sample contains three independent samples and each measurement is carried out at least three times. This gives confidence that the standard deviation of the mean rarely exceeds 5 mg / dl (dL), with a test error of 3 mg / dl.

Results. The change in blood glucose concentration in a test of a normoglycemic volunteer after the cutaneous administration of insulin associated with Transfersom ^® (Transfersulin ^® ) is always slower than achieved by subcutaneous injection of an insulin solution.

The maximum decrease in blood glucose concentration after transdermal administration of Transfersulin ^® usually exceeds 10% of the result obtained after appropriate subcutaneous injection, the area under the curve being at least 20%, as determined based on published reference data. The average decrease in blood glucose concentration, in the case of suspension C, for a period t> 3 hours, is approximately 18 mg / dl (Fig. 11).

 For suspension T, a result was obtained that was approximately 35% worse relative to the data measured for suspension C. The inclusion of phosphatidylglycerol (15 wt.% Relative to phosphatidylcholine) reduces the difference between the C- and T-types of compositions to 25% (data not are given).

 However, even other best non-invasive insulin delivery methods currently available, such as drug electrophoresis (Meyer BR, Katzeff H. L, Eschbach J., Trimmer J., 20 Zacharias SR, Rosen S., Sibalis D. Amer. J. Med. Sd. 1989, 297: 321-325) or transnasal injection delivers less than 5% and less than 10% of the insulin molecules, respectively, into the blood circulation system.

 Example 155.

Highly deformable charged vesicles:
composition, as in examples 72-76.

Recombinant human insulin:
Actrapid ^TM (lyophilisate) as in Examples 72-76 (Novo-Nordisk).

The composition for the test is prepared as described in examples 61-65. The introduction is carried out essentially as described in the previous examples, but the fasting period lasts longer and blood sampling begins earlier. (Thus, the experiment begins with a 12-hour fasting without monitoring, after which there is a fasting period of 12 hours, during which the blood glucose level is checked without any treatment and a period of 16 hours with monitoring, during which the test volunteer he is starving and Transfersulin ^® is injected under the skin. An additional difference is that the application area is only 10 cm ² .

Before administration of insulin, samples are taken randomly in time. After administration of Transfersulin ^® , blood samples are taken every 20 minutes for the first 4 hours and subsequently every 30 minutes. All samples were analyzed using Accutrend (Boehringer-Mannheim, Germany), an automatic diagnostic device. At each time point, three to five readings are taken. The results are shown in figure 12 and correspond to the average value of changes in the concentration of glucose in the blood. Dashed lines give 95% of the margin.

 In the second “without treatment” period, the average concentration of glucose in the blood is 83.2 mg / dl. The decrease in blood glucose concentration in the first hours after cutaneous administration of the drug with the help of highly adapted mixed lipid vesicles is clearly observed. The glucodynamic profile is similar to that measured in previous test series, while the overall effect is slightly stronger, possibly due to a significantly higher concentration of the drug in the latest test compositions.

 Examples 156-158.

Highly deformable charged vesicles:
composition, as in example 153.

Human recombinant insulin
Actrapid ^TM (Novo-Nordisk), batch as shown in FIG. 12.

In these test series, the effect of changes associated with varying batches of insulin is studied using the same Transfersome ^® series. The introduction is carried out as described in the previous examples. The dose per area is similar to that used in the previous examples.

 The average concentration of glucose in the blood is approximately the same as in all three experiments. This is observed despite the fact that the experimental result varies significantly depending on the batch of insulin. One batch works very well, and the other does not work at all. The third gives average results. Small changes from batch to batch for insulin (which are known but usually not indicated and are especially noticeable in the presence of a very large absorbent surface of the carrier, appear to affect the efficiency and / or kinetics of the interaction of the insulin-carrier. It seems that the change in speed drug release, especially sensitive to this phenomenon.Thus, it is important, before conducting serious tests, not only the study of the amount of lipid associated carrier But more important is to determine the release rate of the medicament. A useful for these purposes is measurement glyukodinamiki in tests on animals such as mice and rats, as well as the characteristics of the compositions after injection.

It is clear that glucodynamics in a human volunteer with normoglycemia after the administration of three different Transfersulin ^® batches with identical Transfersomes ^® , but different insulin batches, has a relatively strong effect on the biological activity of the final composition even with small changes in the initial characteristics of the drug (see Fig. 12 )

Claims (58)

 1. A combination of substances, at least two of which exhibit amphipathic properties when in contact with a suitable liquid medium, these two substances are capable of forming extended surfaces, in particular membrane surfaces in contact with said medium, comprising at least one third substance, the molecules of which can associate with the specified surface, characterized in that the said first and second substances with amphipathic properties differ in their solubility in the specified medium, and the substance, which the second one is more soluble in the specified liquid medium, forms less extended surfaces than the second specified substance, while the first, more soluble substance is selected from lipids or lipid-like materials, the second is selected from boundary- or surfactants, and the third substance has one or several chain molecules that are more likely to associate with extended surfaces formed by a combination of the first two substances than with an extended surface formed by one of them.  2. The combination according to claim 1, characterized in that the surface formed by the first two substances, as well as the molecules of the third, carry an electric charge.  3. The combination according to claim 2, characterized in that the extended surfaces formed by the combination of the first two substances, as well as the molecules of the third substance, are both negatively or both positively charged.  4. The combination according to claim 2 or 3, characterized in that the relative concentration of charged components associated with the surface has a value of 5-100 rel. pier %, more preferably 10-80 rel. pier % and most preferably 20-60 rel. pier % of the concentration of all surface-forming amphipathic substances combined. 5. The combination according to any one of paragraphs. 2-4, characterized in that the average charge density on the surface has a value of 0.05-0.5 C • m ^-2 , preferably 0.075-0.4 C • m ^-2 , and particularly preferably 0.10-0.35 CL • m ^-2 .  6. The combination according to any one of paragraphs. 2-5, characterized in that the concentration and composition of the background electrolyte, which preferably contains mono - or oligovalent ions, are selected so as to achieve the maximum positive effect of charge-charge interactions on the desired association.  7. The combination according to claim 6, characterized in that the concentration and composition of the background electrolyte correspond to an ionic strength between I = 0.001 and I = 1, preferably between I = 0.02 and I = 0.5, and more preferably between I = 0 , 1 and I = 0.3.  8. The combination according to any one of paragraphs. 1-7, characterized in that the solubilities of these two first substances in the specified environment differ at least 10 times, preferably at least 100 times.  9. The combination according to any one of paragraphs. 1-8, characterized in that the average radius of the space formed by these extended surfaces has a value of 15-5000 nm, preferably 30-1000 nm, more preferably 40-300 nm and most preferably 50-150 nm.  10. The combination according to any one of paragraphs. 1-9, characterized in that the surfaces have an average curvature, defined as the reciprocal of the average radius of the space surrounded by surfaces, corresponding to the average radius of 15-5000 nm, often preferably 30 and 1000 nm, more preferably 40 and 300 nm, and most preferably 50 -150 nm.  11. The combination according to one of paragraphs. 1-10, characterized in that it includes at least one first or second amphipathic substance, forming an extended surface with another amphipathic substance, increasing the average or local curvature of the specified surface.  12. The combination according to claim 11, characterized in that the concentration of said curvature increasing substance is less than 99% of the saturation concentration or the concentration above which the surface cannot be formed.  13. The combination according to p. 12, characterized in that the concentration of the specified increasing the curvature of the surface of the substance has a value up to at least 0.1%, preferably up to 1-80%, more preferably up to 10-60% and most preferably up to 20-50 % of the corresponding concentration.  14. The combination according to any one of paragraphs. 9-13, characterized in that the surface is on a solid substrate having an appropriate curvature or size.  15. The combination according to any one of paragraphs. 1-14, characterized in that the surface formed by the first two substances, has the form of a membrane, mainly two-layer, surrounding suspended or dispersed in a liquid medium liquid droplets of substances, said at least two substances having a solubility difference of at least 10 times preferably at least 100 times in a preferably aqueous liquid medium.  16. The combination according to p. 15, characterized in that the average diameter of the homoaggregates of the more soluble substances or heteroaggregates of both amphipathic substances is less than the average diameter of the homoaggregates of the less soluble substance.  17. The combination according to any one of the preceding paragraphs, characterized in that the total content of all amphipathic substances that can form a surface has a value of 0.01-30 wt. %, especially 0.1-15 wt. % and most preferably 1-10 wt. % of the total dry weight of the combination.  18. The combination according to any one of paragraphs. 11-16, characterized in that the surface formed by the combination contains charged membrane components in the range of relative concentrations from 1 to 80 mol. %, preferably from 10 to 60 mol. % and most preferably 30-50 mol. %  19. The combination according to any one of the preceding paragraphs, characterized in that the first of these substances is a biocompatible polar or non-polar surface-forming lipid.  20. The combination of claim 19, wherein said lipid is a lipid or lipoid from a biological source or a corresponding synthetic lipid, or a modification of such a lipid, and preferably is a glyceride, glycerophospholipid, isoprenoid lipid, sphingolipid, steroid, sterol, sulfur or a carbohydrate-containing lipid or any other lipid capable of forming bilayers, and in particular a semi-protonated liquid fatty acid, and preferably selected from phosphatidylcholines, phosphatidylethanolamines, phosphorus idylglycerols, phosphatidylinositols, phosphatidic acids, phosphatidylserines, sphingomyelins or sphinophospholipids, glycosphingolipids, in particular cerebroside, ceramidopolyhexoside, sulfatide, sphingoplasmalogen, gangliosides, or synthetic di-diolidene di-dioliden , dilauroyl, dimyristoyl, dipalmitoyl, distearoyl, or the corresponding sphingosine derivative, or any other glycolipid, or diacyl, dialkenoyl or dialkyl lipid.  21. The combination according to any one of the preceding paragraphs, characterized in that said surfactant is a nonionic, zwitterionic, anionic or cationic surfactant.  22. The composition of claim 21, wherein the surfactant is selected from the group consisting of long chain fatty acid or alcohol, an alkyl tri (di) methyl ammonium salt, an alkyl sulfate salt, a monovalent salt of cholate, deoxycholate, glycocholate, glycodesoxycholate, taurodeoxycholate or taurocholate, acyl or alkanoyl dimethyl amine oxide, in particular dodecyldimethyl amine oxide, alkyl or alkanoyl-N-methylglucamide, N-alkyl-N, N-dimethylglycine, 3- (acyldimethylammonio) alkanesulfonate, N-acyl-ethyl-phenyl-phenylethylene-sulfylethylene olia, in particular octaethylene glycol octa-phenyl ether, acyl polyethylene ether, especially non-ethylene dodecyl ether, in particular isoacyl polyethylene glycol ether, octaethylene glycol isotridecyl ether, in particular ethyl ethylene glycol, ethylene ethylene glycol, ) or polyethylene glycol-20-sorbitan monooleate (Tween 80), acylpolyhydroxyethylene ether, in particular polyhydroxyethylene lauryl ether, - myristoyl, -acetyl stearyl or an oleoyl ether, such as polyhydroxyethylene-4, or 6, or 8, or 10, or 12, etc., lauryl ether (as in the Brij series) or in the corresponding ester, for example, a series of polyhydroxyethylene-8- stearate (Myrj 45), laurate or-oleate, or in 40 ethoxylated castor oil (Chemophor EL), sorbitan monoalkylate (e.g. Arlacel or Span), especially sorbitan monolaurate (Aglacel-20, Span-20), acyl- or alkanoyl-N-methylglucamide, in particular or decanoyl- or dodecanoyl-N-methylglucamide, alkyl sulfate (salt), for example in lauryl or oleyl sulfate, sodium deoxycholate i, sodium glycodesoxycholate, sodium oleate, sodium taurate, a fatty acid salt such as sodium elaidate, sodium linoleate, sodium laurate, lysophospholipid, such as n-octadecylene (= oleyl) -glycerophosphatidic acid, -phosphorylglycerol or phosphor n β-acyl, for example lauryl or oleyl glycerophosphatidic acid, β-phosphoryl glycerol or β-phosphoryl serine, n-tetradecyl-glycerophosphatid acid, β-phosphoryl glycerol or β-phosphoryl serine, corresponding palmitoleyl-, eloidoyl-phosphene, corresponding vaccidolysis Strongly or also surface-active polypeptide.  23. The composition according to any one of the preceding paragraphs, characterized in that the second amphipathic substance is identical to the third substance.  24. The combination according to any one of the preceding paragraphs, characterized in that the first amphipathic substance is phosphatidylcholine and / or phosphatidylglycerol, and the second is selected from the group consisting of lysophospholipid, such as lysophosphatidic acid or methylphosphatidic acid, lysophosphatidylglycerol, or partially lysophosphatidylglycerol -methylated lysophosphatidylethanolamine, a monovalent salt of cholate, deoxycholate, glycocholate, glycodeoxycholate or any other sufficiently polar derivative of sterol, laurate, mi ristate, palmitate, oleate, palmitoleate, elaidate or any other salt of a fatty acid and / or surfactant of the Tween-, Myrj- or Brij-, or Triton series, fat-sulfonate or-sulfobetainic, -N-glucamide or sorbitan (Aglacel or Span) a surfactant.  25. The combination according to any one of the preceding paragraphs, characterized in that the third substance includes repeating subunits, especially in the form of chain molecules, such as oligomers or polymers, in particular with an average molecular weight of more than 800 daltons, preferably more than 1000 daltons and most preferably more than 1500 daltons.  26. The combination according to p. 25, characterized in that the third substance is of biological origin and is preferably biologically active.  27. The combination according to any one of the preceding paragraphs, characterized in that the third substance is associated with a membrane-like extended surface, in particular by introducing into the surface or interface between the membrane and a liquid medium in contact with the specified membrane.  28. The combination according to any one of the preceding paragraphs, characterized in that the content of chain molecules has a value of 0.001-50 Rel. % of the total mass of these extended surfaces, and mainly 0.1-35 rel. %, more preferably 0.5-25 rel. % and most preferably 1-20 rel. %, moreover, the specific value of the ratio tends to decrease with increasing molar mass of these chain molecules.  29. The combination according to one of paragraphs. 25-28, characterized in that the chain molecule is a protein and at least a portion of the specified molecule is associated with the specified surface, provided that this part contains at least three segments or functional groups capable of binding to the specified surface.  30. The combination according to one of paragraphs. 26-28, characterized in that the chain molecules are polynucleotides, such as DNA or RNA, in its natural form or after chemical, biochemical or genetic modification.  31. The combination according to one of paragraphs. 25-28, characterized in that these chain molecules belong to the class of polysaccharides with at least partial ability to interact with these extended surfaces either in natural form or after some chemical, biochemical or genetic modification.  32. The combination according to any one of paragraphs. 25-31, characterized in that the chain molecule can act as adrenocorticostatic, b-adrenolytic, androgenic or antiandrogenic, antiparasitic, anabolic, anesthetic or analgesic, analeptic, anti-allergic, antiarrhythmic, anti-atherosclerotic, anti-asthmatic and / or anti-bronchospasm, or antipsychotic, antidiabetic agent, antidote, antiemetic, antiepileptic, antifibrinolytic, anticonvulsant, anticholinergic gent, enzyme, coenzyme or corresponding inhibitor, antihistamine, antihypertensive agent, biological inhibitor of drug activity, antihypotonic, anticoagulant, antifungal, anti-myasthenic agent, agent against Parkinson's disease or Alzheimer's disease, anti-inflammatory, antipyretic, anti-rheumatic respiratory, anti-inflammatory stimulating, bronchodilator, cardiotonic, chemotherapeutic agent, coronary dilator, cytostatic , diuretic agent, ganglioblocker, glucocorticoid, anti-influenza agent, hemostatic, hypnotic agent, immunoglobulin or its fragment or any other immunologically active substance, biologically active carbohydrate, contraceptive, migraine anti-migraine agent, mineralocorticoid, morphine antagonist, muscle relaxant, narcotic, neuro-therapeutic agent , a neurotransmitter or antagonist thereof, a peptide (derivative), an agent for treating eye diseases, a (pair) sympathomimetic or (pair) sympatholytic agent, Christmas tree (derivative), anti-psoriasis or neurodermatitis drug, mydriatic, psychostimulating, rhinological agent, any sleep-inducing agent or antagonist, sedative agent, antispasmodic, tuberculostatic, urological, vasoconstrictor or vasodilator, virostatic agent or any agent for treating wounds , or any combination of the above agents.  33. The combination according to any one of the preceding paragraphs, characterized in that the third substance is a substance that regulates growth.  34. The combination according to any one of the preceding paragraphs, characterized in that the third substance has immunoregulatory properties and includes antibodies, cytokines, lymphokines, chemokines and, accordingly, active parts of plants, bacteria, viruses, pathogens or other immunogens or parts, or modifications of any of them.  35. The combination according to any one of the preceding paragraphs, characterized in that the third substance is a biocatalyst.  36. The combination according to p. 35, characterized in that the biocatalyst is an enzyme or coenzyme.  37. The combination according to any one of the preceding paragraphs, characterized in that the third substance is a recognition molecule, including adhesins, antibodies, catenins, selectins, chaperones or parts thereof.  38. The combination according to any one of the preceding paragraphs, characterized in that the third substance is a hormone, especially insulin.  39. The combination according to claim 38, characterized in that it contains 1-500 int. units insulin / ml, in particular 20-400 int. units insulin / ml and most preferably 50-250 int. units insulin / ml, preferably recombinant human insulin or insulin similar to human.  40 The combination according to any one of the preceding paragraphs, characterized in that the third substance is interleukin, which is suitable for use in humans or animals and includes IL-2, IL-4, IL-8, IL-10.  41. The combination according to p. 40, characterized in that it contains 0.01-20 mg of interleukin / ml, in particular 0.1-15 mg and most preferably 1-10 mg of interleukin / ml.  42. The combination according to any one of the preceding paragraphs, characterized in that the third substance is interferon, which is suitable for use in mammals and contains alpha, beta and gamma interferons.  43. The combination according to claim 42, characterized in that it contains up to 20 rel. wt. % interferon, in particular 0.1-15 mg interferon / ml and most preferably 1-10 mg interferon / ml  44. The combination according to any one of the preceding paragraphs, characterized in that the third substance is a neural tissue growth factor, preferably recombinant human NGF.  45. The combination according to claim 44, characterized in that it contains up to 25 mg of nerve growth factor (NGF) per 1 ml of suspension or up to 25 rel. wt. % NGF as an agent, in particular 0.1-15 rel. wt. % protein and most preferably 1-10 rel. wt. % NGF.  46. The combination according to any one of the preceding paragraphs, characterized in that the third substance is an immunoglobulin (Ig), mainly in the form of an intact antibody, part thereof or a biologically acceptable or active modification thereof.  47. The combination according to p. 46, characterized in that it contains up to 25 mg of immunoglobulin (Ig) per 1 ml of suspension or up to 25 wt. % Ig relative to the total lipid content, preferably with 0.1-15 rel. wt. % protein and most preferably 1-10 rel. wt. % immunoglobulin.  48. A method of obtaining a combination of substances, comprising contacting two amphipathic substances, the first of which is selected from lipids or lipid-like materials, and the second from border-active or surface-active substances, the first substance being less soluble in a liquid medium, than the second substance, and forms a surface larger than the surface formed by the second substance; the formation in the process of contact of the specified first and second substances joint extended surface using mechanical fragmentation; the introduction of a third substance having chain molecules and capable of associating to a greater extent with an extended surface formed by a combination of the first two substances than with surfaces formed by these substances separately; and obtaining a combination of substances in which the molecules of the third substance are associated with an extended surface formed by a combination of the first and second substances.  49. The method according to p. 48, characterized in that the mechanical fragmentation is carried out by filtration, pressure change or mechanical homogenization, shaking or mixing.  50. The method according to p. 48, characterized in that the combination of the first and second substances is adsorbed on a suitable solid substrate, for which they are brought into contact with it, and then with the liquid medium by adding one substance after the other or both at the same time, and then a third substance is introduced, which is subsequently associated with a surface on a solid substrate.  51. The method according to one of paragraphs. 48-50, characterized in that the adsorbing extended surfaces or their precursors suspended in a liquid medium or on a solid substrate, are first obtained using operations that include sequential mixing of at least one first substance, at least one second substance and then added the third substance.  52. The method according to p. 48, characterized in that said at least one first substance, at least one second substance and said at least one third substance, as well as possibly other conventional ingredients that together form said composition, are mixed according to separately with at least one hydrophilic liquid, and then the resulting solutions are combined to subsequently cause, preferably under the action of mechanical energy, the formation of particles with which at least one third substance is associated.  53. The method according to p. 52, wherein the third substance is selected from the group consisting of antidiabetic agents, growth factors, immunomodulators, enzymes, diagnostic molecules, adrenocorticostatic and / or adrenolytic agents.  54. The method according to one of paragraphs. 52-53, characterized in that the said substances are used either as such or dissolved in a physiologically compatible polar liquid, which may be water or miscible with water, or in a solvation promoting agent, together with the polar solution.  55. The method according to p. 54, characterized in that the polar solution contains at least one border-active or surfactant.  56. The method according to one of paragraphs. 52-55, characterized in that the formation of these extended surfaces is induced by adding a substance to the liquid phase, evaporating it, by injection or dialysis, or by mechanical action, such as shaking, mixing, vibration, homogenization, sonication, shear, freezing and thawing or filtering using suitable displacing pressure.  57. The method according to one of paragraphs. 52-56, characterized in that the formation of these extended surfaces is induced by filtration, and the filter material has a pore size of 0.01-0.8 microns, preferably 0.02-0.3 microns and most preferably 0.05-0.15 microns, moreover, several filters are used, in series or in parallel.  58. The method according to one of paragraphs. 52-57, characterized in that said agents and carriers become associated, at least in part, after the formation of an absorbent extended surface.  59. The method according to one of paragraphs. 52-58, characterized in that the said extended surfaces with which the molecules of the third substance are associated are obtained immediately before use of the composition from a suitable concentrate or lyophilisate.

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