Method for developing, testing and using associates of macromolecules and complex aggregates for imp

专利摘要:
The present invention relates to a variety of amphiphilic polymers (polypeptides, proteins, etc.) or other chain molecules (e.g., partially hydrophobized suitable polynucleotides or polysaccharides, etc.) modified with polar and / or charged amphiphilic materials as needed. Appropriate principles and procedures for the growth, testing, preparation and use of combinations with aggregates which consist of a mixture of and form extended surfaces that can be freely suspended or supported are described. The agglomerates can be used after incorporating the described method with chain molecules that exhibit some activity or useful function, which can be used, for example, in vitro or in vivo, for example in drug delivery, symptomatology or bio / hydrolysis. It is suitable. As a detailed example, a cyst-shaped droplet mixture consisting of lipids containing (bound) insulin, interferon, interleukin, nerve growth factor, calcitonin, immunoglobulins, and the like is described.
公开号:KR20010033518A
申请号:KR1020007007020
申请日:1998-10-23
公开日:2001-04-25
发明作者:케에베그레고르
申请人:케에베 그레고르；이데아 악티엔게젤샤프트；
IPC主号:

专利说明:

TECHNICAL FOR DEVELOPING, TESTING AND USING ASSOCIATES OF MACROMOLECULES AND COMPLEX AGGREGATES FOR IMPROVED PAYLOAD AND CONTROLLABLE DE / ASSOCIATION RATES}
[33] In addition, by adding a charged surfactant to the surface of the present invention, to provide a means to accelerate the bonding process between the surface and the protein, and to control the bonding ratio of the polymer-membrane to some extent. This contradicts the above mentioned fact that surfactants inhibit the binding of proteins, which is well known. On the other hand, at least partially removing the surfactant from the surface accelerates the polymer desorption process and desorbs some of the polymer. This also violates known facts. The adsorption of polymers to soft deformable surfaces, in particular corresponding membranes, of the present invention is stronger than the degree of adsorption to less deformable surfaces. Soft membranes are more hydrophilic than those that are not, and mutual repulsion is predominant, which directly violates what was expected.
[34] Accordingly, the object of the present invention is to specify conditions for maximizing the binding force between large, sometimes amphiphilic molecules of polymers such as proteins, or other kinds of suitable chain molecules and complex adsorbent surfaces.
[35] It is another object of the present invention to define beneficial factors that control the adsorption of polymers to the surface of the composite or the corresponding desorption rate from the surface.
[36] It is yet another object of the present invention to provide a method for manufacturing in a form suitable for the (bio) engineering and medical field.
[37] Another object of the invention is to describe a form which is particularly suitable for practical use in the resulting form; The present invention is used, for example, in the fields of medicine and veterinary medicine, such as, but not limited to, symptomatology, separation and (raw) processes, biotechnology, gene proliferation, product stabilization, concentration and transport. .
[1] The present invention relates to a combination of materials which exhibit amphiphilicity and which can form a film-like surface when in contact with an extended surface, in particular an amphiphilic liquid medium. More specifically, the present invention relates to the combination of the surface with other amphiphilic materials at the molecular level, wherein such other amphiphilic, materials bound to the surface typically have repeating subunits such as oligomers and polymers. It is a huge molecule, which often comes from biologically active agents.
[2] The invention also relates to the various uses of the surface and the combinations, as well as to the combination between the macromolecule and the surface and a method of making the surface.
[3] Amphiphilic chain molecules and related polymers, such as proteins, are not absorbed in the same amount but in most cases in various forms, on any surface. The present invention describes the technique and suggests new principles for controlling and utilizing polymers that bind to smooth, complex surfaces. This will be very important for use in future biology, biotechnology, pharmacy, therapeutics and symptom fields.
[4] Adsorbing / coupling (macro) molecules to the absorbent surface (adsorbent / adsorbate combination) is done in a multi-step process:
[5] i) The first step is to redisperse the adsorbate, preferably to accumulate at the adsorbent / solution interface. This step is generally high speed, in which the diffusion rate is controlled.
[6] ii) In the second step, the adsorbate molecules are hydrophobic and bind to the soft (membrane) surface. The process consists of multiple steps, such as partial molecular bonding and continuous rearrangement, at least some of which are often slow.
[7] The likelihood that a polymer specifically binds to a ligand attached to an interface of "soft" lipid membranes decreases closer to the interface, which is Cevc, G., Strohmaier, L., Berkholz, J., Blume, G. Stud. . Biophys. 1990, 138: 57ff. This phenomenon is caused by the same non-coulomb, hydrogen bonding forces that prevent colloidal collapse of adjacent lipid membranes. The overall force decreases due to the hydrophilicity and the firmness of the lipid fluid interface (Cevc, G., Hauser, M., Kornyshev, A. A. Langmuir 1995, 11: 3103-3110).
[8] It has been estimated that the degree of adsorption of non-specific proteins to bilayer lipids (Cevc, et al., Op. Cit .: 1990) is proportional to whether they can be used as hydrophobic binding sites for proteins in the membrane. have. It has been found that the protein bound to the membrane is increased by mechanically making defects in the lipids of the bilayer (eg by sonication) or by inducing lipid phase transfer.
[9] In general, it is known that the more hydrophobic the surface, the higher the degree of adsorption of the lipidic polymer. For example, K. Prime and GM Whitesides (Science, 1991, 252: 1164-1167) used self-assembled monolayer long-chain alkanes with end groups that affect hydrophobicity, which are called "rules" or I was convinced that it was "principle." Thus, to this day "hydrophobic attraction" is considered a major force in the adsorption of proteins.
[10] On the other hand, it is generally known that the net macroscopic level of interaction between hydrophilic polymers such as proteins immersed in a neutral pH aqueous solution and hydrophilic surfaces such as glass or montmorillonite clay is controlled by strong repulsive forces. have. Therefore, under conditions where the rules can be applied in the macroscopic state of van der Waals, Lewis acid-base, and electrical double layer interactions, the adsorption of hydrophilic proteins onto surfaces of hydrophilic minerals is unlikely to occur normally. Quiquampoix et al, Mechanism and Consequences of Protein Adsorption on Soil Mineral Surfaces, Chapter 23 in Proteins at Interfaces (PAI), TA Horbett and JL Brash, eds., ACS Symposium Series 602, 1995, New York: 321-333). Even if the hydrophilic protein is less adsorbed than the hydrophobic surface, some of the hydrophilic protein is adsorbed onto the glass from the solution; The protein is also adsorbed onto the Montmorillonite clay surface. To illustrate this rare phenomenon, the negatively charged immersion in an aqueous medium, through a polyvalent counterion (e.g. calcium), to which the protein binds to the (negative) charged hydrophilic protein. The fact that it is possible to bind uniformly to hydrophilic mineral surfaces has been proposed and proved by experimental data. Other fine charge effects relate to the formation of hydrogen bonds, salts in proteins, and binding of counterions. For example, it has already been suggested that "structural rearrangements in protein molecules, dehydration of adsorbent surfaces, redispersion of charged groups and polarities of protein surfaces may affect the adsorption of proteins." Haynes, CA et al, Colloids Surface B: Biointerface, 2, 1994: 517-566), in this context, under conditions where the protein carries a substantial net negative charge When -LA (alpha-lactalbumin) is strongly adsorbed on PS (polystyrene), coulomb interactions, although important, generally do not modulate the adsorption of proteins to solid surfaces. Another recent study concluded that "there is no clear established fact to date on the degree of charge effect on protein adsorption" (Reversibility and the Mechanism of Protein Adsorption, W. Norde and C. Haynes, Chapter 2 in (PAI), op.cit .: 26-40).
[11] On soft surfaces such as membranes, the fact that the first step in protein adsorption is now at least subject to electrostatic-drive and / or charge control (eg Deber, CM; Hughes, DW; Frasez, PE) Pawagi, AB; Moscarello, MA Arch. Biochem. Biophys. 1986, 245: 455-463; Zimmerman, RM, Schmidt, CF, Gaub, NHEJ Colloid Int. Sci. 1990, 139: 268-280; Hernandez-Caseidis, T .; Villalaain, J .; Gomez-Fernandez, JC Mol. Cell.Biochem. 1993, 120: 119-126.). Frontline experts also concluded that electrostatic forces play an important role in the binding of secretory phospholipase to various lipid aggregates (Scott, DL; Mandel, AM; Sigler, PB; Honig, B. Biophys. J. 1994, 67). : 493-504).
[12] To date, experts in the field have believed that hydrophobic attraction is the major determinant of final protein adsorption, even though the ionic force that binds to the entropy obtained due to the morphological changes of the protein upon adsorption also plays a slight role.
[13] In general, proteins are not adsorbed on the surface having the same charge but strongly adsorbed on the surface having the opposite charge. The pH dependence in protein adsorption reflects this fact. The charge effect can sometimes be hindered by potential factors such as small polyvalent counterions, which can crosslink surface sites with proteins and similar charges that normally repel each other.
[14] The final form of the adsorbed protein rarely coincides with the original form. This is because most models of protein adsorption result in more advanced transitions from the reversibly adsorbed state, resulting in molecular rearrangement or relaxation of the protein on the surface. Polymer rearrangement upon adsorption is often fatal, even more so in the denaturation of proteins. From the fact that enzymes and antibodies retain at least some biological activity in the adsorption state and the fact that biological activity is of some importance in maintaining their original structure, the morphological denaturation of the adsorbed protein is often defined within time and range factors. The conclusion can be drawn. Folding of proteins is greatly affected by hydrophobic interactions. Both phenomena, such as protein binding and morphological changes, are sensitive to the presence of specific affinity agents such as surfactants and phospholipids. By adding the molecule, it was assumed that the adsorption of the protein was reduced or reversed.
[15] Thus, to minimize the adsorption and loss of nonspecific proteins, proteins are frequently mixed with surfactants in the isolation of proteins. In one particular study, as the surface concentration of the implanted fluoric surfactant increases, the adsorption of the protein decreases to an appreciable extent. Monomers with a minimum of EG units (4) are characterized by being extremely inactive with respect to blood components, and the number of ethylene-glycols (EG) in the monomer side chain of the surfactant was 4, 9 and 24 (Analysis of the Prevention of Protein Adsorption by Steric Repulsion Theory, TB McPherson et al., Chapter 28 in PAI, op.cit .: 395-404).
[16] The homopolymer is covalently attached to the surface, which increases the interfacial thickness and hydrophilicity, thereby lowering the utility of the hydrophobic bonding sites on the lower surface, which also reduces the likelihood of protein binding to the modified surface. Rather, it shows denaturation at the surface.
[17] It is also consistent with the findings mentioned above that sometimes surfactants containing short polymer fragments at one end may interfere with or even partially reverse binding to various surfaces. This phenomenon is related to protein solubility or replacement, possibly depending on the surface forces of the surfactant and the relative magnitude of the binding force to the surfactant-protein; usually these factors work.
[18] In another experiment, the addition of boric nonionic surfactant (alkali-polyoxyethylene ether) to an aqueous phase at pH 7.0 in a concentration range of 10 ^-4 wt% resulted in substantial protein separation from the air / water interface ( T. Arnebrant et al, op. Cit.).
[19] Intensive studies have been conducted on the preadsorption and removal of proteins by surfactants (Protein-Surfactant Interaction at Solid Surfaces, T. Arnebrant et al. Chapter 17 in PAI, op. Cit .: 240-254) . It is divided into three types of manpower:
[20] i) binding to specific sites in proteins such as alpha-lactoglobulin or albumin serum by electrostatic or hydrophobic attraction;
[21] ii) mutual adsorption of proteins and surfactants without significant morphology change;
[22] iii) binding of the protein followed by morphological changes to the intersorbed surfactant;
[23] For example, removal of proteins from methylated (hydrophobic) silica surfaces is such that for other surfactants, the protein is removed by replacement due to the higher surface activity of the surfactant. It can be concluded that the surfactant headgroup effect is best expressed on hydrophilic surfaces, but less important on hydrophobic surfaces (Protein-Surfactant Interaction at Solid Surfaces, T. Arnebrant et al. Chapter 17 in PAI, op. Cit. ,: 240-254).
[24] The same conclusion applies to other lipids. The amount of plasma protein adsorbed on the plastil surface is reduced by pretreatment with DPPC liposome suspension; The same tendency is observed when insulin adsorbs on the catheter surface.
[25] Unexpectedly so far, polymers and surfactants that adsorb on soft surfaces composed of a mixture of bipolar, especially lipids, adsorb more effectively than lipid aggregates that do not contain any surface active molecules. To put it more simply, molecular mixtures that form stable membranes—generally not necessarily in the form of lipidous cysts (liposomes) —and / or at least one strong amphipathic, ie relatively water soluble, mixture and interface of phospholipids A bilayer-unstable component (often a surfactant), such as an active agent, consists of at least one bilayered lipidic substance that stabilizes, such as cholesterol, such as protein, rather than a pure phospholipid surface, or consists of only phospholipids, especially liposomes, such as cysts. It is easier to combine with amphiphiles. It was also unexpectedly found that the relative number of amphiphilic polymers (proteins) bound is higher on the surface bearing the net charge with the same sign as the net charge of the adsorption body. This is inconsistent with existing published data, which are known to require opposite charges to build up electrostatic bonds on objects that attract each other and become stronger.
[26] One of the necessities for the above described improvements to the molecular (eg drug-carrier) bodies is the general adsorptivity of the adsorbent surface. This adsorption facilitation enables the adsorption of polymers:
[27] i) First, high concentrations are placed near the adsorbent surface, in part due to counter-charge-charge and other interaction forces.
[28] ii) Next, take advantage of the binding / non-electrostatic interaction with the adsorbent surface. (The latter is generally a hydrophobic process that requires hydrogen bonding sites, which is generated or by surface-flexibility and / or adaptability. Can be done.)
[29] Controlled (polymeric) drug-carrier combinations that meet these requirements and are most suitable for practical use.
[30] In addition, each process step for adsorption of proteins to soft surfaces is variable, but depends on the proximity and majority of hydrophobicity at the membrane-soluble interface phase or inner surface. Thus, the motility of the hydrophobic bonds between the polymer and the bonding surface, which is affected by the number of accessible bonding sites, increases in the presence of the surface active component and the ductility of the membrane.
[31] The rate at which the adsorbing (high) molecules can structurally fit into multiple binding sites is also important. For example, the hydrophobic interaction of a non-charged transfersome membrane is an important factor for insulin-surface binding. The basic multistage coupling usually requires substantial system rearrangement, but requires long adsorption to complete. The optimal incubation time for the Transfersome®-insulin complex may result in somewhat longer results.
[32] The adsorption overview described in the previous paragraph is consistent with the basic adsorption scenario described in the specific data. Notwithstanding this fact, various differences and even discussions which have been clearly distinguished from the known facts have been disclosed so far.
[38] As a solution to this problem of the present invention, it is defined in the attached independent claim.
[39] Justice
[40] By definition, as used herein, "bond" is a complex between two or more other molecules, and at least one of them is the other or a number of well-defined surfaces, regardless of why the complex structure does not exclude covalent bonds. Form aggregates with (s). Bonds between different classes of molecules can be encapsulated (eg, contained in cysts consisting of surface-forming molecules), insertion (eg, included in the surface and subsurface aggregate layers), or adsorption (on the surface of aggregates). Can be said to be based on; On this principle two or more combinations are also possible.
[41] As used herein, in the context mentioned above, "adsorbate", "adsorbing (high) molecule", "bonding (high) molecule", "bonding (high) molecule" and "adsorbent" or "surface bonding", etc. Are used alternately, which describes the bonds between molecules that do not form extended surfaces under selected conditions.
[42] By "carrier" is meant an agglomerate capable of binding to one or more polymers used for any practical purpose, such as in this application, whether natural or originated thereof, or a carrier to a human or animal body.
[43] By "lipid" in the sense of the present invention is meant any substance which possesses characteristics similar to those of fat. Usually, this type of molecule has an extended nonpolar region (chain X), and in most cases also has a water-soluble, polar, hydrophilic group, also called the head group (Y). Basic structural formula 1 of the material is as follows.
[44] XY _n (1)
[45] N is equal to or greater than zero. When n = 0, the lipid is called apolar lipid; n If 1, the lipid is polar. In this context, all amphiphilic substances such as glycerides, glycerophospholipids, glycerophosphinolipids, glycerophosphonolipids, sulfolipids, sphingolipids, isoprenoidides, steroids, sterins or sterols, etc. And all lipids containing carbohydrate moieties are simply referred to as lipids. See PCT / EP91 / 01596 for a more precise definition.
[46] By "edge-active" material or "surfactant" herein is meant any material that increases the propensity of the system to form edges, protrusions or other heavily curved structures and defective areas. Other molecules and inter-surfactants that promote the solubilization of lipids in the presence of more commonly used surfactants in addition to the commonly used surfactants are excluded from this category; Also excludes molecules that cause or promote the formation of defects (at least partially hydrophobic) in the adsorbent (hetero) group. Direct surfactant action, (partial) molecular separation (partial) catalysis, or other forms of surfactant-induced change in suitable molecules often contribute to this effect. As a result, many solvents, as well as asymmetric and amphiphilic molecules and polymers such as a plurality of oligos and polycarbohydrates, oligos and polypeptides, oligos and polynucleotides and / or derivatives thereof, are in addition to conventionally used surfactants. It is included in the above-mentioned category. A very widely used list of surfactants, suitable solvents (also called so-called inter-surfactants), and many other suitable edge active materials are described in relatively large numbers in PCT / EP91 / 01596, wherein See this for example. A more complete list is provided in the Industrial surfactants handbook (Michael Ash, Irene Ash, eds., Gower Publishing, 1993).
[47] A "chain molecule" or "polymer" is a linear or branched molecule that retains a state of groups exhibiting unequal affinity for at least two or more "adsorbing surfaces". Particularly required in view of presenting or synthesizing the corresponding alternatives of the invention is that at least one of said groups is surely (partially) filled at the donor solution and / or at the adsorbing surface. The surface-affinity for each group is often due to their different amphiphiles, namely different hydrophilicity / hydrophobicity. Other groups can be arbitrarily distributed along the chain, but in most cases a number of physically related (eg, multiple hydrophilic or one or more hydrophobic) groups will be located on one chain segment.
[48] As used herein, "polymer" includes the following.
[49] Sugars, starches, celluloses, etc. (see PCT / EP91 / 01596 for a clearer definition of carbohydrates) represented by the basic formula C _X (H ₂ O) _y . It must be used very often to gain intimacy. For example, by attaching carbohydrates that bind hydrophobic moieties to (partially) hydrophobic surfaces, or to non-coulomb interaction forces (e.g., hydrogen bonds) with more hydrophilic joining surfaces. This is made possible by introducing the necessary groups to produce.
[50] Oligo or polynucleotides such as homo or hetero chains of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their chemical, biological or molecular biological (gene) variants (more detailed definitions of See list listed in PCT / EP91 / 01596).
[51] Oligopeptides or polypeptides consisting of 3-250, often 4-100, usually 10-50 identical or different amino acids, naturally overlap with amide bonds, but in the case of prothenomics this is according to different polymerization schemes. May change, even partially or wholly cyclic; Alternatively it is possible to use pure compounds or racemic mixtures (see PCT / EP91 / 01596 for a more detailed and complete definition).
[52] Long polypeptide chains are generally called proteins, regardless of their detailed form or the accuracy of the polymerization reaction. Most of the time, proteins bind to surfaces relatively efficiently. Therefore, we do not cite relevant substances here, but refer to PCT / EP91 / 01596 and the recently published papers in a partial list.
[53] For purposes of explanation only, some relevant groups are briefly summarized below.
[54] Enzymes include oxidoreductases (which include various dihydridegenases, (per) oxidases, (superoxide) dismutases, etc.), transferases (acyl-transferases, phosphorylases, and Other kinases), transpeptidase (esterases, lipases, etc.), lyases (decarboxylase, isomerase, etc.), various proteases, coenzymes, and the like.
[55] Variable or highly variable fragments, such as immunoglobulins, Fab- Fab2-fragments, classified as IgA, IgG, IgE, IgD, IgM with subtypes, in native form or in chemical, biochemical, or genetically amplified forms Single chain antibodies, or portions thereof, can be obtained from the present invention. These include IgG-gamma chains, IgG-F (ab ') 2 fragments, IgG-F (ab), IgG-Fc fragments, Ig-kappa chains, light chains of Ig-s (eg kappa and lambda chains). And also smaller immunoglobulin fragments that are variable or highly variable regions or variants of any of these substances or fragments.
[56] In addition to the above antibodies, immunologically active molecules (endotoxins, chitokines, lymphokines and other large immunomodulators or biological carriers) also belong to the heterologous chain molecule group. Phytohamaglutin, lectin, polyinosine, polycytidylic acid (poly I: C), erythropoietin, "granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 1-18, interferon (alpha, beta or The same is true for gamma and their (bio) synthetic variants), tumor necrosis factor, (TNF-s); sufficiently large, amphipathic tissue and plant extracts, their chemical, biochemical or biological derivatives and substitutes, their Some belong to the same group, etc. As a result, all of the above molecules can be bound appropriately and efficiently with the complex surface as described herein.
[57] Other biologically relevant examples include basic fibroblast growth factor (BFGF), endothelial cell growth factor (ECGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin, and insulin-like growth factors ( LGF I and LGF II, etc.), neuro-growth factors (NGF, NGF 2.5s, NGF 7s, etc.), progenitor-derived growth factors (PDGF), and the like, which affect local or global growth.
[58] Particularly useful derivatives for achieving the object of the present invention are variants, which include many such as aryl, alkyl, alkenyl, alkenoyl, hydroxyalkyl, alkenylhydroxy or hydroxyacyl chains having 1 to 24 carbon atoms, Often (bio) chemically, biologically, by reaction utilizing an adsorbent substituted with at least three nonpolar (hydrophobic) residues, or suitably, a property in which a large number of other non-coulomb interactions between adsorbent and adsorbate are desired Or genetically modified. When the polymer is hydrophobized, relatively few (1-8 or preferably 1-4) carbon atoms per side chain are preferred. The associated scientific literature provides sufficient information on how the chain molecules are hydrophobized for different purposes. The purpose of this detailed description is that the strong holding force of the adsorbates has already been described in other publications (e.g. Torchilin, VP; Goldmacher, VS; Smirnov, VN Biochem. Biophys. Res. Comm. 1978, 85). : 983-990), which is excluded not only because of the characteristics of the prior art but also because it is easy to cause relatively weak reversible bonds.
[59] In the art, it is already known that the addition of surfactants to membranes made of amphipathic materials alters the properties of the membranes. It has also already been proposed that by injecting the component into a reduced droplet surrounded by a corresponding membrane, the component may be used to pass well through a defined hole in the barrier and to allow the component to be well suspended and suspended in a suitable liquid medium. This is described in great detail in the previous application PCT / EP 91/01596, PCT / EP 96/04526.
[60] For the purpose of penetrating the barrier aperture, a process selectively carried out to optimize the cysts with highly compliant membranes is introduced to control or control the degree of binding between the chain molecules and the membrane. Generally does not match. In addition, the three-dimensional conformity of the membrane surface surrounded by the cysts (and variants of the cyst itself) is not necessarily associated with the bonding process, for example when the surface with the binding polymer body is a solid-support, Thus it does not retain the three-dimensional compliance of the unsupported membrane.
[61] For the purpose of controlling and / or enabling the bonding process of the polymer body to bind with the surface, the present invention focuses on the two main effects described above.
[62] The first important phenomenon is that amphipathic molecules, that is, molecules such as polymers or chain molecules already mentioned, are more soluble in at least one amphiphilic substance and floating liquid medium that have the property of forming an extended surface. Better binds to an extended surface consisting of at least one or more materials of lesser nature to form a more extended surface. That is, in the presence of a material having a property of destabilizing the surface, the surface solution interface may be formed from a less soluble surface-forming material in the presence of a second material having a property of destabilizing a more soluble surface than the above-mentioned material. Compared to the corresponding material formed, it attracts more of the adsorbing polymer. In view of the present invention, it is assumed that the mutual surface stimulus occurs in two dimensions, or the surface increases when it expands. For example, if the surface of the cyst contains membrane flexibility, an average cyst diameter of between 20 nm and several hundred nm, such a phenomenon will occur if the surface is shaken or shaken. Lipid micelles that do not retain the diameter in at least one direction do not meet the requirements; If so, such a surface is not considered to extend to the meaning used in the present invention.
[63] The second, more soluble, surface unstable material is generally an edge-active material or surfactant.
[64] The second effect described hereafter is a chain molecule that bonds more easily and better with an electrically charged polymer or an equally charged surface (e.g., both negative or both positive), in which case the latter Is a composite and consists of at least two amphiphilic materials, one of which is characterized by destabilizing a surface formed of a more soluble and less soluble material than the other. In other words, although it is generally known that similar charges repel each other, charged polymers or chain molecules can be cathodic or bonding processes if the surface flexibility and the possible rearrangement between molecules are possible because of the flexibility of the surface. The two materials involved in can better bond with the equally charged surface in either of the cases where they carry a net positive charge. Based on this clear fact, if the negatively charged polymer binds to the positively charged surface, and vice versa, with electrostatic attraction, the binding will be easier and more powerful. It has been estimated.
[65] The two effects described above are effectively combined as defined in detail in the third independent claim.
[66] It can be defined in terms of the differential solubility of the amphiphilic, surface-forming material, which is involved in the selection of polymers or chain molecules, which together form a membrane or surface that takes the form of a cyst suspended in a liquid medium. do. In general, the effect of the present invention becomes more pronounced when the difference in solubility between the materials involved is increased, for example, when the surface tension of the binding polymer is increased. The more soluble film component should be at least 10 times, preferably at least 100 times, soluble than the less soluble surface forming component. therefore. If an amphiphilic surface-forming substance such as phospholipids is combined with a second substance such as a surfactant in a suitable liquid medium such as water, the second component uses a surfactant that is more soluble in water than phospholipids (quality). Is much better.
[67] On the other hand, selectivity can be defined in terms of surface curvature. Characteristic surface curvature by applying the above-mentioned examples of phospholipids (used as basic surface-forming substances) mixed with a surfactant (second component which makes the surface unstable and more soluble) in water (used as a liquid medium) To some extent. Generally speaking, the (average) curvature is defined as the inverse mean radius of the area sealed to the intended surface. In general, the addition of surfactants is expected to increase the curvature of the mixed lipid vesicle surface as compared to the curvature of phospholipid cysts that do not contain any surfactant. Although the saturation concentration of the surfactant does not represent the stability of the curved surface, the optimum surfactant concentration is generally selected from 99% or less based on the saturation concentration; Most are selected from 1 to 80 mol% based on saturation concentration, more preferably from 10 to 60 mol% based on saturation concentration, most preferably from 20 to 50 mol% based on saturation concentration Is chosen from.
[68] On the other hand, if it is impossible to reach the saturation concentration in each system, the amount of surfactant used is less than 99% of the dissolved concentration since the surface is deformed before the saturation concentration reaches saturation after adding the surfactant. This is common. In addition, optimal concentrations of surfactants in the system, such as, for example, such concentrations of mixed lipid aggregates dissolved at expanded surfaces replaced by much smaller average surfaces, often result in concentrations that limit the morphology of the adsorbent surface. It is in 1%-80% as a reference | standard, and most are in 10%-60%, Preferably they are in 20%-50%.
[69] In addition, it is possible to define a mixture of materials that are simple and practically useful in terms of the average curvature of the surface. As mentioned in claim 7, the surface has a value between 15 nm and 5000 nm, preferably between 30 nm and 1000 nm, more preferably between 40 nm and 300 nm, and most preferably between 50 nm and 150 nm. It has an average curvature (defined as the inverse average radius of the area sealed to the surface) corresponding to the average radius having a. However, it should be emphasized that the curvature of the adsorbent surface is not always determined by the properties of the adsorbent membrane. When using a solid-supported surface of the invention made from an optional mixture of amphipathic materials, the average curvature of the surface is usually determined by the solid surface curvature.
[70] Moreover, it is possible to define the invention in terms of the relative concentration of charged components closely related to the surface, when at least similar bonding forces between charges are used. The relative concentration of charged components closely related to the surface is in the range of 5 mol% to 100 mol%, more preferably 10 mol% to 80 mol%, based on the concentration of the total surface-forming amphipathic material obtained at the same time. And most preferably at 20 mol% to 60 mol%. In terms of net surface charge density, the surface has a value of 0.05 Cb / m ² to 0.5 Cb / m ² , more preferably a value of 0.075 Cb / m ² to 0.4 Cb / m ² , and most preferably It is characterized by a value of 0.01 Cb / m ² to 0.35 Cb / m ² .
[71] It is desirable to select concentrations and components of conventional electrolytes consisting of polyvalent ions, in order to maximize the positive effects of charge-charge interactions on the desired bonds. In general, the bulk ion strength at I = 0.001 to I = 1 is maintained, preferably at values of I = 0.02 to I = 0.5, and even more preferably at bulk values of I = 0.1 to I = 0.3. Keep it up
[72] Another effectively used definition of the present invention is the membrane adsorbent surface surrounded by a thin droplet of fluid. The membrane is often a bimolecular layer, and at least two or (self) agglomerated parents having a solubility difference of at least 10 times, preferably at least 100 times, in the (preferably water soluble) liquid medium used to suspend the droplets. In the form of an attractive substance. In such a case, the selectivity of the material forming the membrane is greater than the average diameter of the homo-aggregate of the more soluble material or the diameter of the hetero-aggregate consisting of two materials than the average diameter of the homo-aggregate containing only the less soluble material. It can be specified by making it small.
[73] In particular, the composition is mainly used for the manufacture of a form that is applied to the human body or animal body for medical purposes, in which case the total content of all amphiphilic substances that can form the surface in the system, based on the total dry weight Therefore, 0.01 weight%-30 weight% are preferable, More preferably, 0.1 weight%-15 weight% are good, Most preferably, it is the case of 1 weight%-10 weight% content.
[74] For example, surface-forming or surface-supporting materials, such as materials capable of forming extended surfaces, are biologically compatible polar or nonpolar lipids, particularly when the adsorbent surface has a structure similar to a bimolecular layer. You may select from. Specifically, the main surface-forming substance is a lipid, but any lipid that is a suitable biological source or a corresponding synthetic lipid or a complex lipid derived from a variant of the lipid, of which glycerides, glycerophospholipids, iso Preferred are phosphatidylethanol, which is a group of phosphatidylcholines that can form bimolecular layers such as prenoidride, sphingolipid, steroids or sterols, sulfur or carbohydrate-containing lipids, or semi-quantized fluid fatty acids. Amines, phosphatidylglycerols, phosphatidylinositols, phosphatidyl acids, phosphatidylserines, as well as sphingomyelin, sphingo-phospholipids, glycosphingolipids (e.g. Sphingoplasmagen), gangliside or others Synthetic lipids such as glycolipid, dioleoyl, dirinorail, dionorenoyl, diaraquidoyl, dilauroyl, dimyristoyl, dipalmitoyl, distearoyl are preferred, or Sphincosine derivatives, such as glycolipids, diacyls, diallkenoyls or dialkyl lipids.
[75] In addition, surfactants are effectively used as materials that make the surface unstable and more soluble, and nonionic, zwitterionic, anionic or cationic detergent groups may be usefully used; In particular, long-chain fatty acids or alcohols, alkyl-tri / di / methyl-ammonium salts, alkylsulfonate salts, monovalent salts of cholates, deoxycholates, glycocholates, glycodeoxycholates, taurodeoxycholates, or tau Acyl or alkanoyl-dimethyl-aminooxides such as locates, dodecyl-dimethyl-aminooxides, alkyl or alkanoyl-N-methylglucamides, N-alkyl-N, N-dimethylglycines, 3- (acyldimethyl Ammonio) -alkanesulfonates, N-acyl-sulfobetaines, polyethylene-glycol-octylphenyl ethers such as nonaethylene-glycol-octylphenyl ether, polyethylene-acyl ethers such as nonaethylene-dodecyl ether, octaethylene glycol Polyethylene glycol-isoacyl ethers such as isotridecyl ether, polyethylene-acyl ethers such as octaethylene dodecyl ether, polyethylene glycol-20-monolaurate (twin 20) or polyethyleneglycol-sorbitan-acyl ether, such as polyethyleneglycol-20-sorbitan-monolate (twin 80), polyhydroxyethylene-lauryl, -myristoyl, -cetylstearyl or -oleyl Polyhydroxyethylene-acylethers or the corresponding esters such as ethers and polyhydroxyethylene-4- or 6 or 8 or 10 or 12 (in the Breeze series) are polyhydroxyethylene-8-stearates 45), -laurate or -oleate type, and polyethoxylated castol oil 40 (Cremoper EL) is a sorbitan-monoalkylate such as sorbitan-monolaurate (e.g. arrasel Span), acyl or alkanoyl-N-methylglucamide, such as decanoyl- or dodecanoyl-N-methylglucamide, and alkyl-sulfate, such as lauryl- or oleyl-sulfate Oxycholate, Sodium Glocodeox Fatty acid salts such as cholate, sodium oleate, sodium taurate, sodium elladate, sodium linoleate, sodium laurate, n-octadecylene (= oleoyl) -glycerophosphatidyl acid, -phosphorylglycerol or -phosphoryl Lysophospholipids such as serine, n-acyl-glycero-phosphatidyl acids such as lauryl or oleyl-glycero-phosphatidyl acid, -phosphorylglycerol or -phosphorylserine, n-tetradecyl-glycero-phosphatidyl acid , -Phosphorylglycerol or -phosphorylserine, corresponding palithioeroyl-, eladoyl-, basenyl-lysophospholipids or corresponding short-chain phospholipids or other surface-active polypeptides .
[76] The concentration of the charged film component is in the relative range of 1-80 mol% based on the amount of the total film-forming component, preferably in the range of 10-60 mol%, most preferably 30-50 mol% Will be in the range of.
[77] It is preferable to select phosphatidylcholine and / or phosphatidylglycerol as the surface-supporting substance and lysophospholipid such as lysophosphatidyl acid or methylphosphatidyl acid, lysophosphatidylglycerol or lysophosphatidylcholine, or partially N-methylated lysophosphatidylethanol Monovalent salts of amines, cholates, deoxycholates, glycocholates, glycodeoxycholates or sufficiently polar sterol derivatives, laurates, myristates, palmitates, oleates, palmitolates, eladates or some other Fatty acid salts and / or tweens, mylizes or breezes, in addition to tritons, fatty sulfonates or fatty sulfobetaines, -N-glucamide or -sorbitan (arlacel or span) surfactants forming an expanded surface It is chosen as a material with poor ability.
[78] The average radius of the region surrounded by the extended surface is in the range of 15 to 5000 nm, but preferably in the range of 30 to 1000 nm, more preferably in the range of 40 to 300 nm, most preferably Is in the range of 50 to 150 nm.
[79] In general, a third material that binds to an extended surface formed by combining two other materials (sometimes three, four, five, etc.) consists of molecules with repeating subunits, especially in the form of chain molecules. The third material may be an oligomer or a polymer, in particular an amphiphilic polymeric material having an average molecular weight of at least 800 Daltons, preferably at least 1000 Daltons and in most cases at least 1500 Daltons. The substance is a substance taken from or similar to a organism, that is, it is preferable to have a biological activity such as a bio-component.
[80] It is desirable to insert the third material, in particular into the membrane, such as the interface (s), which become an integral part of the membrane between the membrane and the liquid medium, so as to bond with the extended surface similar to the membrane of the invention.
[81] The content of the above-mentioned substances (molecules) or the corresponding chain molecules is generally between 0.001 and 50% by weight, based on the mass of the adsorbent surface. Often, the content is expressed using similar relative units, whereby the specific ratio decreases as the molar mass of the adsorbed (chain) molecules increases, although the content is in the range of 0.1 to 35% by weight. Preferably it is 0.5 to 25 weight%, Most preferably, it is in the range of 1 to 20 weight%.
[82] If it consists of at least three fragments or functional groups that have properties to bind to the adsorbing surface, the substances can bind to the adsorbing surface as long as the adsorbing polymer or chain molecule is a protein or part of a protein. have.
[83] The polymers or chain molecules according to the invention can be added to polynucleotides or polysaccharide groups, such as DNA or RNA, which have at least a surface-retracting property after being modified in their native form or with some suitable chemical, biochemical or genetic modification. There is a tendency to bond with extended surfaces made from belonging amphiphiles.
[84] Chain molecules that bind to the extended surface are, for example, adrenocorticostaticum, -Adrenoritum, Androgen or Antiandrogen, Antiparacitycum, Anabolicum, Anaesticum or Analgesicum, Anaepicum, Antialgecum, Antiarheisumicum, Antialtroskretium, Anti Astomaticum and / or broncospamoritycum, antibioticum, antidreprecicum and / or anticycoticum, antidiabeticum, antiemethicum, antiepirepicum, antifibrinoticum, Anticobulboom, anticholinergicum, enzymes, coenzymes or the corresponding inhibitors, antihistminicum, antihypertonicum, biological inhibitors of drug activity, antihifotonicum, anticorant, antimycoticum, Antimiastenicum, anti-Morbus Parkinson's or Morbus Alzheimer's preparations, Antiflozisticum, Antipyreticum, Antirelecticum, Anticepticum, Anaphylaxis or Respiratory Involvement Stimulant, broncholyticum, cardiotonicum, chemoterapeticum, vascular dilator, chitostaticum, diureticum, gangliolium blockers, glucocorticoids, anti-flu preparations, haemostaticum, hypnoticum, immunoglobulins Or fragments thereof or other immunologically active substances, bioactive carbohydrates (derivatives), contraceptives, anti-mygraine preparations, mineralocorticoids, morphine-antagonists, muscle relaxants, narcoticcum, neuroteraputicum, neurorepes Ticcum, a neurotransmitter or part of its antagonist, peptide (derivative), optamicum, (para) -simpatimomimeticum or (para) simpaticoritycum, protein (derivative), psoriasis / neudermitis drug, Midriaticum, psychostimulants, linoleumcums, sleeping pills or antagonists, sedatives, spasmoritycum, tubulokulstaticum, eurologicum, vasoconstrictors or vasodilators, viraltaticum or wound-healing substances, May have various physiological functions and actions, such as combinations of such materials.
[85] The present invention can also be usefully utilized when the third material is a growth promoter.
[86] The third substance selected from the group consisting of the following substances which are more usefully used includes, for example, antibodies, chitokin, lymphokines, chemokines and the corresponding active sites of plants, bacteria, viruses, patogens and other immunogens. Or immunomodulators such as variants thereof, enzymes or coenzymes or other types of biocatalysts; Cognitive molecules such as inter alia adherin, antibodies, catenin, selectin, capperon or parts thereof; Hormones and especially insulin. In particular, when insulin is used as the active substance, the amount of insulin contained in the composition of the present invention is 1 to 500 IU (insulin / milliliter), more preferably 20 to 400 IU, most preferably 50 to 250 IU. In the form of a medicament, human recombinant insulin or humanized insulin is preferred.
[87] Other useful uses of the present invention include the use of various chitokines such as interleukin, interferon, and the like, and the interleukins which can be used in humans or animals are suitable for IL-2, IL-4, IL-8, IL-10, IL. There are -12, and such interferons that can be used for the same purpose include, but are not limited to, IF alpha, beta and gamma.
[88] The amount of interleukin contained in the binder is mainly 0.01 mg to 20 mg (interleukin / ml), but especially 0.1 to 15 mg, most preferably 1 to 10 mg. Keep it within the concentration range of the drug.
[89] The relative amount of interferon contained in the binder is mainly up to 20% by weight, in particular 0.1-15 mg (interferon / ml), but most preferably 1-10 mg, if necessary, the final dilution for practical use. Keep the drug in the concentration range possible.
[90] In another embodiment of the present invention, neuronal growth factor (NGF) which binds to the surface of the present invention as a third active substance is described. Human recombinant NGF is preferred in the form of the substance, and the optimal concentration range used is up to 25 mg neuronal growth factor (NGF) / ml in suspension or 25 relative weight%, in particular 0.1-15 relative weight% Protein and most preferably 1 to 10% by weight, which may be diluted before use if necessary.
[91] For the purpose of immunoglobulin (Ig) regulation, the techniques of the present invention described herein can be used in the form of intact antibodies, biologically acceptable and active variants of some or some of the antibodies. As an amount relative to total lipids, up to 25 mg (immunoglobulin (Ig) / ml suspension) or up to 25% by weight of immunoglobulin is used, preferably 0.1-15% by weight protein, most preferably 1 It is recommended to use a relative weight of immunoglobulin at -10%.
[92] The present invention describes a process for the preparation of the above-defined combinations, in particular in the form of active substances, such as biologically, cosmetically and / or pharmaceutically active substances, which method comprises at least the difference in solubility in a suitable liquid medium. It consists of selecting two amphipathic materials, which can form an extended surface, at least when they are in contact with the medium, in particular in the form of a film, and bonded to each other. This method of using an extended surface formed in support of a combination with the surface and in combination with a material capable of attracting the active ingredient is a preferred selection criterion, in which the prerequisites extend more on their own than other materials. That the surface must be more attractive to the component than at least two surfaces forming only the surfaces of the at least two materials selected, which have a difference in solubility in the two liquids and / or the appropriate liquid medium, and, if combined, at least That the surface must be able to form an extended surface, in particular a film-like surface in contact with the liquid medium, and that the surface consisting of a combination of the two materials is more attractive and forms an extended surface than other surfaces It must be able to bind the active ingredient better than the surface formed from the two substances. And finally the components as well as the surface carry a net charge and, on average, both the component and the surface should be negatively charged or both should be positively charged.
[93] Preferred methods of forming the extended surface of the present invention include filtration, pressure change or mechanical homogenization, shaking, stirring, mixing or other controlled mechanical crushing in the presence of the material molecules to be combined with the surface formed during the process. There is a mechanical operation to mix the corresponding materials such as
[94] When an optional combination of surface forming materials is adsorbed onto a suitable supporting solid surface, or otherwise permanently contacts the solid surface, it is brought into contact with the liquid medium by adding different or multiple materials at the same time. It is preferred that at least one post surface-forming process takes place in the presence of the above components, and combines with the continuous solid-supported surface.
[95] Regardless of whether suspended in a liquid medium or supported in a solid phase, the adsorbing surface or their precursors are first prepared by a process of substantially mixing the surface forming molecules, provided that the following treatments do not damage the surface from which they are made. As a result, it is preferable to add the binding molecules to the surface by additionally performing stirring, mixing or incubation.
[96] In the present invention, at least one amphiphilic substance, at least one hydrophilic fluid, at least one edge-active substance or interface, with various components being non-invasive, particularly through intact skin tissues of the human body or animals or animals. Preferred is a method of producing an active material and a surface capable of binding to the material molecules in a composite composed of at least one material. In addition, these components are in a form suitable for non-intrusive components, whereby other conventionally used components may be added in an amount necessary and appropriate to achieve desirable properties and final production stability.
[97] In carrying out the method, each of the selected components is mixed, the components together / dissolved in the solution as needed, and the resulting mixture (s) or solution (s) are combined, finally preferably as described above. Through mechanical manipulation, the material or surface is combined with the component.
[98] Amphiphiles suitable for the purposes described herein may be used as such, or dissolved in a physiologically compatible polar fluid such as water, mixed with the solvent or at least one edge-active substance or interface. It may also be dissolved in the solvation-modifying component together with a polar solution which preferably consists of the active agent.
[99] Another preferred method of forming the component-derived surface is to add the substance in the fluid phase. Alternative methods include evaporation from reverse phase, injection or dialysis or shaking, stirring, shaking, homogenizing, sonication (eg exposing to ultrasound), shearing, freezing and thawing or simple Mechanical stress such as filtration under moderate driving pressure. At the time of filtration, as a filter medium, the size of the filter medium is preferably 0.01 to 0.8 µm, preferably 0.02 to 0.3 µm, and most preferably 0.05 to 0.15 µm. Various filters may be used in succession, or may be used in parallel in order to obtain a desirable surface forming effect and to maximize speed or ease in manufacturing.
[100] After forming the adsorbing surface, it is preferred to at least partially bond the substance or carrier.
[101] Before being used in its form for practical purposes, it is possible to form a binder immediately between the substance molecules and the surface to which they bind. You can then start with a suitable concentrate or lyophilizer.
[102] The present invention describes a method for preparing a component-carrier, particularly for the purpose of delivery of a drug or for other types of medical or biological applications. Thus, it can also be used in terms of barrier air permeation; In this case, it is carried by droplets which can deform very well through the voids in the barrier, in which the average diameter of the barrier voids is much smaller, smaller than the average diameter of the droplets or cysts. It is desirable to provide a surface that binds in the form of a membrane formed by amphipathic molecules surrounded by reduced droplets. However, as described above, although different from the optimal compositional characteristics, often defined as cyst membrane compliance to pore passages, and often in practice, this is achieved between optimal binding properties and optimal membrane compliance. It is necessary.
[103] In addition, the combination of the present invention is used in the field of biotechnology, gene propagation as well as separation techniques for process or diagnostic purposes. Here, in other applications, including enzymatic processes and catalysis, the binding surface may be useful in that it may support a solid rather than take the form of a bladder such as a membrane. For example, the surface of the present invention is fixed to the solid support in order to intentionally fix the active molecule that binds this kind of surface onto the solid support intentionally, and then simply treat, fix, separate, concentrate, etc. Do the process. It is possible to stabilize at least partially amphiphilic surface-form constituents, such as chain molecules, in a catalysis process involving the molecule while the protein, polypeptide, polynucleotide or polysaccharide and / or surface are bound. Thus, it is contemplated that the techniques of the present invention may be used to prepare columns filled with catalysts that are activated, very well bound or optionally reactive polymers. As an example of this, in a solution, it is composed of a surface supported by a solid with non-covalently bound active molecules surrounded by a solid support, the reaction of the solution with the active polymer while passing through the floating polymer It is a chemical reaction conducted by passing the appropriate pair-reactant (s) through the column that occurs. In another example, at least some of the molecular solution separated from the solution is filled in a column or suspended and centrifuged, precipitated, suspended (stirred) on a solid supported adsorbent surface in order to separate the fluid by binding to the substrate surface. Contact with the solid particles in a suitable manner other than by sedimentation, precipitation only, or electromagnetic adsorbent particle separation.
[104] Another use of the present invention relates to controlling the reversibility and / or motility of binding or separation between molecules that bind to the surface and the compatible surface of the complex formed according to the present invention by binding to a suitable amphiphilic material. Larger surface charge densities and / or larger surface ductility and / or larger surface defect densities can accelerate bonding. Thus, the corresponding reducing force may be used to lower the binding rate and also to induce partial or complete separation.
[105] Formation temperature and storage temperature are rarely out of the range of 0 ~ 95 ℃. Since various interesting components, in particular various kinds of polymers, are sensitive to temperature, temperatures below 70 ° C., in particular below 45 ° C., are preferred. The use of non-aqueous solvents, cryonas or heat stabilizers enables use in temperature ranges other than those temperatures mentioned above. In general, most are done at room temperature or physiological temperature, but it is also possible to use them in other temperature ranges, which may be suitable for the particular form. In another embodiment, the holding force of the adsorbing surface compliance at higher temperatures is a possible reason for this; It keeps the material in active form at low temperatures.
[106] Even characteristic forms are quite compliant with most sensitive system components. Storage at low temperatures (about 4 ° C.) is as efficient as using inert gases (eg nitrogen).
[107] The shaping described above can be done when using a specific process for adsorbents or adsorbates, but either is important (the adsorbents made using phospholipid are “Liposomes” (Gregoriadis, G., ed., CRC). Press, Boca Raton, FI., 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 material may also be diluted or concentrated (eg by ultra centrifugation or ultrafiltration).
[108] When or before using the material, additives may be introduced to increase the chemical or biological stability of the combination, the polymer binding force or its reversal, the mobility during separation / bonding, and to facilitate control and mixing. .
[109] Interesting additives include solvents that optimize various systems (eg, chemical stabilizers (e.g., antioxidants and other scavengers, buffers, etc.) at concentrations that do not exceed the limits by favoring or maintaining the properties of the system. ), Adsorption promoters, biologically active co-molecules (eg, fungicides, virus growth inhibitors) and the like.
[110] Suitable solvents for use for the abovementioned purposes include substituted or unsubstituted, for example hydrogenated, aliphatic, cycloaliphatic, aromatic, or aromatic-aliphatic carbohydrates such as benzol, toluol, methylenechloride, dichloromethane or chloroform Alcohols such as methanol or ethanol, propanol, ethylene glycol, propanediol, glycerol, erythritol, alkanesane acid esters such as acetic acid, acid alkyl esters such as diethyl ether, dioxane or tetrahydrofuran, and mixtures thereof However, it is not limited to this.
[111] It may also be convenient to adjust the pH value of the adsorbent / adsorbate mixture after preparing it or before using it. This prevents decay of each system component and / or conjugate. It may increase the biological activity or physiological compatibility of the resulting mixture. In order to neutralize the mixture for application in the field of biology in a vivo or vitro state, depending on the type and purpose of the application, a pH value of 3-12, more preferably a pH value of 5-9, most preferably Biologically suitable acids or bases in the pH range of 6 to 8 are used. For example, dilute aqueous solutions of physiologically acceptable acids, hydrochloric acid, mineral acids such as sulfuric acid or phosphoric acid, and organic acids such as carboxylic alkanoic acid, for example acetic acid. Physiologically acceptable bases include, for example, diluted sodium hydroxide, suitably ionized phosphoric acid, and the like.
[112] Lipids and surfactants which are mentioned briefly or in detail are known. Lipids and phospholipids that bind to polymers to form appropriate aggregates are described, for example, in 'Phospholipids Handbook' (Cevc, G., ed., Marcel Dekker, New York, 1993), 'An Introduction to the Chemistry and Biochemistry of Fatty acids and Their Glycerids' (Gunstone, FD, ed.) And references. Reports on chemical surfactants are described in Mc Cutcheon's, Emulsifiers & Detergents (Manufacturing Confectioner Publishing Co.) and other related references (see, for example, Hadbook of Industrial Surfactants, M. Ash & I. Ash, eds., Gower, 1993). Combinations of related active agents are described in 'Deutsches Arzneibuch', The British Pharmaceutical Guide, European Pharmacopoeia, Japanese Pharmacopoeia, The United States Pharmacopoeia and the like. Relevant polymers are listed in the manufacturer's catalog, appropriate scientific periodicals, and specific references for industry or research purposes.
[113] The present invention describes some relevant properties of the conjugate, for example, with some selected polypeptide / protein and phospholipid / surfactant mixtures.
[114] The following experiments were conducted for the purpose of measuring the binding force of insulin on the complex cysts. A cyst composition of another composition was used. Variants include various surfactants and lipids, various lipid / detergent ratios, other total lipid content, and various types and concentrations of insulin, which have introduced net charge into / into cysts.
[115] As a first example, complex lipid cysts consisting of phospholipids / bioactive surfactants were bound to insulin at various protein / lipid ratios to maximize binding. A monocomponent cyst (liposome), which has been used conventionally, was used as a reference.
[116] Examples 1 to 27
[117] Very well deformable, flexible cysts (transfersomes ^TM ):
[118] Starting Suspension:
[119] 874.4 mg of phosphatidylcholine extracted from soy beans,
[120] 125.6 mg sodium cholate, and
[121] consisting of 9 ml phosphate buffer at pH 7.1
[122] 10% by weight total lipids (TL).
[123] Final Suspension A
[124] The lipid and
[125] Consisting of 0.1, 0.5, 1, 2, 3, 4 mg insulin / 100 mg TL
[126] 5 wt% TL
[127] In order to dilute to the required concentration, the insulin stock solution (4 mg / ml Actrapid ^™ Novo-Nordisk) was mixed with the following buffer.
[128] Unit: mg mg / 100 mg lipidBufferInsulin solution (4 mg / ml; Arctrapide) 4-3 ml 30.75 ml2.25 ml 21.5 ml1.5 ml One2.25 ml0.75 ml 0.52.265 ml0.375 ml 0.12.925 ml0.075 ml
[129] Final suspension A was prepared by mixing 2.5 ml of starting lipid suspension (10% TL) and 2.5 ml of appropriate insulin dilution.
[130] Final Suspension B:
[131] The lipid and
[132] TL content of 5% to 0.25% by weight consisting of 4, 5, 6.67, 10, 20, 40, and 80 (mg insulin / 100 mg TL).
[133] To obtain various insulin / lipid ratios, reference was made to the following pipetting table:
[134] Unit: mg mg / 100 mg lipidFinal TL (wt%) obtainedStarting Suspension (10% Lipid)Buffer 453 ml- 542.4 ml0.6 ml 6.6731.8 ml1.2 ml 1021.2 ml1.8 ml 20One0.6 ml2.4 ml 400.50.3 ml2.7 ml 800.250.15ml2.85 ml
[135] Final Suspension B was prepared by mixing 2.5 ml Aktrapid HM (4 mg / ml insulin) with 2.5 ml of a moderately diluted lipid suspension.
[136] Final suspension C
[137] The substrate and
[138] TL content of 2.5% to 0.125% by weight consisting of 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 80 and 60 mg insulin / 100 mg TL.
[139] To obtain the cited insulin / lipid ratios, reference was made to the following pipetting table:
[140] Unit: mg mg / 100 mg lipidFinal TL concentration (% by weight)Starting lipid suspension, diluted 5% by weight lipidInsulin solution (4 mg / ml; Arctrapide)Buffer 42.52.5 ml1.25 ml1.25 ml 52.52.5 ml1.563 ml0.938 ml 62.52.5 ml1.875 ml0.625 ml 72.52.5 ml2.188 ml0.313 ml 82.52.5 ml2.5 ml- 92.22.222 ml2.5 ml0.278 ml 1022 ml2.5 ml0.5 ml 151.31.333 ml2.5 ml1.167 ml 20One1 ml2.5 ml1.5 ml 300.670.667 ml2.5 ml1.833 ml 400.50.5 ml2.5 ml2 ml 500.40.4 ml2.5 ml2.1 ml 800.250.25 ml2.5 ml2.25 ml 1600.1250.125 ml2.5 ml2.375 ml
[141] In test series C, the suspension was diluted to a volume ratio of 1: 1 with buffer, filtered and lyophilized to make a 5% cyst suspension from 10% of the original suspension.
[142] Preparation of Adsorbent / Adsorbate Mixtures
[143] The buffer was prepared in a conventional manner, and filtered through a 0.2 µm sterile filter (the solution was stored in a glass container for later use). The lipid mixture was suspended in buffer in sterile glass vessel and stirred with a magnetic stirrer for 2 days at room temperature. The suspension was then continuously extruded through etched track polycarbonate membranes (nucleoporous) of fine pore sizes of 400 nm, 100 nm, and 50 nm. At the driving pressure of 0.6 MPa to 0.8 MPa, three passes each time. The cyst suspension thus prepared was lyophilized five times at each temperature of -70 ° C to + 50 ° C. To obtain the desired cyst size, the suspension was re-extruded and passed through a 100 nm filter four times at 0.7 MPa. As a final step, very deformable cysts were sterilized by filtration through a 200 nm pore sterile injectable filter. The cysts were stored in sterile polyethylene containers at 4 ° C. for later use.
[144] Because of the excessively large number of negatively charged amino acids for positively charged amino acids of pI = 5.4 or more, each insulin molecule carries a net charge in the neutral pH range.
[145] Commercially available insulin solution (Actrapid ^™ , manufactured by Novo-Nordisk) was used in the study of the conjugate including the same. As a result, the starting protein solution contained 4 mg insulin / ml and 3 mg m-cresol / ml. Appropriate amount of the solution was added to the suspension of adsorbent cysts to obtain various insulin / lipid ratios. According to the experiment, the resulting carrier / insulin mixture was thoroughly mixed at room temperature and incubated for at least 2 hours.
[146] In test series A, the cyst suspension stock was diluted with acrotropide to obtain the final lipid with a 50 mg TL / ml concentration and various protein / lipid ratios. In test series B, the final lipid concentration changed in the range of 2.5-40 mg / ml depending on the insulin / TL ratio. In test series C, the final lipid concentration changed in the range of 1.25-25 mg / ml. As a comparative example, a series of similar suspensions were prepared using buffer instead of lipid suspension.
[147] Test measurements were made on 4 ml of insulin / cyst mixture respectively. After 2 hours, the lipid cysts were separated from the aqueous sub-phase to determine how much insulin binds to the lipid cysts and how much insulin remains unbound in the aqueous sub-phase. For this purpose, a CENTRISART I-centrifuge tube with a cut-off of 100.000 Da was used. Three tubes were used to dilute each 1 ml of insulin containing suspension and centrifuged at 2000 g for 3 hours (T = 10 ° C.). As a result, any clean surfactant (only, buffer, insulin and some mixed lipids (presumed to contain phosphatidylcholine / cholate)) micelles with insulin concentration and dissolved detergent were measured. They were removed because they proved to contaminate the lipid cysts as they passed through defects in the CENTRISART I-filter All insulin standard measurements mentioned here were performed using an HPLC process.
[148] The original diluent was used as a positive control, and the non-specific insulin adsorbed to the tester was quantified in the negative control. After modifying the non-specific binding, the difference in starting and final insulin concentration of the surfactant was measured. The "lost" insulin was probably associated with the cysts and was expressed in absolute or relative units.
[149] The results of the experiment are shown in FIG. This indicates binding to the cysts in an insulin / lipid ratio of 6 mg / 100 mg TL, less than 80-90% of the added protein. At higher insulin / lipid ratios, the relative efficiency of protein-surface binding decreases, resulting in a binding rate of only 5% for dilution to 2/5 (40 mg / 100 mg). In other words, each 2 mg of 40 mg of insulin, which is very dilute and has a high protein / lipid ratio, binds 100 mg of lipids in the form of highly deformable cysts.
[150] Slightly extending the incubation time or increasing the concentration of the added suspension improves this phenomenon (FIGS. 2 and 3).
[151] Examples 28-45
[152] Commonly used cysts (liposomes), starting suspensions:
[153] 1g phosphatidylcholine extracted from soybean
[154] 9 ml phosphate buffer at pH7.1
[155] Final Suspension A
[156] The lipid and
[157] 0.1, 0.5, 1. 2. 3. 4 mg insulin / 100 mg TL
[158] TL content of 5% by weight consisting of (0.1, 0.5, 1, 2, 3, 4 relative weight%).
[159] Final Suspension B
[160] The lipid and
[161] TL content of 5 to 0.25% by weight consisting of 4, 5, 6.67, 1., 20, 40, and 80 mg insulin / 100 mg TL.
[162] Starting lipid suspensions were prepared as described in Examples 1-27. However, in order to prepare sufficiently small liposomes and to sufficiently monodisperse them, extrusion is further carried out six times through a 100 nm filter.
[163] Very little insulin was bound to the liposomes tested. Only 2-5% of the drug added binds to standard lipid cysts within the range of 4 mg / ml to 100 mg / ml-dilution (data not shown).
[164] In order to examine the effect of the diluted suspension on the highly deformable complex cyst composition and to rule out it experimentally, the following experiment was conducted.
[165] Examples 46-59:
[166] Starting Suspension:
[167] 874.4 mg of phosphatidylcholine extracted from soybean
[168] 125.6 mg sodium cholate (given in 10 vol% TL content)
[169] 9 ml phosphate buffer at pH 7.1
[170] Final suspension:
[171] The final suspension composition is the same as Series B and C of Examples 1 to 27 where the final lipid concentration is reduced.
[172] The measured insulin / lipid ratios are as follows: 4, 8, 10, 20, 40, 80, 160 mg insulin / 100 mg TL
[173] Example 1 for the insulin / lipid ratios described above, except that the diluent was formulated with either actrafeed containing 10 mM cholate and / or cholate containing 5-20 mM cholate (for control and test samples). A cyst suspension was prepared in accordance with the description given in -27. In this way, the final cholate concentration in all samples was 5 mM, which is close to the CMC of this detergent, which prevents the release of cholate from the cyst membrane after dilution.
[174] By preventing the loss of cholate from the cysts, the average charge density of the cyst surface as well as the original active cyst composition is maintained. This improved effect is also reflected in the combination.
[175] In the examples of this continuous test, it was noted that maintaining the concentration of cholate to 5 mM, especially the total lipid concentration, throughout the pipetting process prevented inadvertent solubilization of the cysts.
[176] Results show that up to 10% of the protein / lipid weight ratio of insulin added up to 10% between 80-90% of insulin added is bound to the lipid cyst surface (FIG. 4). This means that the adsorbent-adsorbate binding is almost perfect and the efficiency with which the protein binds is very high. The percent lipid bound with protein slowly decreases with increasing protein / lipid ratio, reaching 7% at 1.6 mg insulin / 1 mg lipid.
[177] The absolute amount of insulin binding to the carrier reaches a maximum of about 0.4 mg insulin / 1 mg lipid, where 15.6 mg of 40 mg of insulin added binds 100 mg total lipid in the form of highly deformable cysts. Turned out. The maximum yield is obtained at a relative ratio of 0.2 mg insulin / 1 mg total lipid, but 14 mg of 20 mg added is measured to bind the mixed lipid cysts. This result is shown in FIG.
[178] Similar results are obtained when cholate molecules are introduced into a mixed lipid cyst suspension with buffer or insulin solution.
[179] Examples 60-71:
[180] Starting suspension (20% TL):
[181] 1099.7 mg of phosphatidylcholine extracted from soybean
[182] 900.3mg Tween 80
[183] 8 ml phosphate buffer pH7.4
[184] Final suspension:
[185] The lipid mixture and
[186] Consisting of 2, 4, 8, 10, 20 and 40 mg insulin / 100 mg TL.
[187] A cyst suspension was prepared in substantially the same manner as described in Examples 1 to 27 except that the stirring time was extended to 7 days. Aktrapide ^™ (Novo-Nordisk) was used as the adsorption source in all cases.
[188] In order to be able to use to fix the insulin concentration at 4 mg / ml, it was prepared at an insulin / lipid ratio of 8 mg / ml to 100 mg / ml with the final total lipid concentration changing. As a comparative example (presumed to have a dilution effect), cysts of similar composition were prepared at various insulin / lipid ratios except that the final total lipid concentration was fixed at 10 mg / ml (1 wt.%). Protein-vesicle binding time was set to 3 hours.
[189] Centrifugation time for separating non-binding insulin from cyst-bound protein was 6 hours (at 1000 g). Details relating to other experiments are the same as in the first test series (Examples 1-27).
[190] Regardless of the fact that the amount of insulin that binds to a membrane containing a nonionic surfactant (Twin-80) is generally less than the amount of insulin that binds to a charged (containing cholate) membrane, the quantitative The properties are similar (see Examples 1 to 27).
[191] At a relative insulin / lipid ratio of 0.04 mg insulin / 1 mg lipid, the amount of insulin bound to the membrane is about 50%. The relative concentration of 0.2 mg insulin / 1 mg lipid when bound to maximum corresponds to only 5.2 mg of protein binding of 20 mg of insulin added as a whole. In other words, an absolute optimization that provides maximum yield in this test series is obtained at 0.04 mg insulin / 1 mg lipid.
[192] Examples 72-76:
[193] Starting suspension (10% TL):
[194] 74.4 mg phosphatidylcholine extracted from soybean
[195] 125.6mg Sodium Cholate
[196] 9 mL of phosphate buffer pH-7 (-7.4; with this buffer, the pH of the starting suspension is in the range of 7.3 to 7.6. Since the preferred pH range is 7.3 to 7.4, the following test series with cholate as surfactant Was used at pH 7.1.)
[197] Insulin A:
[198] Phosphate buffer 4 mg / ml, 8 mg / ml, 10 mg / ml, 20 mg / ml
[199] 30 μl HCL (1M) / ml dissolved dry insulin, and
[200] 30 μl 1M NaOH / 1ml solution
[201] Insulin B:
[202] 4mg Aktrapide / mL phosphate buffer at pH 7.4
[203] Insulin-Cysts Mixture
[204] 5% by weight total lipid concentration
[205] 0.04, 0.08, 0.1 and 0.2 mg dry insulin / 1 mg total lipid
[206] (4, 8, 10, 20 relative weight%)
[207] A cyst suspension was prepared as described in Examples 1-27 using a similar membrane composition. However, in order to increase the ratio of insulin / lipid using dry insulin with a significantly higher final lipid concentration, it is dissolved at a higher concentration than that used in commercially available solutions.
[208] Lyophilized human recombinant insulin is not readily soluble in phosphate buffer, pH 7.4. To prepare the insulin solution, a dry, lyophilized human recombinant insulin “powder” similar to Aktrapide ^™ was first added 2 ml of buffer and vortexed completely. After being temporarily acidified (adding 60 μl of HCL), the solubility of insulin is sufficiently increased to give a clear solution, and 60 μl of NaOH is added to bring the pH back to 7.4 where the insulin is stable ( Hexamer), disintegration / bonding is prevented. A solution to be added by directly dissolving 8 mg of insulin in 2 ml of a buffer at pH 7.4 was prepared.
[209] A cyst suspension (2 mL) and Insulin Liquid-A (2 mL) were thoroughly mixed and incubated for 12 hours at the insulin / lipid ratio given above. In all cases, the final total lipid concentration was 50 mg / ml. For reference, solution B was used. The remaining experiments were carried out as described in Examples 1 to 27.
[210] result:
[211] The binding insulin (which may be temporarily a monomer solution) was made from a dry protein powder (which may at least temporarily be a monomer solution) comparable to that measured by the insulin of the acuteid of Examples 1-27. It is shown that a large amount of insulin can bind to the lipid cyst suspension at a concentration of 50 mg / ml. The maximum binding insulin is found near the value at which the weight ratio of protein / lipid is 1/5, which binds the lipid membrane mixed with about 16 mg of insulin added.
[212] At similar protein concentrations, the same results are obtained when measured with dissolved ad hoc and a commercial insulin solution.
[213] In the following linked experiments, insulin adsorbed to various charged or uncharged fluid, mixed lipid membranes was prepared.
[214] Examples 77-92:
[215] Conventional cysts, SPC liposomes, neutral (TL = 10 wt%):
[216] No net charge, it consists only of zwitterionic phospholipids
[217] 1 g of phosphatidylcholine from soybeans
[218] 9 ml of phosphate buffer at pH 7.4.
[219] Conventional cysts, charged SPC / SPG liposomes (TL = 10 wt%):
[220] Net negative charge of 25 mole% anionic phosphatidylglycerol
[221] 750 mg of phosphatidylcholine extracted from soybean
[222] 250 mg of phosphatidylglycerol extracted from soybean
[223] 9 ml phosphate buffer at pH 7.4
[224] Neutral cysts that can deform very well (TL = 10% by weight):
[225] No net charge, zwitterionic phospholipids and nonionic surfactants
[226] 550mg phosphatidylcholine extracted from soybean
[227] 450mg Tween 80
[228] 9 ml phosphate buffer at pH 7.4
[229] Charged cyst A (TL = 10 wt%), which can be very well deformed:
[230] No net charge due to 25 mole% anionic cholate.
[231] 874.4 mg of phosphatidylcholine extracted from soybean
[232] 125.6 mg sodium cholate
[233] 9 ml phosphate buffer pH 7.1.
[234] Charged cyst B (TL = 10% by weight), which can be very well deformed:
[235] Charged to net negative charge due to 25 mole% (relative amount of PC) anionic phosphatidylglycerol.
[236] Phosphatidylcholine Extracted from Soybean 284.3mg
[237] 94.8mg of phosphatidylglycerol extracted from soybean
[238] 620.9mg Twin 80
[239] 9 ml phosphate buffer at pH 7.4
[240] Insulin-Cysts Mix
[241] 50, 25, 10, 5 mg total lipid / ml final suspension
[242] 0.04, 0.08, 0.1 and 0.2 mg insulin / 1 mg total lipid
[243] (Relative weight percentage of 4, 8, 10, 20 proteins)
[244] All cysts were prepared as described above. Tween-containing cysts were stirred for 7 days. The cholate-containing cysts and liposomes were stirred for 2 days. Aktrapide 100 HM ^™ (produced by Novo-Nordisk) was used as the insulin source. It comes from the final protein, which results in a change in the concentration of the final lipid (50, 25, 10 and 5 mg TL / ml). However, due to SPC-liposomes, 4 relative weight% samples were investigated.
[245] The experimental protocol is as described in Examples 1-27. The incubation time was 3 hours, and in the case of the comparative example, the centrifugation time was 6 hours (at 500 g) in the entire preparation step in order to prepare more easily. The measurement results are shown in FIG.
[246] Even if it is charged with a net negative charge, it is clear from the results that insulin binds best with the negatively charged surface. It is also good to use a very flexible membrane that allows the cysts to deform very well.
[247] It is extremely flexible and has a relative bonding efficiency of 80-90% for making charged membranes. A very large protein membrane bond is obtained when the weight ratio of insulin / lipid is 1/25 in the two types of tested phospholipid-surfactant mixture. Uncharged membranes consisting of phospholipids and nonionic surfactants show 50% relative binding at comparable insulin / lipid ratios. However, insulin added only between 2.5% (Examples 28-45) is calculated to bind to uncharged phosphatidylcholine liposomes. In this case, the worst thing is that the result is overvalued by the protein binding to the charged liposome, which binds to 10-20% of the added insulin when the protein / lipid weight ratio is 1/25. Therefore, a charged conventionally used lipid bilayer is an intermediate between an uncharged liposome membrane and a more flexible but neutral (transfersome ^TM ) membrane.
[248] From this fact, net surface charges (from components that bind to charged lipids or other charged membranes) are associated with membrane ductility (promoted by the presence of other molecules involved in the detergent and adsorbent) to maximize surface- or carrier-protein binding. You can see that there is a connection. In "soft surfaces", the question remains as the charge pushes the surface adsorbed against the adsorbent when the protein becomes easy to insert into the interface region.
[249] Examples 93-95:
[250] Conventional cysts, SPC liposomes, neutral (TL = 10 wt%):
[251] No pure charge, only Zwitterionic phospholipids
[252] 1 g of phosphatidylcholine from soybeans
[253] 9 ml phosphate buffer at pH 7.4
[254] Charged cyst A (TL = 10% by weight) that can deform very well
[255] Net negative charge due to 25 mole% of anionic cholate
[256] 874.4 mg of phosphatidylcholine extracted from soybean
[257] 125.6mg Sodium Cholate
[258] 9 ml phosphate buffer at pH 7.1
[259] Charged cyst B (TL = 10% by weight), which can be very well deformed:
[260] Net charge due to 25 mol% (relative to PC) of anionic phosphatidylglycerol.
[261] 284.3 mg of phosphatidylcholine extracted from soybean
[262] 94.8 mg of phosphatidylglycerol extracted from soybean
[263] 620.9mg Tween 80
[264] 9 ml phosphate buffer at pH 7.4
[265] Insulin-Cysts Mix
[266] 50, 25, 10, 5 mg total lipid / ml final suspension
[267] 0.04, 0.08, 0.1 and 0.2 mg insulin / 1 mg total lipid
[268] (Relative weight percentage of 4, 8, 10, 20 proteins)
[269] Studies on Mobility of Pre-Adsorbed Insulin: Measurements were made over time for phosphatidylcholine tween 80 mixed with membranes. Test cysts were prepared as described in the corresponding Examples above. It took 2 hours to obtain the first data point after mixing the protein solution and lipid suspension. For the neutral high strain film, the next point took 3 hours. All suspensions of the other examples were also incubated for 4-5 days and 5-6 weeks.
[270] result
[271] It was clearly found that the adsorption of insulin to an uncharged SPC / twin mixed membrane was time dependent (shown in FIG. 9 with some representative data). It increased from 30% after time and increased to 50% after 3 hours with an insulin / lipid weight ratio of 1/25. After 4 days, the binding efficiency increased to 64%, but since the binding efficiency was only 58% after 5 weeks, the amount of change was not so important.
[272] The binding of insulin to simple phosphatidylcholine liposomes was measured to increase to 2.5% after 3 hours and to 5% after 6 weeks.
[273] As can be seen from the increase in the amount of protein binding to the membrane from 64% after 2 hours to 76% after 6 weeks, the adsorption of insulin to the charged SPC / SPG / twin is very fast and neutral membrane Stronger than Smaller increments at 2 hours, compared to the amount of binding during the first hour, indicate faster binding motility.
[274] The binding ratio of insulin is much greater than that of charged SPC / cholate mixed membranes. In tests conducted with such charged cysts, it was found that the adsorption of proteins to the mixed lipid membranes had no time dependence. After 2 hours, the binding becomes as high as the incubation for 5 weeks if there is no experimental error. It is believed that the charged, flexible membrane and the adsorbing insulin will be much faster than the uncharged membrane. For reference, it was thought that electrostatic attraction that could not be ignored may also affect the desorption of protein molecules. Very weak and / or slow binding to the phosphatidylcholine membrane indicates that only hydrophobic bonds are not sufficient to achieve high payloads. This is due to the limitation of the insulin molecule's ability to find a suitable binding site on the surface of the lipid bilayer. Unusual, repulsive forces between poorly adsorbed protein molecules and neighboring labile adsorbates can also be important.
[275] Examples 96-100
[276] Very deformable cyst suspensions with different charge densities (TL = 10% by weight):
[277] Charged to net negative charge due to 25, 33, 50, 67, 75 mol% phosphatidylglycerol
[278] 137mg, 205mg, 274mg, 343mg, 411mg phosphatidylglycerol extracted from soybean
[279] 411 mg, 343 mg, 274 mg, 205 mg, 137 mg phosphatidylcholine extracted from soybean
[280] 452mg twin 80
[281] 9 ml phosphate buffer at pH 7.4
[282] 2mg insulin / ml final suspension
[283] Lipid cysts were prepared as described in Examples 93-95. As shown in FIG. 4, increasing the relative concentration of charged lipids in the membrane promoted binding to the cyst-insulin and increased the viscosity of the final suspension.
[284] At higher SPG / SPC molar ratios, the lipid suspensions prepared in Examples 93-95 are more viscous and difficult to handle. Larger relative concentrations of the charged lipid composition increased the relative amounts of cysts that bind insulin. This is shown in FIG.
[285] Changing the charged lipid content affects the binding efficiency of the protein in a complex manner. First, the relative amount of insulin that binds to the cysts increases. It can be seen that when the SPC / SPG is around 50, the relative coupling amount is maximum. Due to the effect of surface charge on the interfacial density and / or protein adsorption motility, a very large SPG content impedes effective insulin binding. Density is not maximized.)
[286] Examples 101-104:
[287] Extremely flexible charged membrane mixed with insulin 1/1 (TL = 10% by weight)
[288] 874.4mg Phosphatidylcholine Extracted from Soybean
[289] 125.6mg Sodium Cholate
[290] 9 ml phosphate buffer at pH 7.1
[291] 4mg insulin / ml starting solution
[292] Other methods are also used to prepare the cysts: In addition to the extrusion and freeze-drying cycles described in Examples 1-27 above, a much simpler protocol (only continuously extrude the suspension) is also tested. . No significant difference in protein adsorption efficiency was found for the mixed lipid membranes (FIG. 8). However, the shape adaptability of lipid cysts as defined by the “pore penetration assessment” was different from that of other batches: The deformation of the prepared cysts in Examples 1 to 27 was found to be very large.
[293] Examples 105-106:
[294] Extremely flexible charged membrane with various additives
[295] (Final suspension)
[296] 437mg phosphatidylcholine extracted from soybean
[297] 63 mg sodium cholate
[298] 1 ml phosphate buffer at pH 7.1
[299] 2 mg insulin / ml (as final suspension)
[300] Additive A
[301] m-cresol 1.5 mg / ml (final)
[302] Additive B
[303] Benzyl Alcohol 2.5mg / ml (final)
[304] The co-solvent added to the transfersomes containing sodium cholate affects the amount of insulin that binds to the final membrane. The relative binding efficiency in the presence of m-cresol is 60% and the efficiency after introducing benzyl alcohol into the test suspension is 90%.
[305] The additives used in Examples 103-104 can also be used as preservatives.
[306] Examples 107-110:
[307] Similar membranes with various insulins taken from various sources:
[308] 437 mg of phosphatidylcholine extracted from soybean
[309] 63 mg sodium cholate
[310] 1 ml phosphate buffer at pH7.1
[311] Ark Tribe feed 100HM ^TM (Novo-Nordisk Inc.),
[312] Human recombinant (product of Novo-Nordisk company) which is originally dry,
[313] Originally taken from Forcin (from Sigma Chemical Industries)
[314] 2 mg insulin / ml, and
[315] Insulin analogue taken from Lispro ^{TM (} from Pleasure)
[316] No significant difference in the various protein adsorption efficiencies for the similar membrane was observed. However, this does not exclude the possibility of various desorption / sorption rates.
[317] In particular, dry insulin, dissolved in acidic buffer and returned to the neutral pH range, adsorbs with the lipid membranes mixed as efficiently as insulin intended to use Aktrapid ^™ (Novo-Nordist) solution.
[318] Examples 111-118:
[319] Soft, uncharged membrane
[320] Starting suspension (10% TL):
[321] 1099.7 mg of phosphatidylcholine extracted from soybean
[322] 900.3mg Tween 80
[323] 19 mL phosphate buffer at pH7
[324] Final suspension:
[325] 8.4 μg IF mixed with lipid mixture as above,
[326] As given in Figure 10, increasing the relative amount of interferon using 1.84 mg TL / ml-18.4 µg TL / ml.
[327] The protein / lipid mixture was mixed at an increased molar ratio and the material was prepared as described in Examples 60-71. The first step is done with centris art separation tubes (cut-off 100 kDa), which is a non-specific protein that is always pre-coated with albumin (taken from a solution containing 40 mg BSA / ml buffer) in this test series. The adsorption rate is reduced to levels below 15%. After incubation with BSA, washing was performed twice with the buffer and filled with an appropriate concentration of interferon solution (prepared by diluting with stock in the same buffer). In order to evaluate the final protein concentration, a commercial ELISA immunoassay for IF was used. In the same procedure, the method as described in Examples 1 to 18 was used to measure the amount of interferon binding to cysts. Thus, the degree of binding of the protein was consistent with the "loss of protein" taken two or three times from the supernatant.
[328] The obtained result is shown in FIG. It is understood that this results in a figure quantitatively similar to that described for binding of insulin.
[329] Examples 119-134:
[330] Very flexible, charged membrane
[331] Starting suspension
[332] 874.4 mg of phosphatidylcholine extracted from soybean
[333] 125.6 mg sodium cholate
[334] 10% by weight total lipid (TL) content of 9 ml phosphate buffer at pH 7.1
[335] Final suspension
[336] Lipid / Protein Mixtures Described in FIG.
[337] (Other data corresponding to those given in Examples 111-118)
[338] From the results of two different experimental series shown in FIG. 10 (filled with diamonds and squares), the negative charge for the efficiency of interferon binding to a highly deformable bilayer, even though it is generally negatively charged for the protein molecule, It can be seen that the membrane has a desirable function.
[339] Examples 135-145:
[340] Starting suspension (10% TL):
[341] Soft, uncharged membrane
[342] SPC / Tw80
[343] 550mg of phosphatidylcholine extracted from soybean
[344] 450mg twin 80
[345] 9 ml phosphate buffer at pH 6.5
[346] Soft, charged membrane
[347] SPC / NaChol
[348] 874.4 mg of phosphatidylcholine extracted from soybean
[349] 125.6 mg sodium cholate
[350] 9 ml phosphate buffer at pH7.1
[351] Final Suspension:
[352] Lipids having a ratio as described above, and
[353] Consists of 10000 IU of Interleukin-2 (IL-2).
[354] The process was carried out with both a given lipid mixture and a protein. Then, the degree of binding was measured. Whereas the amount of IL-2 is determined using proteins that depend on the stimulation of Renca cells in vitro compared to the standard curve. Separation was substantially conducted as given in Examples 119-134. This gave the data given in the table below (the absolute concentration of IL-2 is given in IU units and the relative protein amount is given in%).
[355]
[356] The difference between the starting value and the final value (total recovered protein) is due in part to the loss of protein during cyst / IL-2 separation and the modification of the activity of the protein in the presence of lipids.
[357] Short-term binding of interleukins with varying surface charge densities to previously formulated highly deformable lipid cysts has been found to be less sensitive to charge effects than those listed in the table above (data not shown).
[358] Examples 146-148:
[359] Conventionally used neutral cysts (starting suspension):
[360] 1 g of phosphatidylcholine from soybeans
[361] 9 mM phosphate buffer at pH 6.5
[362] Neutral cysts (starting material) that can deform very well
[363] 550mg of phosphatidylcholine extracted from soybean
[364] 450mg Tween 80
[365] 9 ml phosphate buffer with pH 6.5
[366] Charged cysts (startings) that can deform very well:
[367] 874.4 mg of phosphatidylcholine extracted from soybean
[368] 1% 5.6 mg sodium cholate
[369] 9 ml phosphate buffer at pH 7.1
[370] Calcitonin (for example salmon) mixed with cysts (final suspension)
[371] 100mg total lipid / ml final suspension
[372] 1 mg protein / 100 mg total lipid
[373] The entire lipid suspension was prepared as described above. To the treated cysts were added proteins (short-circulated with a small amount of ¹²⁵ I-labeled protein and purified for a short time before use) and incubated for at least 24 hours; Co-compression was added and passed through a microporous filter during the preparation of the suspension.
[374] To determine the relative efficiency of the polypeptide binding to the membrane, the protein / vesicle mixture was chromatographed using size-exclusion gel chromatography to continuously detect radioactivity. This allows two peaks to each contain in the solution a radiolabeled protein that binds to the cysts. The area under the curve is about 30%, 70% for conventional cysts, 60 to 70% and 40 to 30% for soft membranes in the neutral state, for charged, highly flexible membranes. 80% 20%.
[375] Examples 149-152:
[376] Neutral cysts (starting material) that can deform very well
[377] 550mg of phosphatidylcholine extracted from soybean
[378] 450mg Tween80
[379] 9 ml phosphate buffer at pH 6.5
[380] Charged cysts (starting materials) that can deform very well
[381] 874.4 mg of phosphatidylcholine extracted from soybean
[382] 125.6 mg sodium cholate
[383] 9 ml phosphate buffer at pH7.1
[384] Immunoglobulin G mixed with cysts (final suspension)
[385] 100mg total lipid / ml final suspension
[386] 0.5 mg and 1 mg protein / 100 mg total lipid
[387] Total lipid suspensions were prepared as described above.
[388] The immunoglobulin (monoclonal IgG acting directly on fluorescein) was contained in the material by addition to the already treated cyst suspension. After separating the amount of bound cysts and lost immunoglobulins, the relative contributions from the former were measured by fluorescent quenching in the isolated, original control solution. This allows the final IgG concentration to be maintained in each.
[389] The IgG carrier membrane binding efficiency was measured to be at least 85% for charged and highly deformable cysts and 10% for neutral soft membranes. The small differences observed are presumed to be due to the fact that IgG contains a large amount of hydrophobic Fc regions, which are easily injected into the lipid membrane even when the membrane is softened, adversely affecting the components that occur.
[390] Examples 153 to 154:
[391] Charged cysts that can deform very well, type C:
[392] 130.5 mg of phosphatidylcholine extracted from soybean
[393] 19.5 mg of cholate, sodium salt
[394] 0.1 ml ethanol
[395] Uncharged cysts that can deform very well, type T:
[396] 75mg phosphatidylcholine extracted from soybean
[397] 75mg twin 80
[398] 0.1 ml ethanol
[399] Insulin, Human Recombinant:
[400] 1.35ml Actrapides ^TM 100 (made by Novo-Nordisk)
[401] Formulation Test
[402] Some lipid mixtures were dissolved in alcohol until a homogeneous phospholipid solution was obtained (cave: sodium cholate was not completely dissolved). The mixture was injected into the insulin solution and mixed thoroughly. After the aging treatment for about 12 hours, the resulting suspension, which is composed of "large-sized cysts", is easily filtered with a 0.2 µm filter (Sartorius, Gottingen) Filtered several times).
[403] exam
[404] A male bolontier (75 kg, 42 years old) who did not eat anything was fed nothing for 17 hours before measuring the first glucose concentration. The glucose concentration in the person's blood is to be changed temporarily. A 2-4 ml sample is injected every 10-20 minutes through a soft venous catheter in the arm of the bolontier. After an initial test period of 70 minutes, when the average blood glucose concentration was 78.4, transfer type C was used (45 IU), evenly applying the undamaged skin surface inside the front of the other arm. The painted area was 56 cm 2. Thirty minutes after using the test suspension, the skin surface appeared dry when visually seen, and after thirty minutes the suspension appeared only faint traces.
[405] Standard glucose-dehydrogenase assays (Merck, Gluck-DH) were used to determine sugar in blood. It was measured at least three times with each specimen containing three respective samples. The mean standard deviation measured in this way did not exceed 5 mg / ml.
[406] result
[407] After dosing of insulin in combination with the transfer bit ⓡ (seolrin ^ⓡ transfer), change in blood sugar levels in the blood sugar is normal bolron tier dyeotda always more than that obtained by injection of insulin solution was paha.
[408] The maximum amount of reduction of the concentration of sugar in the blood after administration of the transfer seolrin ^ⓡ using only 10% of what is obtained by reference to the general subcutaneous injection corresponding to surpasses the region of the curve below which at least 20%. In the case of suspension C of t> 3, the average suppressed amount of blood glucose level in blood was about 18 mg / ml.
[409] Suspension T was about 35% poor compared to the data measured by suspension C. Injecting phosphatidylglycerol (relative to 15% by weight relative to phosphatidylcholine) resulted in a reduction of 25% (data not shown) between the C-type and T-type substances.
[410] However, iontophoresis (Meyer, BR, Katzeff, HL, Eschbach, J., Trimmer, J., Zacharis, SR Rosen, S., Sibalis, D. Amer. J. Med. Sci. 1989, 297: 321 325) or other noninvasive insulin delivery methods that are best used to date, such as the use of transnasal sprays, begin to circulate in the blood to less than 5% and 10% of insulin molecules, respectively.
[411] Example 155:
[412] Charged cysts that can be transformed very well:
[413] Compositions Used In Examples 72-76
[414] Insulin, Human Recombinant:
[415] Aktrafeed ^™ (freeze-dried) used in Examples 72-76 (product of Novo-Nordisk)
[416] Test substances were prepared as described in Examples 61-65. One-day dosing was essentially done as described in the previous examples, but the fasting time was further extended and blood sampling started sooner (thus the experiment began with 12 hours of unmonitored fasting, The blood glucose level was monitored without any treatment, followed by a 12-hour fasting time and 16 hours of treatment with TransferSulin® with no subjects being fasted.) The application area was only 10 cm 2. There was a slight difference when this happened.
[417] Samples were taken irregularly before dosing insulin. After dosing Transfersulin®, blood samples were taken every 20 minutes over the first 4 hours and thereafter every 30 minutes. All samples were analyzed with Acutrend (Boehringer-Manheim, Germany). Three to five records were obtained at each time point. The results shown in Fig. 12 correspond to the average value of the blood sugar concentration change. Collided lines provide a 95% confidence limit.
[418] The average blood glucose level in the second "no treatment" period was 83.2 mg / dl. A significant decrease in blood glucose levels is observed within 1 hour following dosing of the drug with mixed lipid cysts with great compliance. The glucodynamic profile is similar to that measured in the test series, and the overall effect is somewhat stronger, perhaps due to the much higher drug concentration in the latter test substance test.
[419] Examples 156-158:
[420] Charged cysts that can be transformed very well:
[421] Composition of Example 153
[422] Insulin, Human Recombinant:
[423] As given in Figure 2 arrangement, the arc trad feed ^TM (Novo-Nordisk Ltd.).
[424] In the test series, the same Transfersome® batch was used to study the effects of changing the internal-batch on insulin. Dosing was carried out according to the above examples. The capacity per area was as used in the above examples.
[425] In all three experiments, the average blood glucose level was about the same. The results of this striking experiment varied widely between insulin batches. One batch was very effective, but the second was not; The third had many immediate results.
[426] Changes from small batches to batches for insulin (although this is known, but most are unknown, especially permanent in the presence of large adsorbing (carrier) surfaces) and / or motility of the insulin-carrier interaction It seems to affect efficiency. It is generally known that the rate of change of the degree of freeness of the drug is particularly sensitive to the phenomenon. Therefore, it is also important to measure the drug release rate, as well as the amount of carrier that binds lipids before serious biological testing. In test animals such as mice and rats, it is also useful for this purpose to measure glucodynamics by characterizing the material after injection. After administration of three different transfersulins®, the glucodynamics of the bolontier with normal blood glucose levels have the same configuration as transfersomes, but other insulins are relatively potent even with small changes in the nature of the original drug with respect to the biological activity of the other substances. The effect is obvious.
[427] The present invention can provide a method for manufacturing in a form suitable for the (bio) engineering and medical arts by defining beneficial factors that control the adsorption of polymers to the composite surface or the corresponding desorption rate from the surface, and also results It describes the properties of a material that is particularly suitable for practical use in a conventional form, for example, in the fields of medicine or veterinary medicine, for example, symptomology, isolation and (bio) process, biotechnology, gene proliferation, sanctions. Although it is used for stabilization, concentration, conveyance, etc., it is not limited to this.

权利要求:
Claims (57)
[1" claim-type="Currently amended] It consists of two or more substances that exhibit amphiphilicity when in contact with a suitable liquid medium and differ in solubility within the liquid medium, and when contacted with the liquid medium increases the extended surface, in particular the membrane surface, such that the amphiphilic third material molecules As a combination with
The two or more materials,
Materials that dissolve better in the liquid medium than other materials form less extended surfaces than the other materials in the combination, and
Wherein the molecules of the third material are selected to bind better with the extended surface formed by the two or more other materials than with the extended surface formed only with the other less soluble material.
[2" claim-type="Currently amended] It consists of two or more substances which are amphiphilic when contacted with a suitable liquid medium and can form extended surfaces, especially membrane surfaces, when contacted with the liquid medium in a bonded state, and molecules of other amphiphilic materials having a net charge The at least two materials characterized in that the surface is purely charged such that the net charge density of the surface and the net charge of the amphiphilic molecules bound to the surface have the same polarity (both negative or positive). Combination.
[3" claim-type="Currently amended] Two or more substances which exhibit amphiphilicity upon contact with a suitable liquid medium and which can form extended surfaces, in particular membrane surfaces, when differing in solubility and at least combined in such a solvent, To combine,
So that the material that melts better in the liquid medium than other materials forms a less extended surface than the other materials in the combination,
Allowing the third material molecule to bind better with the extended surface formed from a combination of the two materials than the extended surface formed only by another less soluble material,
A combination of two or more materials, characterized in that both the third material molecule that is easy to bind to the surface as well as the surface formed by the bound material are both charged to the cathode or to the anode.
[4" claim-type="Currently amended] 4. A method according to any one of claims 1 to 3, wherein at least one material that is self-aggregating to form an extended surface, wherein the amphiphilic material has a higher solubility in liquid than other components, in particular the self-aggregating material, in particular the difference in solubility is A combination of two or more materials comprising a material characterized in that it becomes more flexible when mixed with another material that is at least 10 times, preferably at least 100 times, in the liquid medium.
[5" claim-type="Currently amended] The method of claim 1, wherein the curvature comprises at least one amphiphilic material that can self-aggregate to form an expanded surface, which when combined with the surface increases the curvature of the surface. Combination of two or more substances, characterized in that the concentration of the substance to increase is no more than 99% of the concentration that can not form a saturation concentration or surface.
[6" claim-type="Currently amended] The method of claim 4 or 5, wherein the concentration of the more soluble or increasing curvature is 0.1% or more, preferably 1 to 80% or more, more preferably 10 to 10% of the relative concentration defined in claim 5. A combination of two or more substances, characterized by at least 60%, and most preferably 20-50%.
[7" claim-type="Currently amended] The method according to claim 5 or 6, wherein the average curvature of the surface (inverse of the average radius) is 15 nm to 5000 nm, preferably 30 nm to 1000 nm, more preferably 40 nm to 300 nm, and most preferably Is a combination characterized in that it corresponds to an average radius of 50 nm to 150 nm.
[8" claim-type="Currently amended] The combination according to any one of claims 5 to 7, wherein the surface is supported by a solid, in particular by supporting a surface having a suitable curvature or size.
[9" claim-type="Currently amended] 9. The method according to any one of claims 2 to 8, wherein the relative concentration of charged components associated with the surface is from 5 to 100 relative mole percent, more preferably 10, based on the concentration of the total surface forming amphiphilic material obtained together. ~ 80 relative mole%, most preferably 20 to 60 relative mole%.
[10" claim-type="Currently amended] 10. The method of claim 2 or 9, wherein the average charge density of the surface is 0.05 Cb / m ² to 0.5 Cb / m ² , preferably 0.075 Cb / m ² to 0.4 Cb / m ² , more preferably. Preferably 0.10 Cb / m ² to 0.35 Cb / m ² .
[11" claim-type="Currently amended] 11. The method according to any one of claims 2 to 10, wherein the background electrolyte composition and concentration, including unit or polyvalent ions, are selected to maximize the effect of the interaction between charges at the necessary binding, Preferably the combination corresponds to an ionic strength (I) of 0.02 to 0.5, more preferably 0.1 to 0.3.
[12" claim-type="Currently amended] 12. The method according to any one of the preceding claims, wherein the poorly soluble in the liquid medium and preferably the surface-forming and / or charge-carrying amphiphilic material is a lipid or lipid-like material and is well soluble in the liquid medium. Preferably increase the surface curvature, flexibility or compliance and / or wherein the charge-bearing material is the same as the surfactant or third binding material.
[13" claim-type="Currently amended] The method according to any one of claims 1 to 12, comprising a molecular arrangement in the form of fine droplets suspended or dispersed in a liquid medium and coated in one or more layers with two or more types of self-aggregating amphiphiles. The two or more substances have solubility of 10 or more, preferably 100 or more times in an aqueous liquid medium, such that the average diameter of the homogeneous aggregate of the well-soluble substance or the heterogeneous aggregate of the two substances is not well dissolved. A combination of two or more substances, characterized by being smaller than the diameter of the homogeneous aggregate of the substances.
[14" claim-type="Currently amended] The method according to any one of claims 1 to 13, wherein the total content of all the amphiphiles which can form the surface is 0.01 to 13, based on the total dry weight of the aggregate, especially when the combination is used in humans or animals. 30% by weight, preferably 0.1 to 15% by weight, most preferably 1 to 10% by weight of the combination.
[15" claim-type="Currently amended] The material according to any one of claims 1 to 14, wherein the substance forms a more expanded surface, which contains at least one (bio) compatible polar or nonpolar surface-supporting lipid, wherein the surface preferably has a dual structure. Combination characterized in that having.
[16" claim-type="Currently amended] The method according to claim 15, wherein the extended surface-forming substance is a lipid or lipoid or a corresponding synthetic lipid derived from an organism, preferably glycerides, glycerophospholipids, isoprenoidides, sphingolipids, steroids, steroids. Lean or sterols, sulfur or carbohydrate-containing lipids or any other lipids capable of forming a bilayer, in particular semi-quantized fluid fatty acids, preferably phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidyl acid, phosphatidylserine, Sphingomyelin or sphingophospholipids, glycosphingolipids (e.g. cerebromide, ceramide polyhexide, sulfatides, sphingosplasmagen), gangliosides or other glycolipids, in particular dioleoyl- , Dilinoleyl-, dilinoleyl-, dilinolenoyl-, diarachidoyl-, Synthetic lipids of dilauroyl-, dimyristoyl-, dipalmitoyl-, distearoyl or the corresponding sphingosine derivatives, or of other glycolipids, diacyl-, diallkenoyl- or dialkyl-lipids Combination characterized in that the variant.
[17" claim-type="Currently amended] The method according to any one of claims 12 to 16, wherein the surfactant is a nonionic, zwitterionic, anionic or cationic surfactant, especially a long chain fatty acid or alcohol, alkyl-tri / di / methyl-ammonium. Salts, alkylsulfonate salts, cholates, deoxycholates, glycocholates, glycodeoxycholates, monovalent salts of taurodeoxycholate or taurocholate, in particular acyl- or alkanoyls such as dodecyl-, dimethyl-aminooxide -Dimethyl-aminooxide, alkyl- or alkanoyl-N-methylglucamide, N-alkyl-N, N-dimethylglycine, 3- (acyldimethylammonio) -alkanesulfonate, N-acyl-sulfobetaamine Polyethylene-acyl ethers, in particular polyethylene-glycol-octylphenyl ethers such as nonaethylene-glycol-octylphenyl ether, nonaethylene-dodecyl ethers, in particular poly- such as octaethyleneglycol-isotridecyl ether Tylene glycol-isoacyl ethers, especially polyethylene-acyl ethers such as octaethylene dodecyl ether, such as polyethylene glycol-20-monolaurate (twin 20) or polyethylene glycol-20-sorbitan-monolate (twin 80) Polyethyleneglycol-sorbitan-acyl ether, polyhydroxyethylene-4 or 6 or 8 or 10 or 12, such as lauryl ether (in bridge series) or for example polyhydroxyethylene-8-stearate (mirror 45 In the corresponding esters or polyethoxylated caster oil 40 (Cremoper EL), such as), -laurate, or -oleate type, in particular polyhydroxyethylene-lauryl, -myristoyl-, -cetylste Polyhydroxyethylene-acyl ethers such as aryl or -oleoyl ethers, especially sorbitan-monoalkylates such as sorbitan-monolaurate (Alacel 20, span 20) (e.g. In the market), alkyl-sulfates (salts) such as acyl- or alkanoyl-N-methyl glucamides, such as decanoyl- or dodecanoyl-N-methylglucamide, lauryl- or oleoyl-sulfate, Sodium deoxycholate, sodium glycodeoxycholate, sodium oleate, sodium taurate, sodium ellidate, sodium linoleate, fatty acid salts such as sodium laurate, n-octadecylene (= oleoyl) -glycerophosphatidyl Lysophospholipids such as acids, -phosphorylglycerol or -phosphorylserine, the corresponding palmitoel oil-, elliodoyl-, basenyl- lysophospholipids or the corresponding short-chain phospholipids or other surfaces Combination characterized in that it is an active polypeptide.
[18" claim-type="Currently amended] 18. The membrane component according to any one of claims 12 to 17, wherein the surface contains a charged component at a relative concentration of 1 to 80 mol%, preferably 10 to 60 mol%, more preferably 30 to 50 mol%. The combination characterized in that.
[19" claim-type="Currently amended] 19. The phosphatidylcholine and / or phosphatidylglycerol of claim 11, wherein the phosphatidylcholine and / or phosphatidylglycerol is a surface-supporting substance and lysophosphatidyl or partially N-methylated, such as lysophosphatidyl acid or methylphosphatidyl acid, lysophosphatidylglycerol or lysophosphatidylcholine One lysophosphatidylethanolamine, cholate, deoxycholate-, glycocholate, monovalent salts of glycodeoxycholate, any other sufficiently polar sterol derivatives, laurate, myristate, palmitate, oleate, palatelate, Ellidate or some other fatty acid salt and / or -sulfobetaine, -N-glucamide or -sorbitan (arlaccel or span) surfactants are combinations characterized in that they are difficult to form extended surfaces.
[20" claim-type="Currently amended] The method according to any one of claims 1 to 19, wherein the average radius of the region enclosed by the extended surface is 15 to 5000 nm, preferably 30 to 1000 nm, most preferably 40 to 300 nm, most preferably. 50-150 nm combination.
[21" claim-type="Currently amended] 21. The method according to any one of claims 1 to 20, wherein the third material capable of bonding with the expanded surface is a repeating subunit in the form of a chain molecule such as an oligomer or a polymer, and is preferably 800 Daltons or more. A combination comprising repeating subunits having an average molecular weight of at least 1000 Daltons, most preferably at least 1500 Daltons.
[22" claim-type="Currently amended] 22. The combination according to claim 21, wherein the third substance is taken from an organism, preferably bioactive.
[23" claim-type="Currently amended] 23. The combination according to any one of claims 1 to 22, wherein the third material is inserted at the interface between the membrane and the liquid medium in contact with it and engages with the membrane-like surface.
[24" claim-type="Currently amended] 24. The method according to any one of claims 1 to 23, wherein the relative content of the third molecule and the corresponding chain molecule is 0.001 to 50%, preferably 0.5 to 25%, more preferably relative to the mass of the adsorbent surface. Since 1 to 20%, the specific ratio is a combination, characterized in that the decrease with increasing the molar mass of the chain molecule.
[25" claim-type="Currently amended] The chain molecule according to any one of claims 21 to 24, wherein the chain molecule is a protein, provided that a part of the chain molecule contains at least three functional groups or fragments having a characteristic of binding to the surface. Combination, characterized in that part is combined with the surface.
[26" claim-type="Currently amended] The combination according to any one of claims 21 to 24, wherein the chain molecule belongs to a polynucleotide group such as DNA or RNA after chemical, biochemical or genetic modification or in natural form.
[27" claim-type="Currently amended] The method according to any one of claims 21 to 24, wherein the chain molecules belong to the group of polysaccharides which undergo at least partially interaction with the surface after chemical, biochemical or genetic modification or in natural form. The combination characterized by the above.
[28" claim-type="Currently amended] The chain molecule according to any one of claims 21 to 27, wherein the chain molecule is adrenocorticostaticum, -Adrenorylticum, Androgen or Antiandrogen, Antiparacitycum, Anabolicum, Anaesticum or Anagesticum, Analepicum, Antiallergicum, Antiarmitumum, Antiarterocreroticum, Anti Asmaticum and / or Broncospamoritycum, Antibioticum, Antidrefreshboom, and / or Antipsychotic, Antidiabeticum, Antidot, Antiemeticum, Antiepirepicum, Antifibrinori Ticcum, anticonfociboom, anticholinergicum, enzymes, coenzymes or the corresponding inhibitors, antihistaminecum, antihypertonicum, biological inhibitors of drug activity, antihyponickum, anticoagulant, antimycoticum , Anti-miastenicum, anti-Morchiparkinson or Morbus Alzheimer's, antipologistikum, antirereticum, antiumeticum, anticepticum, analeticum involved in breathing Respiratory stimulants, broncholyticum, cardiotonicum, chemoteraputicum, vasodilators, kinostaticum, diureticum, gangliolium-blockers, glucocorticoids, antifluid component, hemostaticum, hypnoticum, immune Globulins or fragments thereof or other immunologically active substances, bioactive carbohydrates (derivatives), birth control pills, anti-mygranular ingredients, mineralo-corticoids, morphine-antagonists, muscle relaxants, narcoticcum, neurotherapeutic, neuro Lepticum, neurotransmitters or antagonists, peptides (derivatives), optamicums, (para) -symffericomimethicum or (para) sympericoticum, proteins (derivatives), psoriasis / neudermitis drugs , Midriaticum, psychostimulant, linoleumcum, sleep-inducing agent or antagonist, stabilizer, spasmorinticum, tumercumotaticum, urolojicum, vasoconstrictor or vasodilator, viraltaticum or Any wound-healing substance, or that which may act as a combination of the component combination according to claim.
[29" claim-type="Currently amended] The combination according to any one of claims 1 to 28, wherein the chain molecule or component of the third substance is a growth regulator.
[30" claim-type="Currently amended] 30. The method according to any one of claims 1 to 29, wherein the third component is an antibody, chitokin, lymphokine, chemokine and corresponding active site of plant, bacteria, virus, patogen or other immunogen or Some of them and combinations, characterized in that it possesses immunomodulatory power, including variants.
[31" claim-type="Currently amended] 31. The combination according to any one of claims 1 to 30, wherein said third substance component is an enzyme, a coenzyme, or some other kind of biocatalyst.
[32" claim-type="Currently amended] 32. The combination according to any one of the preceding claims, wherein the third substance component is a recognition molecule comprising interalia adherin, an antibody, catenin, serectin, capperon or part thereof.
[33" claim-type="Currently amended] 33. Combination according to any one of the preceding claims, characterized in that the component is a hormone, in particular insulin.
[34" claim-type="Currently amended] 34. The composition according to any one of the preceding claims, preferably containing 1-500 IU insulin / mL of human body type, in particular 20-400 IU insulin / mL, most preferably 50-250 IU insulin / mL. Combination characterized by doing.
[35" claim-type="Currently amended] 35. The interleukin according to any one of claims 1 to 34, containing interleukin at 0.01 mg to 20 mg interleukin / mL, preferably 0.1 mg to 15 mg interleukin / mL, most preferably 1 mg to 10 mg interleukin / mL, and IL- 2, IL-4, IL-8, IL-10, IL-12 including the interleukin is characterized in that suitable for use in humans or animals after dilution to the actual required drug concentration range if necessary.
[36" claim-type="Currently amended] IF IF, including, but not limited to, alpha, beta, gamma, preferably containing 0.1-15 mg interferon / ml and most preferably 1-10 mg interferon / ml, up to 20% by weight The final combination of the interferon of the drug concentration as necessary to the actual required concentration range, and then the combination, characterized in that it can be suitably used in humans or animals.
[37" claim-type="Currently amended] 37. The method according to any one of claims 1 to 36, after dilution prior to use, if necessary, preferably to human recombinant NGF up to a 25 mg nerve growth factor (NGF) / mL suspension or up to 25 relative weight percent by component. NGF, in particular 0.1-15 relative weight percent protein and most preferably 1 to 10 relative weight percent NGF.
[38" claim-type="Currently amended] 38. The protein according to any one of claims 1 to 37, wherein the suspension is 25 mg or less of immunoglobulin (Ig) mg / mL or 25 relative weight%, more preferably 0.1 to 10 weight% protein relative to total lipids and Most preferably, a combination containing 1 to 10% by weight of immunoglobulin, wherein the component can be used in the form of an antibody, in part of an antibody, or in the form of an active variant that can be applied to an organism.
[39" claim-type="Currently amended] In the preparation of biological, cosmetic and / or pharmacologically active agents:
Selecting two or more amphiphilic materials which, when combined with and in contact with a suitable liquid medium, can form an extended surface, in particular a membrane surface, and differ in solubility in the liquid medium; And
Causing the surface formed by the combination of materials to bind the active agent to a larger surface than the surface formed solely of low solubility material in the liquid medium and to form an extended surface than the surface formed of only one material; Characterized in the manufacturing method.
[40" claim-type="Currently amended] 40. The method of claim 39, wherein the combination of surface forming materials is produced by filtration, pressure change, mechanical homogenization, shaking, stirring, mixing, or other controlled mechanical grinding in the presence of material molecules.
[41" claim-type="Currently amended] 40. The method of claim 39, wherein the selected combination of surface forming materials is adsorbed on a suitable support solid surface or permanently contacted with the solid surface, followed by permanent contact with the liquid medium by the addition or addition of various materials in turn. Wherein at least one step of the surface-forming step is in the presence of a component which in turn binds to the solid-supported surface.
[42" claim-type="Currently amended] 39. The surface of claim 38, wherein the adsorptive surface or its predecessor, suspended or supported in a solid, is mixed with surface-forming molecules, followed by the addition of binding molecules and, if necessary, under conditions that do not damage the surface. Method for producing a first characterized by the step of binding to the surface by stirring, mixing, or incubation.
[43" claim-type="Currently amended] 43. The method according to any one of claims 39 and 42, wherein the surface which binds to the substance molecule corresponds to any one of claims 1 to 37.
[44" claim-type="Currently amended] 44. The method according to any one of claims 39 and 43, wherein the characteristics of the liquid suspension correspond to any one of claims 1 to 37.
[45" claim-type="Currently amended] In the non-invasive preparation of various substances such as diabetes treatment, growth factors, immunomodulators, enzymes, cognitive molecules or adrenocorticostatica, adrenolytica, etc .:
Wherein the surface capable of bonding with the substance molecule is formed from at least one amphiphilic substance, at least one hydrophilic fluid, at least one edge active substance or surfactant, at least one component and, optionally, other conventional components. Characterized in the manufacturing method.
[46" claim-type="Currently amended] 46. The method of claim 45, wherein the one or more edge-active substances or surfactants, one or more amphiphilic substances, one or more hydrophilic fluids and agents are separately mixed, dissolved as needed to form a solution, and the resulting Process for producing a mixture or a solution, preferably made of a material that combines with the material molecules by using mechanical energy.
[47" claim-type="Currently amended] 47. The method of claim 45 or 46, wherein the amphiphilic material is used by itself, dissolved in physiologically compatible polar solvents that can be mixed with water or water, or dissolved in solvating regulators with polar solutions. Manufacturing method characterized in that it can be.
[48" claim-type="Currently amended] 48. The method of claim 47, wherein said polar solution contains at least one edge-active substance or surfactant.
[49" claim-type="Currently amended] 49. The surface as claimed in claim 45 or 48, wherein the surface is shaken, stirred, vibrated, homogenised, sonicated, sheared, lyophilized or convenient, as required, by addition of material into the fluid phase, evaporation from the reverse phase. A manufacturing method characterized in that formed by injection or dialysis to which mechanical stress such as filtration using a driving pressure.
[50" claim-type="Currently amended] The surface of claim 49, wherein the surface is formed by filtration, and the pore size of the filtration material is 0.01 μm to 0.8 μm, preferably 0.02 μm to 0.3 μm, and most preferably 0.05 μm to 0.15 μm. , Characterized in that a plurality of filters may be used sequentially or in parallel.
[51" claim-type="Currently amended] 51. A method according to any one of claims 45 or 50, wherein the material and carrier are allowed to bond at least partially after the adsorption surface is formed.
[52" claim-type="Currently amended] 52. The method according to any one of claims 45 or 51, wherein the surface which binds to the substance molecule is prepared immediately before use from a suitable thickening agent or lyophilizer as necessary.
[53" claim-type="Currently amended] Use of a combination according to any one of claims 1 to 52 for the preparation of drug carriers, drug reservoirs, or for application in other fields of medicine or biology.
[54" claim-type="Currently amended] Use of a combination according to claim 1 or 53 for use in biotechnology or gene proliferation.
[55" claim-type="Currently amended] Use of a combination according to claim 1 or 54 for the purpose of (biological) processing or diagnostics in the field of separation processes.
[56" claim-type="Currently amended] To stabilize surface-bound molecules in the form of at least partially amphiphilic chain molecules, such as (derived) proteins, polypeptides, polynucleotides, or polysaccharides, and to use them in hydrolysis processes associated with the molecules in the surface-bound state. Use of a combination according to any one of claims 1 to 55
[57" claim-type="Currently amended] Kinematically and / or reversibly affects the bonding or separation between the surface binding molecules and the complex and compliant surface such that the higher the surface charge density and / or the greater the surface ductility and / or the greater the surface bonding density Use of the combination according to any one of claims 1 to 56, wherein the speed is higher, or vice versa, the binding speed is slower or partial molecular separation occurs.

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同族专利:

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公开号 | 公开日

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JP2002528406A|2002-09-03|

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NO20003287D0|2000-06-22|

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US20080311184A1|2008-12-18|

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HK1032745A1|2005-08-12|

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KR100464601B1|2004-12-31|

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JP4838936B2|2011-12-14|

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HU0102741A3|2002-12-28|

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HU0102741A2|2002-03-28|

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CA2309633A1|2000-05-04|

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NO20003287L|2000-08-23|

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CN1192766C|2005-03-16|

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EP1039880A1|2000-10-04|

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BR9814415A|2000-10-10|

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CN1283107A|2001-02-07|

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MXPA00006196A|2003-07-21|

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WO2000024377A1|2000-05-04|

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AU765385C|2004-05-20|

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AU1435099A|2000-05-15|

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CA2309633C|2010-12-14|

---

US20080279815A1|2008-11-13|

---

AU765385B2|2003-09-18|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1998-10-23|Application filed by 케에베 그레고르, 이데아 악티엔게젤샤프트

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1998-10-23|Priority to PCT/EP1998/006750

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2001-04-25|Publication of KR20010033518A

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2004-12-31|Application granted

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2004-12-31|Publication of KR100464601B1

优先权:

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申请号 | 申请日 | 专利标题

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PCT/EP1998/006750|WO2000024377A1|1998-10-23|1998-10-23|Method for developing, testing and using associates of macromolecules and complex aggregates for improved payload and controllable de/association rates|